Leica DM IRB User Manual

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Leica DM IRB

Instructions

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Issued in 1998 by:

Leica Microsystems Wetzlar GmbH

Ernst-Leitz-Strasse

D-35578 Wetzlar (Germany)

Responsible for contents:

Marketing MQM, product management, Tel. +49 (0) 6441-292519

Fax +49 (0) 64 41-29 2255

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Leica DM IRB

Instructions

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Copyrights

All rights to this documentation and the software it describes are owned by Leica Microsystems Wetzlar GmbH. Copying of text and illustrations – in full or in part – by printing, photostat, microfilm or other techniques, including electronic systems, is only permitted subject to the express written consent of Leica Microsystems Wetzlar GmbH.

The information contained in the following documentation represents the latest stage of technology and knowledge. We have composed the texts and illustrations with great care. However, as it is impossible to eliminate the risk of error completely, we cannot accept any kind of liability for the correctness of the contents of this manual. Nevertheless, we are always grateful to be notified of any errors.

The information in this manual may be altered without prior notice.

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Contents

Important notes on this manual ....................... 7

General safety information ............................... 8

Intended application .......................................... 10

The microscope and its components ............. 11

Key subassemblies ............................................. 11

Stands ................................................................... 12

Tube mount........................................................... 12

Tube ..................................................................... 12

Brightness adjustment ....................................... 13

Coarse and fine control ..................................... 13

Mains switch ....................................................... 13

Incident light fluorescence device .................. 13

Aperture diaphragm ........................................... 13

Condenser ............................................................ 13

Condenser height adjustment .......................... 13

Specimen stages and accessories ................. 14

Objective nosepiece and objectives ............... 14

IR/R tube adapter ................................................ 14

Eyepieces ............................................................. 14

Transmitted light illumination unit ................... 14

Field diaphragm ................................................... 14

Lamphousings ...................................................... 14

Filters .................................................................... 14

Installation site ................................................... 15

Unpacking ............................................................ 16

Installation ........................................................... 16

Assembly .............................................................. 17

Assembly tools .................................................... 17

Assembly of the

transmitted light illumination column.............. 17

Assembly of condensers ................................... 18

Assembly of IC condenser prisms ................... 21

Condenser top ..................................................... 22

Condenser 0.30 S70............................................. 22

Condenser 0.53 S23 and 0.90 S1 ....................... 23

Assembly of field diaphragm ............................ 24

Assembly of filters and filter holder ................ 24

Assembly of ICT objective prisms,

IC module and IC objective prisms .................. 25

Differences between prisms D and D1 ........... 26

Inserting the analyser/polariser ...................... 26

Inserting the fluorescence module ................. 27

Assembly of the lamp mount, mirror housing, lamphousing,

illumination telescope ........................................ 27

Assembling and exchanging

incident light lamps ............................................ 29

Lamphousing 107 L.............................................. 29

Lamphousing 106 zL ........................................... 30

Assembling and exchanging

Hg and Xe lamps ................................................. 31

Assembly of the tubes and

tube adapter IR/R ................................................ 33

Adaption of the slide overlay device

and the macro dual system ............................... 36

Inserting the eyepieces/graticules ................. 37

Inserting the photoeyepieces ........................... 38

Screwing objectives in and out........................ 38

Assembling the stages, the plane stage

and object guide.................................................. 38

The E version DM IRB/E ..................................... 41

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Contents

Learn mode .......................................................... 43

Installing the objective prisms ......................... 43

Learning the IC objective prisms ..................... 43

Installing the objectives .................................... 44

Parfocality ............................................................ 47

Dry objectives ...................................................... 47

Oil immersion objectives ................................... 47

Exiting the Learn mode ...................................... 47

Individual user adjustments .............................. 48

Installing the fluorescence filter cube ........... 49

Concluding the installation ............................... 49

Motorized objective nosepiece........................ 50

Operating modes................................................. 51

DRY and IMMERSION ........................................ 51

Changing the operating mode .......................... 51

Automatic lowering of the

objective nosepiece ........................................... 52

Brightness adjustment ...................................... 53

Electronic focus .................................................. 54

Operation .............................................................. 64

Operation

of transmitted light interference contrast ..... 78

Operation of incident light fluorescence ...... 81

Operation of filters ............................................. 88

Operation of the slide overlay device ............ 89

Operation of the macro device ........................ 90

Operation of LMC ................................................ 97

Principle of LMC ................................................. 98

Components ......................................................... 99

Assembly/adjustment ........................................ 100

Areas of application .......................................... 102

Care and maintenance ...................................... 103

Troubleshooting .................................................. 105

Basic setting for transmitted light .................. 64

Operation of objectives ..................................... 70

Operation of transmitted light.......................... 71

Operation of phase contrast ............................. 74

Operation of transmitted light darkfield ........ 76

Operation of transmitted light polarisation .. 77

Storage.................................................................. 112

Packaging and transport................................... 112

Technical description ........................................ 113

General technical data...................................... 125

Main wearing and spare parts, tools ............. 127

EU Conformity declaration ............................... 128

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Important notes on this manual

This manual is an integral part of the Leica DM IRB microscope and must be read carefully before you start using the microscope.

This manual contains important instructions and information on the operating safety and maintenance of the system. It must therefore be kept in a safe place.

Text symbols and their meaning:

(1.2)

→ p. 20

The manual is multi-lingual. Due to the spiral binding you can turn the language version you want to the front.

The DM IR is available both as a life sciences microscope and as a metallographic/industrial microscope. In cases where the function and operation are identical, the same text and illustrations are used in both the separate instruction manuals.

Numbers in brackets, e. g. (1.2) refer to illustrations, in this example Fig. 1, item 2.

Numbers with an arrow, e.g. → p. 20 refer to a

particular page in this manual.

Special safety information is indicated by the triangular symbol on the left and is given a grey background.

Caution! Operation errors can damage the

microscope and/or its accessories.

Warning of hot surface.

Explanatory note.

Not part of all configurations.

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General safety information

This instrument of safety class 1 has been built and tested according to EN VDE 0411-1, safety standards for electrical measurement, control and laboratory equipment.

To keep the microscope in this safe condition, it is essential to note the advice and warnings given in this manual.

The mains plug must only be inserted into a grounded outlet.

If an extension cord is used, it must be grounded as well. Any interruption of the ground connector inside or outside the microscope or disconnecting the ground connector can make the microscope potentially dangerous. Intentional interruption is forbidden!

n. b.:

61 010-1/IEC

1010-1/

Make sure that only fuses of the specified type and rating are used as replacements. It is forbidden to use mended fuses or to shortcircuit the fuse holder.

The instruments and accessories described in this manual have been safety-tested and checked for possible hazards. Before modifying the instrument in any way or combining it with non-Leica products not dealt with in this manual, it is essential to consult the Leica agency for your area or the main factory in Wetzlar!

Any unauthorized alteration to the microscope or use for which it was not intended will automatically terminate any warranty claim.

n. b.:

Using the ground connection, any accessories connected to the microscope which have their own and/or a different power supply can be given the same ground conductor potential. Please consult our servicing personnel if you intend to connect units without a ground conductor.

n. b.:

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n. b.:

The electric accessories of the microscope are not waterproof. If water gets inside them, it may cause electrical shock. Do not put the microscope and its accessories too near a water supply or anywhere else where water may get inside them.

Before changing fuses or lamps, always turn the mains switch off and disconnect the mains cable.

n. b.:

Protect the microscope from major temperature fluctuations. These may lead to condensation which can damage the electric and optical components.

Avoid skin contact when using immersion oil! Ask the supplier for a safety information sheet!

n. b.:

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Intended application

The new DM IRB is the logical further development of the successful inverted research microscope from Leica. It is used for examinations of cells and tissue, for micromanipulation and microinjection techniques all the way through to microdissection or confocal microscopy. The DM IRB has universal application potential, incorporating all the contrasting techniques of brightfield, darkfield, phase contrast, DIC, fluorescence and Hoffmann modulation contrast, which are all easy to use and switch between. Variable illumination and imaging light paths, HCS optics, modular accessories and a wide range of peripherals make the large DM IRB research microscope from Leica a versatile and powerful product.

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The microscope and its components

Key subassemblies

The following views of the whole microscope show and name important subassemblies of the microscope and its accessories.

Fig. 1 – 2 1 Binocular phototube, 2 Eyepiece adapter tube, 3 Eyepieces, 4 Tube mount (tube change interface), 5 Tube port for photo/TV

connection, 6 Beamsplitter switch, 7 Mains switch, 8 Brightness adjustment, 9 Lateral TV port, 10 Coaxial coarse and fine drive, 11 Fluorescence module, 12 ICT prism adjustment, 13 Sextuple objective nosepiece, 14 Centring buttons for incident light field diaphragm, 15 Field diaphragm adjustment, 16 Filters, 17 Aperture diaphragm adjustment, 18 Lamphousing mount (or mirrorhousing for two lamphousings), 19 Lamphousing, 20 Stage plate, 21 Analyser, 22 Tube lens module (Bertrand lens and magnification changer), 23 Switch rod for lateral TV port, 24 Transmitted light illumination column, 25 Condenser, 26 Transmitted light lamphousing, 27 Transmitted light field diaphragm, 28 SLR port, 29 Second lamphousing

Fig. 1 View from the right Fig. 2 View from the left

2 1 20 4

24 25

22 23

17 18

29 14

15 9

6 12

8 7

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The stand

There are 5 basic versions of the DM IRB stand, which allow over 50 microscope variants to be configured.

These 5 basic versions are: – Manual or electronic stand – With or without integrated fluorescence axis – With or without SLR front port or bottom port – Lateral photo port 100 % or 80 % – With or without integrated magnification

changer

The variants and their components, differences and uses will be explained individually in this manual. The function and operation of all microscopy techniques and the necessary accessories for the Leica DM IRB will be described and explained in detail in the Opera-

tion section of this manual.

First of all, here is a general overview:

The stand with bottom port cannot be equipped with an SLR front port. This variant is only produced at a customer’s specific request.

The electronic stand additionally offers a motorised objective nosepiece, electronic focusing, IC objective prism coding, LCD display of microscope functions and (optional) motorised filter cube changer (RF4-mot module) with electric dark flap, control panel, etc. All the above-mentioned stands are also available as fluorescence stands with integrated fluorescence axis. All the fluorescence stands (including the manual versions) can be fitted with the RF4-mot module.

Tube mount

The interface between the stand and the tube is called tube mount or tube change interface. The tube mount is compatible with DM IR tubes and the IR/R tube adapter which allows the use of DM R tubes.

Stands

The basic stand has a photo port on the left for the adaption of: TV camera, SLR camera or photomicro system. The variants offered send either 100 % or 80 % of the light to this photo port.

Besides the lateral photo port, the SLR stand also has another port facing the front (front port*) which can be equipped with either an SLR camera or a TV camera with c-mount connection.

Tube

The tube, or its tube lens, produces the primary image together with the objective. DM IR tubes consist of a basic part, the binocular part and the tube change ring. The trinocular tube also has a photo/TV port. A switchable mirror either directs the light 100 % to the eyepieces or 100 % to the photo port, or splits it 50 % /50 %.

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Brightness adjustment

Aperture diaphragm

A 12 V 100 W transformer is built into the stand for stepless regulation of brightness with the brightness control.

Coarse and fine control

The coarse and fine focus control allows fast and precise focusing of the microscope image. Focusing is done by a vertical movement of the objective nosepiece. The vertical movement range is 9 mm.

Mains switch

The mains switch is used for switching the microscope power supply on and off.

Incident light fluorescence device

The variant with incident light fluorescence device contains the integrated fluorescence axis and the lamp mount or a mirrorhousing for adaption of a second lamphousing. The fluorescence stand also comprises the fluorescence module which takes 4 filter cubes. This module is also available as a motorised variant under the name RF4-mot module (see Technical Description).

The aperture diaphragm determines the resolution, field depth and contrast of the microscope image. The best resolution is achieved when the apertures of the objectives and the condenser are roughly the same.

The aperture diaphragm in the illumination light path is not intended for adjusting the brightness of the image. This should only be done by turning the brightness adjustment knob or using the neutral light damping filter.

Condenser

The condenser is a lens system through which the light is collected and focused on the specimen underneath. The condenser is designed to utilise the numerical aperture of the objective.

Condenser height adjustment

The markings on the transmitted light illumination column indicate the height settings of the different condensers.

n. b.:

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Specimen stages and accessories

Transmitted light illumination unit

The specimen stage supports the specimens that are to be examined through the microscope. Several options are available to accommodate the wide variety of specimens examined, such as object guides, extension plates, specimen clips, scanning stage, heating stage, etc.

Objective nosepiece and objectives

The objective nosepiece is used to hold the objectives. L objectives with long working distances, for example, are specially corrected to respect different thicknesses of vessel bases. All microscope objectives from magnification

2.5x to 100x can be used. All objectives from the DM L and DM R range with 25 mm thread are compatible. The performance data of Leica objectives can be found in the chapter “Technical Data; Performance Data” or on the relevant objective lists available from your Leica agency.

IR/R tube adapter

The tube adapter is used to adapt tubes from the DM R range.

The transmitted light illumination unit consists of the lamphousing and the transmitted light illumination column. The transmitted light lamphousing comprises a precentred 12 V 100 W halogen lamp and a filter module for three swing-in filters.

Field diaphragm

The field diaphragm is used to produce Koehler illumination.

Lamphousings

A variety of lamphousings are offered for the DM IRB (for halogen, mercury or xenon lamps). The description and area of application can be found in the operation section of this manual.

Filters

Filters are generally used to enhance the contrast of the specimen and are assembled in the illumination column. A variety of different filters are easily changed.

Eyepieces

A magnified, virtual image of the actual intermediate image produced by the objective is produced with the eyepieces. They act as a magnifier.

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Installation site

The microscope should be used in a dust-free room which is free of oil and chemical fumes and extreme humidity. Also, the workplace should not be exposed to major temperature fluctuations, direct sunlight or vibrations. These may impair measurements or photographs of the microscope image.

Ambient conditions:

Temperature 10 –36 °C Relative humidity 0– 80 % to 30 °C

Microscopes in warm and humid climates need special care to prevent build-up of fungus. Further details are given in the chapters “Maintenance” and “Storage”.

n. b.:

Lamphousings* and power units* must be placed at least 10 cm away from the wall and from flammable objects.

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Unpacking

Installation

Please compare the delivery carefully with the packing note, delivery note or invoice. We strongly recommend that you keep a copy of these documents with the manual, so that you have information on the time and scope of the delivery later when ordering more equipment or when the microscope is serviced. Make sure that no small parts are left in the packing material. Some of our packing material has symbols indicating environment-friendly recycling.

n. b.:

Keep the packing material for storage and transport of the microscope and its accessories.

Try to avoid touching the lens surfaces of the optics. Any fingermarks on the glass surfaces should be removed with a soft leather or linen cloth. Even small amounts of finger perspiration can attack the surfaces of optical instruments within a short time. Further information is given in the Maintenance and Cleaning chapters.

n. b.:

● First take all the components out of the transport and packing material.

● Put the basic DM IRB stand on a desk which has enough room for it.

On no account should the microscope be connected to the power socket yet!

n. b.:

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Assembly

Assembly tools

Installation and assembly of the microscope should preferably be carried out together with a member of Leica sales or service staff. Only a few ordinary screwdrivers are required for assembly, and these are supplied with the microscope.

Fig. 3 Assembly tools 1 Cross-tip screwdriver*, 2 Hexagonal screwdriver, 3 mm, 3 Centring keys, 2 mm*, 4 Centring keys, 1.5 mm*, 5 Allen key, 3 mm*, 6 Allen key, 2.5 mm* (short version)

Assembly of the transmitted light illumination column

Wipe off the interface surface (4.3) with a dry cloth. Tilt the illumination column (4.1) slightly to the back and insert so that the pin (4.2) engages in the groove of the interface surface (4.4).

Erect the TL illumination column and secure with the 4 screws.

When screwing on the TL illumination column, do not hold onto it so that optimal alignment to the optical axis is guaranteed. The angle of tilt of the illumination column can be varied or clamped securely in the vertical position with the knurled screw (5.1).

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The lamphousing for transmitted light illumination for 12 V 100 W halogen lamps with single-lens aspherical collector and heat protection filter is an integral part of the transmitted light illumination column. The halogen lamp is preassembled. The chapter on Troubleshooting includes a description of how to assemble and change halogen lamps.

The cable on the illumination column can then be connected to the 12 V 100 W socket on the back of the microscope stand.

Assembly of condensers

The technical description of the condensers can be found in the chapter “Technical description”. All condensers of the Leica DM IRB are equipped with a 6-position rotating disc (6.2 and

8.3) and can be individually fitted with the

corresponding annular diaphragms for phase contrast (PH), darkfield (DF) or IC prisms for TL interference contrast (ICT) (10). Usually the annular diaphragms are already inserted in the condenser disc in the factory, so you will not normally have to fit them yourself. The condenser disc (11.5) is removed from the condenser by slackening the screw (11.4) on the underneath of the condenser.

Fig. 4 Assembly of transmitted light illumination column 1 Transmitted light illumination column, 2 Pin of TL illu-

mination column, 3 Support surface, 4 Groove of support surface, 5 Drill holes for fixing screws

Fig. 5 Transmitted light illumination column, back view 1 Knurled screw for clamping the transmitted light illumi-

nation column

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Insert the light rings for Phaco (identified by the code numbers 0, 1, 2, 3 and the intercept distance S of the corresponding condenser top, e. g. 2 S1) and the DF diaphragm (identified by D for darkfield and the intercept distance S of the corresponding condenser top, e. g. D S1, see table “Technical Description”) in the slots of the disc as follows:

– Slightly unscrew the two centring screws

(10.11) using the supplied centring key (12.1).

– Insert the diaphragms so that the mount fits

under the spring (10.3) of the slot.

– When the light rings are assembled, their

identification code must be visible i.e. pointing upwards (12.3, 12.4 and 12.5).

– Insert the light rings in the order 0, 1, 2, 3. The

DF diaphragm can only be inserted in a large hole.

– Using the centring keys, screw the centring

screws back in until they no longer protrude over the outer edge of the disc.

– Fit IC condenser prisms if appropriate (see

assembly of ICT objective prisms).

– Insert the plastic labels (10.7) in the disc

(12.2), allocating them to the corresponding diaphragms.

– Mark any empty holes with white labels.

Insert the disc into the condenser with notches (10.6) facing upwards – towards the aperture diaphragm (6.3 and 8.4) – and screw down (11.4).

Fig. 6 Condenser 0.30 S70 1

Condenser top 0.30 S70 (not for use with condenser base (8.1)), 2 Condenser disc, 3 Aperture diaphragm, 4 Filter holder, 5 Condenser clamp screw

Fig. 7

Condenser holder

Condenser slide changer, 2 Condenser centring screws,

3 Condenser height adjustment, 4 Condenser clamp screw, 5 Screw for clamping the condenser holder

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Fig. 8 Condenser 0.53 S23 1 Condenser base, 2 Condenser top 0.53 S23 (inter-

changeable), 3 Condenser disc, 4 Aperture diaphragm, 5 Filter holder, 6 Dovetail guide

Fig. 9 Condenser tops for condenser base (8.1) 1 Condenser top 0.53 S23, 2 Condenser top 0.90 S1, 3 Condenser top P 1.40 OIL S1, 4 Spacer ring for assembling

9.2 and 9.3

Fig. 10 6-position condenser disc, empty 1 Condenser disc with slots for light rings and IC condenser

prisms, 2 Guide groove for IC condenser prisms (2 concealed, 3 Spring, 4 Holes for centring keys, 5 Spaces for label plates, 6 Notches, 7 Label plates, 8 Light ring for darkfield, 9 IC condenser prism with 2 guide cams, 10 Light ring for phase contrast, 11 Centring screws

groove is

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Assembly of IC condenser prisms

The IC condenser prisms are assembled at the factory. The following steps are only necessary in case of a retrofit: Remove the condenser disc (11.5) by slackening the screw (11.4) on the underneath of the condenser. – Using the centring keys (12.1), slightly un-

screw the two centring screws (10.11).

– IC condenser prisms can only be inserted into

the large holes of the condenser disc which have guide grooves (10.2).

– Insert the IC condenser prisms in ascending

order, e. g. K1, K2 and so that the mount fits under the spring (10.3) in the slot and the 2 guide cams engage in the grooves of the condenser disc (10.2).

– When the prisms are inserted, their

identification code, e. g. K10, must be visible and pointing towards the

centre of the disc

(12.6 and 12.7).

– Using the centring keys, screw the centring

screws back in until they no longer protrude over the outer edge of the disc. The prism is adjusted with the left centring screw only (see operation of ICT). The right centring screw must never restrict the adjustment range.

– Assemble the light rings and DF diaphragm if

appropriate (see previous section).

– Insert the label plates (10.7) corresponding to

the relevant IC condenser prism. – Mark any empty holes with white labels. – Remove any finger marks or dust on the

prisms carefully. – Put the condenser disc back in the condenser

with the notches (10.6) facing upwards –

towards the aperture diaphragm (6.3 and 8.4).

Screw down the disc (11.4).

Fig. 11 Condenser 0.90 S1 (bottom up) 1 Condenser base, 2 Condenser top 0.90 S1, 3 Spacer ring, 4 Fixing screw for condenser disc, 5 Condenser disc

Fig. 12 6-position condenser disc, fully equipped 1 Centring keys for centring screws (in working position), 2 Label plates, 3, 4 Light rings for phase contrast, 5 Light ring for darkfield, 6, 7 IC condenser prisms, 8 Hole for brightfield

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Condenser top

The base and top of condenser 0.30 S70 form a self-contained unit (Fig. 6). The condenser top 0.30 S70 (13.4) cannot be used with the condenser base (8.1). The condenser top 0.53 S23 (8.2 and 9.1) is screwed straight on to the condenser base (8.1). A spacer ring (9.4 and 11.3) must be used for assembling the condenser tops 0.90 S1 and P 1.40 OIL S1 (9.2 and 9.3).

Assembly of the condensers to the illumination column

Condenser 0.30 S70

Tilt the TL illumination column to the back (13.1). Insert the condenser 0.30 S70 (13.4) from below into the dovetail guide of the illumination column (13.2), with the condenser top pointing towards the microscope stage. Adjust the height of the condenser until its upper edge is flush with the condenser height marking S70 on the illumination column. Secure the condenser with the supplied hexagonal screwdriver. Erect the TL illumination column.

Fig. 13 Assembly of condenser 0.30 S70 1

Transmitted light illumination column (tilted), 2 Dovetail guide, 3 Condenser height markings S1, S23 and S70, 4 Condenser 0.30 S70, 5 Condenser clamp screw, 6 Field diaphragm clamp screw, 7 Transmitted light lamphousing

Fig. 14 Assembly of condenser 0.30 S70 1 Condenser 0.30 S70 in working position (upper edge of

condenser is flush with condenser height marking S70)

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Condensers 0.53 S23 and 0.90 S1

With the illumination column tilted to the back, insert the condenser holder (15.4) into the dovetail guide of the illumination column from below (15.2). The condenser height adjustment should point to the left. Adjust the height of the condenser holder until its upper edge coincides with the condenser height marking S23 or S1 on the illumination column (16.1), depending on the condenser top used. Secure the condenser holder with the hexagonal screwdriver and clamp screw (15.5). Mount the base part of the condenser with the dovetail guide (8.6) to the slide change mechanism (7.1) of the condenser holder (17). The condenser top should point downwards and the aperture diaphragm control towards the front (17.3). Slacken the clamp screw (17.5) and push the condenser back as far as the stop. Retighten the clamp screw (17.5) slightly.

Fig. 15 Assembly of condenser holder 1 Transmitted light illumination column, 2 Dovetail guide, 3 Condenser height markings S1, S23 and S70, 4 Condenser holder, 5 Clamp screw for securing the condenser holder, 6 Clamp screw for field diaphragm module, 7 Transmitted light

lamphousing

Fig. 16 Assembly of condenser holder 1 Condenser holder in working position for condenser

0.53 S23 (upper edge of condenser holder coincides with condenser height marking S23)

Fig. 17 Assembly of 0.53 S23 condenser 1 Dovetail guide of the condenser, 2 Sliding condenser

changer, 3 Aperture diaphragm adjustment, 4 Condenser top

0.53 S23, 5 Condenser clamp screw

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Assembly of field diaphragm

Assembly of filters and filter holder

To enable Koehler illumination when using condensers 0.53 S23 and 0.90 S1, a field diaphragm has to be assembled. Insert the field diaphragm module (18.1) into the mount (Fig. 18) from below. The diaphragm adjustment (18.2) should point in the direction of the tube. Secure with clamp screw (18.3).

Fig. 18␣ ␣ Assembly of field diaphragm 1 Field diaphragm module, 2 Field diaphragm adjustment, 3 Clamp screw for securing the field diaphragm module

1 2

The Leica DM IRB is equipped with a holder with spaces for 3 filters with 40 mm diameter. The filters are already fitted into the holder at the factory. If you are retrofitting filters yourself, assemble as follows:

● Slacken the clamp screws (Fig. 19.1) and

remove the filter holder.

● Put the filters into the holder (20).

● Mount the filter holder onto the transmitted

light illumination column and secure in position with the clamp screws.

Fig. 19 Assembly of filters 1 Clamp screw for securing the filter holder

Fig. 20 Assembly of filter holder for 3 filters

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Assembling the ICT objective prisms

Assembling the IC module and IC objective prisms

The IC prism disc with the IC prisms ordered by the customer are already assembled in the microscope at the factory. In case you want to retrofit the IC prism disc, please proceed as follows:

Fig. 21 IC objective prism disc without fixing knurl 1 IC objective prism in mount, 2 Code letter (e. g. A), 3 Washer and screw, 4 Stop pin

Remove the front cover (22.2) under the objective nosepiece (23.1) after slackening the Allen screws (22.4). Insert the IC prism disc (22.1) in the mount and tighten with the two Allen screws. n. b.: insert the prism disc with the prism mount pointing downwards.

Retrofitting individual IC prisms:

Please align prisms against the stop pin (21.4) and only screw down lightly to avoid strain. Insert the prisms so that the code letter, e. g. A points upwards and is readable. Label the position of the prism on the front of the ICR prism disc with a label plate (22.5).

Examples of prisms:

Prism A – for objectives N PLAN 5x, 10x. Prisms D and D1 – both for objectives N PLAN 20x, 50x, 100x and HC PL FLUOTAR 5x – 100x.

Fig. 22 Assembly of IC objective prism module 1 IC objective prism module, 2 Cover, 3 Fixing screw, 4 Hole for fixing screws, 5 Label plates, 6 Knurled fixing knob

5 6

Abb. 23 Assembly of IC objective prism module 1 Objective nosepiece, 2 Mount for IC objective prism module, 3 Stop pins

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Differences between prism D and D1

Inserting the analyser

Prism D is the standard prism with greater shearing and therefore higher detection sensitivity for minute topological and refractive index variations in the specimen. Prism D1 has smaller shearing than prism D and a lower detection sensitivity for topological and refractive index variations. However, prism D1 is better at resolving details of fine specimen structures.

Remove the blind slide and insert the analyser

(24.2) from the left as far as the 1

clickstop.

Inserting the polariser

The polariser is inserted into the filter holder of the condenser. In addition a whole-wave compensator is applied to the back of the polariser. It is activated by turning the polariser over, in order to enable colour contrasting in polarisation or interference contrast (the com-

pensator is active when the lambda symbol λ is

visible from above).

Fig. 24 Polariser/analyser 1 Polariser POL , 2 Analyser ICT ↔

↔

Fig. 25 Condenser 0.53 S23 1 Filter holder with polariser inserted (swung out)

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Inserting the fluorescence module

The fluorescence module (Fig. 26) is part of the fluorescence stand, but is also available as a retrofit kit. To retrofit the fluorescence module, remove the blind cover from the stand. The fluorescence module can be fitted with up to four different filter cubes (26.3). They are inserted into the dovetail mount (26.2) of the fluorescence module with their engraving facing downwards (towards the turret plate). The fluorescence module is inserted on a dovetail guide into its slot on the stand by pushing it as far as the stop. One part of the fluorescence module is the anti-glare protection (27.1), which can be inserted between the tube and the stage. Proceed in the same way if you are inserting a motorised filter cube changer instead of the manual filter module. Also read the manual for the electronic version.

Assembly of the lamp mount, mirror housing, lamphousing, illumination telescope

1. Insert the lamp mount or mirror housing in the back panel and screw down with Allen screws. Engage the guide pin of the lamp mount (29.1) in the back panel of the microscope stand (28.2).

2. Hold the lamphousings 107/2, 107, 106 z against the lamphousing mount and secure with the fixing screw (Fig. 31).

3. We recommend using the illumination telescope for gas discharge lamps. This is inserted between the lamp mount and the lamphousing 106 z (30.4) and magnifies the image of the focal point of the lamp by the factor 2x in the entrance pupil of the objective. This results in a significantly higher illumination intensity for fluorescence.

Fig. 26 Fluorescence module 1 Rotatable turret, 2, 4 Dovetail mounts for filter cubes (the

numbers 1␣ –␣ 4 are markings of the assembly positions),

3 Filter cubes, 4 Display of the position in the light path, 5 Switch rod with BG 38 and light stop

Fig. 27 Anti-glare protection

Page 28

4. Connect the lamp plug to the connecting socket in the stand (28.3).

5. Insert light filters, 50 mm Ø into the 2 spaces in the lamphousing mounts (29.4).

n. b.:

Connect the appliance cable to the mains socket on the microscope stand (28.4)!

Fig. 28 Back view of microscope 1 Space for assembling a lamphousing mount or mirror

housing, 2 Hole for guide pin, 3 Socket for lamp plug, 4 Mains socket, 5 Potential equalisation

Fig. 30 Mirror housing and illumination telescope 1 Lever for mirror switching, 2 Lateral lamphousing mount

with fixing screw, 3 Back lamphousing mount with Allen screw, 4 Illumination telescope for gas discharge lamps

3 2

5 4

Fig. 29 Lamp mount 1 Guide pin, 2 Lateral lamphousing mount, 3 Dovetail ring for

mounting to stand, 4 2 spaces for light filters, 5 Allen screws for fixing

Fig. 31 Lamphousing 106 z L 1 Collector adjustment, 2 Vertical lamp adjustment, 3 Horizon- tal lamp adjustment, 4 Mount ring

3 1

Page 29

Assembling and exchanging incident light lamps

Exchanging the 12 V 100 W halogen lamp:

n. b.:

Disconnect the lamp and lamphousing from the power supply. Pull out the mains plug.

Fig. 32a –c Lamphousing 107 L 1 Mount with halogen lamp, 2 Collector

Lamphousing 107 L

Slacken the fixing screw on the cover and lift off the cover (Fig. 32a and 32b). Move the collector to the front and pull the defect 12 V 100 W lamp out of the base towards the front. Without removing its protective cover, put a new lamp into the base, without tilting, as far as it will go.

n. b.:

Leave the protective cover on the lamp until it is in position. Avoid making finger marks or wipe off immediately. Close the lamphousing.

Page 30

Lamphousing 106 z L

Slacken the fixing screw on the lid (33.10). Pull the cut-out plug slightly out of the socket and flip up lid (33.11; 33.1). Move the collector to the front and lift the defect lamp out of the base (33.1; 33.2; 33.3). For convenience, the lamp holder can be removed from the lamphousing as well. To do this, slacken the fixing screws on the lamp holder (33.10) and pull out lamp holder (Fig. 34). Without removing its protective cover, put a new lamp into the base, without tilting, as far as it will go.

n. b.:

It is important to leave the protective cover on the lamp until it is in position. Avoid making finger marks or wipe off immediately. Close the lamphousing.

Fig. 33 Lamphousing 106 z L 1 Lid, flipped up, 2 Collector, 3 12 V 100 W halogen lamp or gas discharge lamp in holder, 4, 9 Cover fixing screws, 5 Reflector, 6, 7, 8 x-y adjustment screw for reflector, 10 Fixing screws for lamp mount, 11 Socket for contact plug

8 9

10 11

2 3

Fig. 34 12 V 100W lamp holder with halogen lamp

Page 31

Assembling and exchanging incident light lamps

Assembling and exchanging Hg and Xe lamps

Power units:

Hg and Xe lamps are powered by separate power units. Please make sure to read the special manuals for these power units.

Fig. 35 Lamp holders for gas discharge lamps 1 Upper clamp, 2 Seal point of the burner, 3 Lower clamp, 4, 6 Lamp holder screws, 5 Socket for cut-out plug, 7 Protective cover

Lamphousing 106 z L

Besides the halogen lamp, the following gas discharge lamps can be used, which each require different lamp holders (Fig. 35) and power units:

Type Average life span

Hg ultra high pressure lamp Xe high pressure lamp Hg ultra high pressure lamp Hg ultra high pressure lamp

150 W

(A.C.)

175 W

(D.C. stabilised)

100 W

(D.C. stabilised/non-stabilised)

100 W

(D.C. stabilised/non-stabilised,

type 103 W/2)

100 h 400 h 200 h

300 h

Hg 50

Hg 100

Xe 75

Hg 100

Stab.

Page 32

n. b.:

Always insert the burner so that

It is extremely important to heed the following advice!

Always disconnect the power unit from the mains before assembling the lamphousing 106 z. Wait for the lamphousing to cool down for at least 15 minutes as otherwise it may explode. Never touch glass parts of the burner with your bare hands as finger perspiration burns in. Wipe off any finger perspiration and dirt with a clean cloth. Adjust the lamps immediately after ignition. Never look directly into the light path (risk of glare). Always wear the supplied gloves and face mask when assembling Xe burners (risk of explosion). Avoid switching on and off frequently, as this greatly reduces the life of the lamp. Hot Hg lamps do not ignite again until they have cooled down. It is best to keep a record of the number of hours a lamp has been in use (hour counter in the power unit) and compare it with the manufacturer’s specifications. Spent burners become discoloured and should be exchanged before the specified life expectancy has expired.

The LH 106 z L is opened by undoing the fixing screws on the lid (11.10). Pull the cut-out plug slightly out of its socket and flip up lid (11.11, 11.1).

n. b.:

the lettering is upright after insertion (different diameters of the metal base for the Hg 100 and Xe 75 burners ensure that these are always inserted the right way up).

2. if the bulb has a seal point (Fig. 35), the burner is turned so that this point will be at the side, not in the light path.

Put the upper pin of the burner between the clamps of the flexible power supply and clamp with screw (33.5). Unscrew the stud (35.3) in the holder slightly, insert the lower end of the metal base and retighten the stud.

To exchange the collector on the lamphousing 106 z: Using the focusing knob (36.1), move the collector to the rearmost position. Pull the focusing knob of the collector outwards so that the collector can be removed.

Make sure that the lamp base and the power unit have the same number. If the lamp base is marked L1, for example, L1 must also be set on the power unit to make full use of the lamp and not to shorten its life.

n. b.:

Page 33

Move the collector to the front position with the focusing knob (36.1).

n. b.:

Remove the protective cover from the burner.

Put the lamp holder with burner inserted into the lamphousing and secure with the screws (10.10). Try moving the collector (36.1): it must not touch the power lead. When closing the lamphousing, make sure that the pins of the cutout plug engage in the sockets (35.11). Retighten the screws of the lid. Push the cut-out plug in as far as it will go. Attach the lamphousing to the microscope and connect to the power unit (compare mains voltage!).

Adjust the burner immediately after ignition.

Assembly of the tubes and tube adapter IR/R

Using a screwdriver, slacken the clamp screw (37.1; 38.1; 39.1) on the side of the tube change mount on the stand, mount the tube or tube adapter IR/R (clamp screw points to the right) and align with edges parallel to the microscope (the Siedentopf binocular points upwards in a V shape). The guide pin in the tube mount of the stand must fit into the groove of the tube change interface or interface of the tube adapter IR/R. Retighten the clamp screw. The procedure is the same for mounting the tube on the IR/R adapter. Similarly, the DM R tubes can be connected via this adapter, e.g. the binocular observation and photo tube HC FSA 25 PE (41.1), viewing angle

30°.

With side port for reflecting measurement

scales and µ marks into the microscope image

(slide overlay) and for connecting the MACRODUAL ZOOM device. Field of view index up to 22. Eyepiece diameter 30 mm for HL PLAN 10x/20 or 22 eyepieces. Eyepieces with larger field of view numbers are not recommended for use on the DM IRB.

Fig. 36 Lamphousing 106 z L with Hg 100 W lamp 1 Collector focusing, 2 Lamp adjustment, vertical, 3 Lamp

adjustment, horizontal, 4 Lamp holder Hg, 5 Reflector adjustment (not visible)

The tube adapter IR HC is mounted on the tube change mount of the stand and stabilised by tightening the clamp screw.

Page 34

n. b.:

Hold on to the tube adapter until the clamp screw is tightened. Then insert the HC FSA 25 PE tube in the change mount of the tube adapter and fasten with clamp screw.

Photo port for 1 photo/TV connection (43.2) Photo port for 2 photo/TV connections (43.1)

Switchable 100 %/100 % (25.3)

All Leica DM R trinocular tubes have the following beamsplitting system: 100 % vis., 100% photo or 50 %/50 %.

The following tubes from the Leica DM R range are also adaptable:

Bino HC BSA 25 (42.1) Trino HC FSA 25 P and PR (42.2)

(P + PR = with and without back reflection)

Fig. 38 HCI 3T 22 trinocular tube with 45° viewing angle Light path: 100 % vis – switch rod

150 %– 50 % – switch rod

100 %– photo – switch rod Field of view index up to 22, eyepiece diameter 30 mm for HC PLAN 10x/20 or 22 eyepieces, interpupillary distance setting: 55 – 75 mm

1 Clamp screw, 2 Eyepiece port, 3 Siedentopf binocular part, 4 Photo/TV exit, 5 Switch rod

Fig. 37 HCI B22, binocular tube with 45° viewing angle, field

of view up to 22 mm, eyepiece diameter 30 mm for HC PLAN 10x/20 or 22 eyepieces, interpupillary distance setting: 55 – 75 mm 1 Clamp screw, 2 Eyepiece port, 3 Siedentopf binocular part

Fig. 39 HCI BV22, ergo binocular tube with 15°– 50° viewing angle, field of view index up to 22, eyepiece diameter 30 mm for HC PLAN 10x/20 or 22 eyepieces, interpupillary distance setting: 55 – 75 mm 1 Clamp screw, 2 Eyepiece port, 3 Siedentopf binocular part

4 3

5 1

Page 35

Fig. 40 1 Tube adapter R/IR HC, 2 Clamp screw

Fig. 41 Tubes from the DMR range 1 HC FSA 25 PE, 2 Side port for optical overlay, 3 Tube adapter IR HC, 4 Clamp screw for mounting the

adapter to the stand, 5 Clamp screw for mounting the tube to the adapter, 6 Photo/TV port

Fig. 42 Leica DM R HC tubes 1 HC BSA 25, 2 HC FSA 25 P + PR, 3 Beamsplitter switch rod, 4 Mount for photo adapter tube, 5 Clamp for photo adapter

tube, 6 Clickstop position for Pol eyepieces, 7 Socket for control cable (PR tube only)

1 23

2 5

Fig. 43 Photo adapter tube 1 Photo adapter tube with 2 exits, 2 Photo adapter tube with 1 exit, 3 100 % ↑ / 100 % switch rod, 4 Clamp screw

Page 36

Adaption of the slide overlay device and the macro dual system

With the Leica DM IRB inverted microscope, the slide overlay and macro devices can only be adapted onto the FSA 25 PE tube. This tube has a side flange (44.1) for mounting the reflection optics. These reflection optics are used for mechanical and optical adaption of the slide overlay device and the macro dual zoom system. The slide overlay device consists of the reflection optics (44.2), the illumination unit with 6 V 4 W halogen lamp (44.8), the standard 5 x 5 cm slide holder (44.6) and the control (44.5) for focusing the slides. The halogen lamp is powered by a separate transformer (Fig. 45). Mount the reflection optics (44.3) onto the tube flange (44.1) with the coupling ring (44.2), ensuring that the guide pin engages in the groove, and screw down. In the same way, screw the slide overlay device with coupling ring to the reflection optics, again watching the position of the guide pin.

Fig. 44 Slide overlay device on the FSA 25 PE tube (with tube adapter/45) 1 Tube flange, 2 Coupling ring of reflection optics, 3 Reflection optics, 4 Coupling ring of slide overlay device, 5 Knurled focusing ring, 6 Slide holder 5 x 5 cm, 7 Filter slot, 8 Illumination adapter tube of lamphousing

2 4 6

1 3 8

Fig. 45 Transformer

Fig. 46 Macro device on FSA 25 PE tube with tube adapter/45 1 Tube flange, 2 Coupling ring, 3 Reflection optics, 4 Coupling ring, 5 Macro adapter, 6 Screw ring, 7 Zoom setting ring 1 : 4, 8 Scale of zoom factor, 9 Scale of magnification factor of the

working distance, 10 Scale of object distance from the bottom edge of the mirror housing, 11 Mirror housing

1 3 5

7 9

1110

642

Page 37

Screw the reflection optics (46.3) to the tube flange with the coupling ring (46.2). Align the macro adapter (46.5) to the macro dual zoom and secure with the screw ring (46.6). Screw the macro adapter and macro dual zoom to the reflection optics with the coupling ring. Check that the guide pin engages in the groove.

Inserting the eyepieces

The eyepieces are inserted into the eyepiece tubes.

Use the following eyepieces only:

HC PLAN 10x/20 HC PLAN 10x/20 M HC PLAN 10x/22 HC PLAN 10x/22 M HC PLAN 12.5x/16 M Widefield 16x/14 B (M) Widefield 25x/9.5 B (M)

(a spacer ring is required for the widefield objectives)

Information on the diameter, the visible area of the specimen and the total magnification of the microscope can be found in the chapter “Technical data; performance data”.

Inserting graticules*

Be very careful to keep the optical surfaces clean. Any dust particles and finger marks will show up in the image.

The graticule diameter for all HC PLAN eyepieces is 26 mm.

Eyepieces 10x/20 M and 12.5/16 M only:

● Screw the retaining sleeve out of the under-

neath of the eyepiece.

● Insert the graticule with the coated side

facing downwards (towards the objective). If there is any lettering on the graticule it must be imaged the right way round when viewed in the later observation direction.

● Screw the retaining sleeve back in.

Eyepieces 10x/22 M only:

● Screw out the underneath of the eyepiece.

● Remove the retainer ring inside it with a blunt

blade.

● Insert the graticule with the coated side

facing downwards (towards the objective). If there is any lettering on the graticule it must be imaged the right way round when viewed in the later observation direction.

● Screw the retainer ring back in.

Important!

You can retrofit graticules yourself to the HC PLAN eyepieces in the above list.

Graticules can only be inserted in eyepieces with an adjustable eyelens = ond eyepiece should be an M type as well.

M type. The sec-

Page 38

Inserting the photoeyepieces*

The HC PLAN observation eyepieces (slot-in diameter 30 mm) are designed for direct visual observation. For the adaption of photo-micrographic equipment with a fixed magnification factor, e.g. DM LD and MPS systems, and for special TV adaption systems, special eyepieces with a slot-in diameter of 27 mm and the engraving HC...PHOTO are used (note the adapter!) See special manual for further details on adapting photo and video equipment.

If any positions remain unoccupied, close them with a screw cover to prevent dust penetrating the microscope optics.

Please note that the front lenses of the objectives point upwards and are therefore more exposed to contamination than those on upright microscopes. Therefore check fairly frequently that the front lens is clean.

A constantly updated optics sheet outlines the current range of objectives that can be used on the DM IRB/E. Ask your Leica agency for a copy.

Screwing objectives in and out

For the electronic version of the microscope, the DM IRB/E, the objectives are screwed in during the initial installation (see relevant chapter). For the manual version, proceed as follows:

Remove the screw covers from the objective threads. Screw the objectives into the openings in the eyepiece so that the magnification can be changed in steps (e.g. in the order 4, 10, 20, 40).

Fig. 47

Assembling the stages, the plane stage and object guide

Plane stage

The plane stage is fixed to the microscope with 3 screws (48.4). The object guide can be mounted to either the right or the left of the plane stage (48.2).

Fig. 48 Plane stage 1 Insert ring, 20/40 mm diameter, 2 Drill holes for mountable

object guide, 3 Drill holes for specimen clips, 4 Drill holes for securing the stage

3 3

Page 39

3-plate x/y stage

To assemble the square insert plate:

The 3-plate x/y stage no. 19, size 247 x 230 mm, x-y adjustment range 60 x 40 mm, is delivered in separate packaging and assembled as follows:

This stage is usually delivered with the DM IRM, so the description of its assembly has been taken from the DM IRM manual!

1. Screw the 3 Allen screws out of the stage support surfaces and wipe any remains of packaging or dust, etc. from the stage with a clean cloth.

2. Align the stage with the x-y drive (49.1) at the front right and lay it so that its undersurface rests on the stage support surfaces.

3. Align the drill holes in the stage over those in the support surface. If the drill holes are covered, please adjust the upper stage plate with the x-y stage drive.

4. Screw down the stage with Allen screws.

1. Insert the corner of the insert plate that is marked red (50.5) at an angle from above into the corner of the stage that is also marked red and is fitted with a spring (50.5).

n. b.:

Only press the spring at the side!

Do not press the square insert plate onto the spring from above, as the insert will then not be aligned plane-parallel to the stage and can be bent.

2. Drill holes (50.2) for attaching small biological specimen clips.

3. Insert the round stage inserts into the opening (50.1).

Fig. 49 3-plate x/y stage no. 19 without inserts 1 Stage drive, 2 Rear fixing holes, 3 Front fixing hole (not

visible, concealed by stage plate), 4 Corner with red dot and spring

Fig. 50 3-plate x/y stage 1 Insert ring, 20/40 mm diameter, 2 Drill holes for specimen

clips, 3 Drill holes for securing the stage, 4 Coaxial drive for specimen positioning with universal joint, 5 Red markings

3 5 2 1 2

Page 40

Rotary stage and insert frame for coverslips

Connecting the microscope to the mains

The rotary stage is secured with 3 screws. The rotary mount has to be moved to make all the screw holes accessible. Align the screws (51.2).

n. b.:

Washers (51.3) should be used as well for the back drill holes. Only screw the screws in lightly, as the rotary stage first has to be pressed into the centre. This is done by inserting the centration aid (51.4) into the rotary stage. Engage the Bertrand lens by turning the knurl and focus with the lever. Move the stage until the bright circle is in the centre of the field of view. Then secure the stage in position, disengage the Bertrand lens and remove the centration aid. To secure specimen slides in the frame inserts (52.1), press on the middle of the leaf spring (52.2) and slide in the coverslip in the direction of the arrow. Clamp the frame insert in the object guide (51.1).

When you have installed all the components as described, you can connect the microscope to the power supply with the mains cable.

If you have the manual version of the DM IRB, installation is now complete and you can jump to the “Operation” chapter.

If you have a DM IRB/E model (i.e. the electronic version), you have to set up the system.

The following chapter describes how to set up the electronic version of the DM IR: the DM IRB/E.

Fig. 51 Rotary stage 1 Object guide, 2 Screws for securing the stage, 3 Washers, 4 Centration aid

Fig. 52 1 Frame insert for coverslips, 2 Leaf spring

Page 41

The E version DM IRB/E

Features of the Leica DM IRB/E

The Leica DM IRB/E offers the following additional functions:

For users who want to program the Leica DM IRB/E microscope themselves, a free software development kit called “Leica SDK“ is available for Windows 3.11, Windows 95 and Windows NT on request.

– Motorised, sextuple objective nosepiece – Electronic focusing – Coding of the IC objective prisms* – Motorised fluorescence filter cube change

with electrically operated dark flap*

– Control panel for remote control of the

microscope components*

– Footswitch* (in connection with control panel

or DM STC stage drive)

– LC display of microscope functions.

This manual applies to the Leica DM IRB/E inverted research microscope with Eprom version numbers:

Master (M) 2.40 Nosepiece (R) 2.30

Z drive (Z) 2.40 Deviations will therefore naturally occur for previous or subsequent versions.

* The EPROM version number is displayed by simultaneously

pressing the “LEARN“ and “CHANGE“ keys (Fig. 53b), and by pressing the “CHANGE” key afterwards the 3 EPROMS can be read out in succession.

Important note:

Before using a brand new Leica DM IRB/E microscope for the first time, an initial installation has to be carried out.

Function and operation

Straight after the Leica DM IRB/E microscope is switched on, the system will be initialised. This takes a few seconds. A message to this effect will appear in the LC display on the front of the microscope.

Page 42

Assembly and initial installation

The assembly of the individual components, such as transmitted light illumination column, condenser, etc. has been completed.

n. b.:

The objectives should not be screwed in at this point. The best time to do this is when executing the learn mode. Generally you are free to choose the order in which the separate steps of the learn mode are carried out. However, for the first installation we recommend you keep to the following order: Check that the focus threshold and the lower threshold are deleted, i. e. neither of the two symbols may appear in the display.

If necessary, delete them by sustaining the relevant keys (␣␣␣␣ , Fig. 53b) for longer than 1 sec. Using the lower focus key, behind the handwheel on the right, move the nosepiece to the lower stop.

Fig. 53a

see Fig. 53b

Fig. 53b Controls

LEARN STEP

Lower Z position

-1.86mm S1 10 0 xPH3 HH I

CHANGE

Focus position

Focus stepwidth (S0, S1, S2, S3, SC)

Page 43

Learn mode

After switching on, the microscope is in the normal operation mode.

↔

10 0xPH3 l

Normal operation mode

The learn mode is switched on with the “LEARN” key. The objective nosepiece rotates through 180° so that the current objective is in the most accessible position (furthest to the right on the outside). This function also serves for cleaning, assembling, immersing, etc. the objective.

Learn:

PARF OB J L /R

E X I T

The “EXIT” option in the display flashes. If you press the “LEARN” key again, the current objective is rotated back into the observation position without any parameters being changed. Besides this, you automatically switch back to the normal operation mode.

Installing the objective prisms

If your system is not equipped for interference contrast, skip this section and the next and continue reading at “Installing the objectives”. The IC objective prisms are normally put in the turret at the factory. If you are retrofitting TL interference contrast, refer to the instructions on page 16 of the DM IRB manual.

Learning the IC objective prisms (IC turret)

Using the “CHANGE” key, select the ICT option in the Learn mode. Keep pressing the “CHANGE” key until the second learn menu appears and the ICT position in the display flashes. Confirm by pressing the “LEARN” key.

Learn:

I CT FLUO

Input menu of learn mode

E X I T

Page 44

Turn the IC turret (situated under the objective nosepiece) until it clicks into the brightfield position (H). Operate the focus handwheel until the letter H appears on the display panel as well. Turn the IC turret by a quarter of a rotation into the next clickstop area. The message “IC prism 2” appears in the display. Read the marking on the turret and set the electronic display to the same code by turning the handwheel. Do this for all four positions. Empty positions should be coded “–”.

Installing the objectives

Select the “Objectives” option in the Learn mode (OBJ) by pressing the “CHANGE” key; the “OBJ” option now flashes. Confirm your choice with the “LEARN” key.

Ob j e c t i ve 1 :

PHO EX I

Learn mode: Objective data – Magnification

↔

I C Pr i sm 1 :

H E

Learn mode: IC turret

Conclude the Learn mode for the IC turret by pressing the “CHANGE” key. “EXIT” flashes in the display; confirm with the “LEARN” key.

X IT

Select objective “1” by pressing the objective change keys (behind the focus handwheel on the left).

Page 45

Now screw the objective with the lowest magnification into the nosepiece opening which is furthest to the right.

Display: Objective magnification

By turning the focus handwheel, select the number in the electronic display that corresponds to the magnification of the objective.

Display: Phase contrast

By pressing the “CHANGE” key, select the display field for phase contrast.

Ob j e c t i ve 1 :

Learn mode: Objective data – Phase contrast

By turning the focus handwheel, select the display that corresponds to the engraving on the objective (PH1, PH2 . . .). The symbol “--” is for brightfield objectives.

PHO

EX I

↔

Display: IC objective prism

By pressing the “CHANGE” key, select the display field for the IC objective prism.

Ob j ec t i v e 1:

5xPHO EX I

Learn mode: Objective data – IC coding (code letter)

By turning the focus handwheel, select the display that corresponds to the top line of engraving on the objective (A, B, C, D, E, F). The symbol “H” (Hellfeld, = brightfield), is for objectives that are not suitable for IC. The choice of objective prism that can be set here is confined to the IC prisms that are actually on the IC turret and that have been learned.

↔

Page 46

Operating modes: Dry/Immersion

To ensure simple yet safe objective change, the objectives have to be classified in one of the following three categories:

1. Dry objectives (D) = all dry objectives with a short working distance (< = 3 mm).

2. Immersion objectives (I).

3. Combined objectives (C) = dry objectives with a long free working distance (> 3 mm), objectives which can be used for scanning purposes as well through an oil layer.

By pressing the “CHANGE” key, select the display box for the operating mode. Now select the valid objective category for the objective you are using (D, I, C) by turning the focus handwheel.

Ob j e c t i v e 1:

↔

To learn further objectives:

Select objective no. 2 with the upper objective nosepiece key. Screw the objective with the next highest magnification into the nosepiece opening which is now furthest to the right.

By turning the handwheel, select the magnification display that matches the objective, as you did for the first objective. Proceed in the same way for setting the Phaco display, the IC prism display and the operating mode. Then repeat the setting procedures for the other objectives. Nosepiece positions that are not occupied by an objective are given the code “--”. This has the effect that these positions are not travelled to in standard mode.

Conclude the Learn mode for the objective parameters by pressing the “CHANGE” key. “EXIT” flashes in the display; confirm with the “LEARN” key.

5xPHO EX I

Learn mode: Objective data – Operating mode

Now all the objective parameters for the first objective have been learned, and the other objectives can be installed.

Before selecting the “Parfocality” option in the Learn mode, you should take the following steps: – If you want to use a specimen holder on your

stage, fit it now. – Put a specimen on the stage. – Switch to the highest magnification and focus

the image of the specimen. – Set the focus position with the

(→ Fig. 53b). You can now begin with learning the parfocality.

key

Page 47

Parfocality

Oil immersion objectives

Select the “Parfocality” option (“PARF”) in the Learn mode by pressing the “CHANGE” key; the “PARF” option now flashes. Confirm your choice with the “LEARN” key.

1 : 5x S1 EX I

ADJUST & LEARN

Learn mode: Parfocality

Dry objectives

Select the dry objective with the highest magnification by pressing the objective nosepiece keys (behind the focus handwheel on the left). Focus the specimen with the focus handwheel. Use the “STEP” key to select the suitable focus stepwidth, also using the focus keys if necessary (behind the handwheel on the right). When the focus position is set, the z drive stops near the focal plane. Confirm the learned focus position by pressing the “LEARN” key. “ADJUSTED” appears in the display. Now select the dry objective with the next lower magnification. Focus the specimen again with the focus handwheel and confirm with “LEARN”. Repeat this procedure until you have reached the smallest dry objective. For low-power dry objectives (5x, 10x) it is advisable not to correct the focus any further, as these objectives are focused immediately after switching on.

Once the parfocality has been learnt for all the dry objectives it can be done for the immersion objectives. Please remember that if the specimen is very small and lightweight it must be fixed onto the stage to prevent it being moved by the adhesive force of the oil. Select the oil immersion objective with the objective changing key. Apply a drop of immersion oil to this objective. To do this, you can move the objective to the most easily accessed outer position with one of the objective changing keys. Return the objective to the working position and focus. Confirm the learnt focus position by pressing the “LEARN” key. “ADJUSTED” appears in the display. Proceed similarly for any other immersion objectives.

Conclude the parfocality setting by selecting the “EXIT” option with the “CHANGE” key and confirming with “LEARN”.

Exiting the Learn mode

To leave the Learn mode, select “EXIT” and confirm with “LEARN”.

Page 48

Individual user adjustments

Select the option “L/R” in the Learn mode by pressing the “CHANGE” key and confirm your choice by pressing the “LEARN” key.

Switches:

EX I

Z-drive Objective Nosepiece move keys keys ment when the Chosen function

right left up

right left down

left right up

left right down

right handwheel is

turned clockwise

Ob j e c t . < > F oc us

Focus <> Obj ec t .

b j ec t . F ocu s

Learn mode: User adjustment

This option allows you to choose whether you want to operate the objective nosepiece on the left or the right side of the microscope. The function of the focus keys then also shifts to the other side of the microscope.

It is also possible to reverse the rotation direction of the handwheel and its effect on the focusing direction.

↔

Possible combinations for user adjustment

By turning the focus wheel, choose the one of the four possible combinations that suits you best.

The standard setting made at the factory is as follows (= combination 1 in the above table):

– The nosepiece is operated on the left side of

the microscope; accordingly the keys for

lowering and refocusing are on the right.

– Rotation direction of the handwheel and fo-

cusing movement:

If the handwheel on the right of the micro-

scope is rotated clockwise, the objective

nosepiece is moved upwards, i. e. the objec-

tive moves towards the sample.

Conclude the Learn mode for user adjustment by pressing the “CHANGE” key. “EXIT” flashes in the display; confirm with “LEARN”.

Page 49

Installing the fluorescence filter cube

Select the “FLUO” option in the Learn mode by pressing the “CHANGE” key. Confirm by pressing the “LEARN” key.

Fil terblock 1:

EX I T

Learn mode: Fluorescence

Pull out the filter cube drawer on the left side of the microscope stand and put the filter cube you want to use in the holder of the fluorescence turret plate in the light path. The filter cube must click noticeably in position. Now select the corresponding filter cube name in the LC display by rotating the focus handwheel.

I 3

Learning other filters: Select the next filter cube position by pressing one of the “FLUO” keys on the control panel of the FLUO module. Insert the filter cube and select the corresponding name in the display. Repeat the procedure for any other filters. Unoccupied positions are given the code “-”.

To conclude the Learn mode for the fluorescence module, press the “CHANGE” key. “EXIT” then flashes in the display; confirm with “LEARN”. Now leave the learn mode by pressing “EXIT” and confirming with “LEARN”.

Concluding the installation

Installation is now complete. You are back in the normal operation mode.

Before you start work, you should set the focus threshold with one of your specimen slides. (with the key, → Fig. 53b).

Page 50

Motorized objective nosepiece

The electronic nosepiece control allows easy and safe change of the objective magnification. Objectives are changed with 2 push buttons (objective changing keys) which are easily accessed behind the focusing handwheel on the left of the microscope.

Left side of microscope

The upper key is pressed to increase the magnification, the lower key to decrease the magnification. Short pressure on the key switches to the next lower or higher magnification. If you sustain the key for longer then 0.3 sec., the display jumps to the next higher or lower magnification every 0.5 sec. The nosepiece is not actually turned until you choose a specific magnification by releasing the key. All you have to do to switch from any higher magnification down to survey magnification, for example, is to sustain the lower objective key for approx. 3 sec. The selected objective is turned into the light path in the direction that involves the shortest travel distance.

n. b.:

When installing the system (see “Individual user adjustments”, → p. 48) it is possible to operate the objective change function on the right side of the microscope instead of the left.

Page 51

Operating modes

DRY and IMMERSION

The Leica DM IRB microscope is equipped with a switch function between the operating modes “Dry” and “Immmersion” (IMM) to ensure straightforward, error-free operation. This prevents

– dry objectives from being immersed in oil by

mistake

– immersion objectives from being used without

immersion oil by mistake.

Changing the operating mode

The operating modes are switched by simultaneously pressing the keys “lower z position”

and “focus position” on the control panel of the microscope. This means that immersion cannot be travelled to in Dry mode and dry objectives with a short working distance cannot be travelled to in Immersion mode.

To ensure smooth operation, immersion oil must be applied to all oil immersion objectives in the nosepiece that are to be used before you start work.

Contamination of the dry objectives is prevented by the fact that the objective nosepiece is always lowered before objectives are changed.

When switching from one operating mode to the other, please proceed as follows:

Starting in the DRY mode: (Display at the bottom right in the LD display: D) Press the keys “lower z position” ”focus position” microscope simultaneously to switch from Dry to Immersion.

The objective nosepiece is lowered and the message “Change Objective” appears in the display. The corresponding oil immersion objective is switched into the light path with the objective changing key (normally the upper objective changing key). From now on, only oil immersion objectives or objectives of the “Combined” category are travelled to. (“I” now appears in the LC display at the bottom right).

on the control panel of the

and

Page 52

The procedure for switching to the Dry mode is analogous: Again, press the keys “lower z position”

and “focus position” on the control panel of the microscope simultaneously to switch from “Immersion” to “Dry”.

The objective nosepiece is lowered and the message “Change Objective” appears in the display. You now have the opportunity to put a new specimen slide (without immersion oil) on the stage. Then, using the objective changing key, switch the appropriate dry objective into the light path (normally the lower objective changing key). From now on, only dry objectives or objectives of the “Combined” category are travelled to. (“D” now appears in the LC display at the bottom right).

To learn objective categories, see “Installing the objectives” (→ p. 44).

Automatic lowering of the objective nosepiece

In order to be able to operate the objective changing keys easily and without touching the stage in situations where space is difficult, e.g. if there are small object inserts in the stage and/or if the specimen plane is relatively high above the stage level, the objective nosepiece is lowered before it is rotated. The end position for this lowering (= lower z position _↓) can be chosen by the user. If the lower z position is not set, the objective nosepiece is lowered by the maximum possible distance.

Page 53

Brightness adjustment

Instead of a potentiometer, Leica DM IRB/E microscopes are equipped with an incremental transducer for brightness adjustment. This means that the adjustment wheel is automatically moved from clickstop to clickstop and therefore has no end stops.

After it is switched on, the microscope is in normal operating mode. In the first line of the LC display, the current Z position is given in µm or mm on the left. You can switch to the lamp voltage display by pressing the “CHANGE” key on the control panel. The lamp voltage is displayed in volt from

2.5 – 12 V. If you press the “CHANGE” key again you return to the display of the Z position.

The lamp intensity is adjusted by rotating an adjustment wheel on the left side of the microscope. If you hadn’t already switched to the lamp voltage display, this will happen automatically when you move the wheel. Similarly, the display will switch to the Z position if the Z position is changed with the handwheel or by pressing the key.

To switch off the transmitted light illumination, the brightness is first reduced to 2.5V. Then rotate beyond the lower value. To switch on again, rotate briefly in the opposite direction.

For photomicrography we recommend a setting of 10.5V.

Page 54

Electronic focus

The electronic focus offers the user the following advantages:

– Extremely sensitive focusing, especially for

high magnifications.

– Fine focusing selectable in 4 steps; coarse

focusing can be switched on “blind” at any time. The stepwidths desired by the user (corresponding to gear ratio and sensitivity) are allocated to every single objective and automatically reset as soon as the particular objective is used.

– Fast lowering of the objective nosepiece and

exact repositioning to the previously set focal plane.

– Electronic parfocality of all objectives through

intelligent linking of motorized objective nosepiece and electronic focus drive.

The controls of the electronic focus are:

– The focusing handwheels, conventionally

positioned on both sides of the microscope.

– Two keys (focus keys) for fast lowering of the

objective nosepiece and returning to the focal plane. The keys are in a convenient position in front of the right handwheel. If both focus keys are pressed simultaneously, coarse focusing is switched (SC). The coarse focus is switched off again the moment that the two keys are pressed simultaneously again, or a different focusing speed is switched with the STEP key.

– Key for switching the focusing speed (“STEP”,

S0 = 0.05 µm, S1 = 0.1 µm, S2 = 0.7 µm, S3 = 1.5 µm) on the front of the microscope. The micrometer values always indicate the smallest stepwidth that can be carried out.

Page 55

– Key for defining the “lower Z position”.

Pressing the key for longer than 1 sec. deletes the threshold; another press of the key for longer than 1 sec. sets the current Z position as “lower threshold”.

– Key

(= focus position). Pressing the key for longer than 1 sec. deletes the threshold, another press of the key for longer than 1 sec. sets the current Z position as “upper Z threshold”.

for defining the “upper Z threshold”

Coded IC objective prisms (option)

The objective IC prisms are arranged on a turret underneath the objective nosepiece. To facilitate allocation and thus the setting of the objective prisms to the objectives, the LC display on the front of the microscope indicates both the IC prism required for the objective in the light path and the currently effective IC prism on the turret. The latter flashes if the combination is wrong.

If using the microscope without the stage plate, please note that when you replace the front fixing screw if the stage plate, it must not be screwed in fully. If it is screwed in too far it will block the focus motor. The message BLK then appears in the LC display on the front of the microscope.

n. b.:

Page 56

Motorised fluorescence filter cube change (option)

A control panel is connected to the Leica DM IRB for motorised filter cube change.

Mot. filter cube module

filter moduleShutter

F-bus

RS 232 control F-bus panel

Back of Leica DM IRB

Connection for motorised fluorescence filter cube change

F-bus

out

Control panel for mot. filter cube change

Mains unit

Three keys are used for operation:

The two “FLUOR” keys are used to switch to the adjacent filter cube. If you switch one of these keys twice, you switch by two filter cube positions.

The “SHUTTER CLOSED” key is used for opening and closing the electric shutter. The LED indicates whether the shutter is closed or not (LED lights up when closed).

Page 57

Control unit*

An electronic control unit can be connected for the remote control of individual microscope components such as objective nosepiece and Z drive.

It is operated with keys on the front of the control unit.

The control unit can also be combined with a dual footswitch.

Connecting the control unit

When using a Leica DM IRB microscope, the unit must be powered by an external power unit. The switch on the back of the unit must therefore be at EXT, and voltage is supplied via an ordinary 7 V plug-in-power supply.

(Mains unit: 7␣ –␣ 15 V, pole direction␣␣␣␣ ,

I = > 100 mA). The supplied connecting cable is connected to the MICROSCOPE socket on the control panel and CONTROLPANEL on the microsope. The CONTROLPANEL interface on the microscope is active from Eprom version number 2.4 upwards.

The connection port is on the back of the unit.

POWER

15 V DC EXT INT

Connection port of the control unit

MICROSCOPE

FOOTSWITCH

Optionally, a dual footswitch, order no. 505096, can be connected to the FOOTSWITCH socket.

Page 58

Keys on the control unit

The control unit is operated by keys.

MAG FLUOR

FILTER LAMP FIELD

OFF

SPEED

COND

FOOT

Keys on the control unit

X – Y

LAST

SHUT

LAST

FOCUS

STEP

The keys can be used to operate the following microscope components:

Component Name on the unit

Filter FILTER ((in preparation)

Lamp LAMP

Condenser COND

function not

Field diaphragm FIELD

possible on Leica DM IRB/E

Aperture diaphragm AP

Objective change MAG

Incident light fluroescence axis FLUOR

Z drive FOCUS

x-y stage X-Y

Footswitch FOOT

Several keys are allocated to each component. These are grouped together on the key panel.

Page 59

The control unit is operated in a similar way to the key-mode operation of the Leica DM IRB microscope.

Significance of individual keys:

This is also the reason why the nosepiece rotation is slightly delayed after the keys are pressed. The LAST key is used to switch to the objective that was used last.

LAMP

Brightness can be adjusted with the arrow keys: Up arrow → brighter, down arrow → darker. The ON/OFF key is used to switch the lamp off and on. When it is switched on again, the brightness that was set before it was switched off is reset.

MAG

The arrow keys are pressed to change the magnification. The objective nosepiece rotates to the next higher magnification within the same operating mode (IMM/DRY) when the upwards arrow key is pressed once. Similarly, the next lower magnification is switched to when the downwards arrow is pressed. If the same key is pressed more than once in quick succession, the nosepiece is rotated by the corresponding number of valid positions.

FLUOR

The arrow keys are used to switch from one filter cube to the next. Pressing the keys several times causes the corresponding position to be jumped to. The LAST key positions the filter cube that was set before the last keystroke. The SHUT key is used to close the dark flap.

Page 60

FOCUS

X-Y

Here the arrow keys control the movement of the Z drive. The movement speed for the fine focusing is selected with the STEP key. For safety reasons, the movement range is limited to 400 µm above the focus position and 400 µm below the lower Z position. If both arrow keys are pressed at the same time, the system switches to coarse focusing (SC). The Z drive then moves at a higher speed. Here, the movement range is limited by the set threshold (lower Z position) and at the top end by the focal plane (focus position), to prevent collision damage. The coarse focus is switched off the moment the two arrow keys are pressed simultaneously again or a specific focus speed is selected with the STEP key. The AF key is not assigned yet.

The arrow keys define the direction in which the stage is to move. Two different speed modes can be preselected (Slowmode/Fastmode) with the SPEED key. The travel speed is increased as a function of the speed mode with a predefined ramp: the longer one of the arrow keys is pressed, the faster the stage moves. The speed mode determines the maximum speed.

FOOT

The left footswitch is assigned a function via key 1, the right footswitch via key 2. After the microscope is switched on, the footswitches have the default function Magnification change. To assign a different function, first press key 1 or 2 and then the desired function key. An acoustic signal is given for correct inputs.

Page 61

Examples for the use of the footswitches

Example 2

Example 1

Switching between two magnifications with a footswitch:

Set a magnification and switch to a second magnification you would like to use. Assign the LAST function for magnification change to the left, by first pressing key 1 and then the LAST key (at MAG). Now switch between the two magnifications with the left footswitch.

Switching between two fluorescence filters with a footswitch:

Set a filter cube with the arrow keys in the FLUO box and switch to a second filter cube you would like to use. Assign the LAST function for filter cube change to the right footswitch, by first pressing key 2 and then the LAST key (at FLUO). Now switch between the two fluorescence filters with the right footswitch.

Page 62

Person sensor (option)

Switch position AUTO

The dark flap can be opened automatically via a sensor when you look through the microscope and closed again when you look away. This is achieved by mounting the person sensor (order no. 505 101) onto the right eyepiece tube and connecting the cable on the sensor to the SHUTTER socket on the control unit for motorised filter cube change.

SHUTTER

FLUO R

CL OSED

3 4

Person sensor port

The diode next to SHUTTER CLOSED indicates whether the dark flap is currently open (diode off) or closed (diode on). You can control the dark flap manually by pressing the key above SHUTTER CLOSED or with the SHUT key on the control unit.

When the microscope is switched on the dark flap is closed at first. To activate the person sensor you first have to open the dark flap manually.

The dark flap is opened when you look through the eyepiece. When you move away, it will be closed again after about 3 seconds.

Switch position LIGHT ON

The dark flap remains open (e. g. for photomicrography). If the flap is still closed, it must be opened first manually. It will then remain open.

Switch position SENSOR OFF

The person sensor is switched off. The position of the dark flap remains unchanged.

n. b.:

The shutter key on the microscope or on the control unit can be operated at any time. The SHUTTER CLOSED diode indicates whether the flap is currently open or closed. If the dark flap is closed before the automatic functions are switched on, it has to be opened manually before the automatic functions take effect.

LIGHT

Front view of the person sensor

FLUO

AUTO SENSOR

OFF

Page 63

Front controls

There is an LC display and five control keys on the front of the microscope.

Z position stepwidth

- 1 . 86

mm S1

100xPH3

Objective data

LC display

The display gives information on the following functions:

– Z position in µm or mm. – Set stepwidth for the fine focusing (S0, S1,

S2, S3 and coarse focusing = SC, can be switched on and off by simultaneously pressing both focus keys).

– Lower Z position set (symbol visible =

threshold set).

– Focus position set (symbol visible = threshold

set).

– Objective data (corresponding to the objec-

tive engraving). – magnification – Phaco (PH1, PH2, . .. ) – required objective IC prism (option).

– Currently switched IC prism or brightfield

position. – Fluo cube (option). – Operating mode (dry or immersion).

lower Z position set

focus position set

focus keys on the right side of the microscope

HH D

current IC prism or brightfield

At the factory the microscope is set so that the fast focusing is controlled with the two keys on the right side of the microscope, indicated by the symbol on the right in the upper line of the display. Similarly, if this symbol appears on the left side of the display, it means that the focusing is controlled with the two keys on the left of the microscope.

n. b.:

When the microscope is installed the influence of the rotation direction of the handwheel on the focusing direction of the objective nosepiece can be reversed (→ p. 48).

fluo cube

operating mode dry/imm

↔

Page 64

Operation

n. b.:

Basic setting for transmitted light

Switching on the halogen lamp

When using acids or other aggressive chemicals, special care should be taken. Avoid direct contact of these substances with optical and mechanical components.

Switch on the 12 V 100 W lamp at the mains switch (55.7). Adjust the brightness with the dial. The numbers are not absolute parameters, but merely serve for reproducible setting. The white dot on the dial indicates the setting for approx. 3200 °K for photography on artificial light film and for TV microscopy.

Fig. 54␣ –␣ 55 1 Binocular phototube, 2 Eyepiece tube, 3 Eyepieces, 4 Tube mount (tube interface), 5 Tube port for photo/TV connection, 6 Beamsplitter switch, 7 Mains switch, 8 Brightness adjustment, 9 Lateral TV port, 10 Coaxial coarse and fine drive, 11 Fluorescence module, 12 ICT prism adjustment, 13 Sextuple objective nosepiece, 14 Centring buttons for incident light field

diaphragm, 15 Field diaphragm adjustment, 16 Filters, 17 Aperture diaphragm adjustment, 18 Lamphousing mount (or mirrorhousing for two lamphousings), 19 Lamphousing, 20 Stage plate, 21 Analyser, 22 Tube lens module (Bertrand lens and magnification changer), 23 Switch rod for lateral TV port, 24 Transmitted light illumination column, 25 Condenser, 26 Transmitted light lamphousing, 27 Transmitted light field diaphragm, 28 SLR port, 29 Second lamphousing

Fig. 54␣ ␣ View from right side of microscope Fig. 55␣ ␣ View from left side of microscope

2 1 20 4

24 25

13 19

22 23

17 18

29 14

15 9

6 12

21 8

Page 65

Adjustment specimen

For initial microscope adjustment we recommend you use a specimen that has both high and low contrast areas.

It is easier to focus incident light fluorescence specimens in transmitted light first.

Focusing the specimen

(For the DM IRB version, please read the section about the operation of the E focus and objective nosepiece first. Here, an example of manual operation is given for each case.)

Focus the specimen you want to examine. To do this, the objective nosepiece should be lowered first. The objective is turned into the light path by rotating the black knurled knob on the nosepiece. The objective should click audibly into position.

Please be careful with high objective magnifications when focusing or making x-y adjustments!

When using objectives with a high magnification and a short working distance (from 50x), the specimen and the stage insert may be lifted and tilted. When scanning the specimen, the front lens of the objective may knock against the edge of the stage insert. Lower the coarse and fine drive if possible when turning the nosepiece and changing the objectives, in order to avoid contact between the front lens and the stage insert.

Caution!

n. b.

Focus the specimen with the coarse and fine drive, which changes the height of the objective nosepiece. The stage height remains unchanged. The total vertical travel of the nosepiece is 7 mm. In air, the focusing range extends from 2 mm below the stage surface to 5 mm above it.

One drum interval of the fine focusing corresponds to about 2 mm of the objective nosepiece.

Caution with special objectives! Here there may be contact between the stage insert and the front lens the moment the objective is moved over the edge of the inner hole of the stage insert!

Page 66

Checking of various microscope components

Operation of L objectives with correction mount

Engage or disengage the filters (54.16) according to the required brightness. If necessary, disengage the Bertrand lens by turning the knurled knob (54.22), pos. 1. Disengage the analyser (55.21), if necessary, by pulling it out partway. Disengage the filter systems, if necessary, by rotating the turret (55.11). Push in the switch rod(s) for the beamsplitter (54.23). Clamp the transmitted light illumination arm with the knurled wheel (5.1).

Fig. 56␣ ␣ Examples of objectives 1, 2 Objectives with correction mounts (Corr) for adjusting

to different vessel base thicknesses (e. g. 0.1 – 1.3 mm and 0 – 2 mm), 3 Objective with built-in iris diaphragm (1.30 = maximum aperture, 0.60 = minimum aperture), 4 Magnification colour code, 5 Knurled ring for adjusting the correction mount, 6 Knurled ring for adjusting the built-in diaphragm

Roughly set the correction mount to the thickness of the base of the vessel on the stage by turning the knurled ring. Focus the specimen with the coarse and fine drive. Then operate the correction mount until you achieve the greatest image contrast, using the fine focus if necessary.

Setting the tubes and eyepieces

Eyeglass wearers must remove (for 10x/25) or push back (for 10x/20 and 10x/22) the anti-glare protection of the eyepieces, but it should always be left on for viewers not wearing eyeglasses.

● Set the interpupillary distance on the tube by pulling apart or pushing together the eyepiece tubes until only one image can be seen with both eyes.

● Note your personal interpupillary distance.

● If using ergotubes, set the viewing angle

(15°–50°) as well by tilting the binocular viewing port. To avoid symptoms of fatigue, vary the viewing angle from time to time.

● Close any tube exits you are not using to prevent stray light disturbing the image.

123

Page 67

Binocular tube HCI B22 or HCI BV22

For eyepieces with inserted graticule only*:

● Greatly defocus the specimen or remove from the light path.

● Exactly focus the graticule by adjusting the eyelens with a relaxed eye (the eye relaxes best if you look out the window at a distant object for a moment).

● Focus the specimen, only adjusting the eyepiece with graticule.

● Then close this eye and focus the specimen by adjusting the second eyepiece only.

Only if neither eyepiece has a graticule inserted:

● Greatly defocus the specimen or remove it from the light path.

● Adjust the eyelens until the edge of the field of view appears sharp. When you adjust the eyelens a white line becomes visible round the basic part of the eyepiece. This indicates the correct position of the eyelens for viewers with normal or corrected eyesight.

Eyeglasses with multirange lenses (bifocal and progressive) must be removed for microscopy.

● Focus the specimen through the eyepieces.

Only when one eyepiece is without an adjustable eyelens:

● Exactly focus the specimen through this eyepiece first (close your other eye).

● Then focus the image by adjusting the eyelens of the second eyepiece.

Fig. 57␣ ␣ HCI B22 binocular tube, 45° viewing angle, field of view no. up to 22, eyepiece diameter 30 mm for HC PLAN 10x/20 or 22 eyepieces, interpupillary distance setting: 55␣ –␣ 75 mm 1 Eyepiece tube, 2 Eyepiece, 3 Anti-glare protection

Page 68

To correct defective eyesight:

● Look through the right-hand eyepiece tube with your right eye and sharply focus the specimen with the fine drive.

● Then look at the same area of the specimen with your left eye and rotate the left eyepiece tube until you obtain a sharp image. Do not use the fine drive for this.

● If using eyepieces with adjustable eyelenses, do not compensate for defective eyesight by adjusting the eyepiece tube, but by adjusting the eyelens of the eyepiece.

Fig. 58

HCI BV22, ergo binocular tube with 15°–50° viewing angle, field of view no. up to 22, eyepiece diameter 30 mm for HC PLAN 10x/20 or 22 eyepieces, interpupillary distance setting: 55 –75 mm 1 Clamp screw, 2 Tube port, 3 Siedentopf binocular part

Trinocular tube HCI 3T22

● Set the beamsplitter at visual observation by pushing in the switch rod. The switching positions are indicated by symbols on the side of the tube.

100␣ % vis – switch rod

150␣ % – 50␣ % – switch rod

100␣ %␣ –␣ photo – switch rod

● The eyepieces are set in exactly the same way as on the binocular tube.

● Compensate defective eyesight by adjusting the eyelens of the eyepiece.

Fig. 59 HCI 3T22, trinocular tube with 45° viewing angle Light path: 100% vis – switch rod

150 %– 50 % – switch rod

100 %– photo – switch rod Field of view no. up to 22, eyepiece diameter 30 mm for HC PLAN 10x/20 or 22 eyepieces, interpupillary distance setting: 55 –75 mm

1 Clamp screw, 2 Tube port, 3 Siedentopf binocular part, 4 Photo/TV port, 5 Switch rod

5 1

4 3

Page 69

Operation of the side photo/TV port

Operation of the front photo/TV port

The delivery comprises two alternative outfits for the lateral photo/TV exit (Fig. 59a).

One outfit has a beam split of 1 100% visual 80 % side 2 120 % visual 80 % side

The second version has a beam split of 1 100 % visual 110% side 2 110 % visual 100% side

If the switch rod (60.2) for the side port is pulled out, the beam split version no. 2 is active. If the switch rod is pushed in, beam split no. 1 applies.

Stands either with or without SLR front port can be supplied.

The beam split is as follows:

The side port is switched off, i. e. 100 % of the light goes to the visual light path:

If the switch rod (60.3) for the SLR exit is pulled, 50 % of the light goes to the SLR and 50 % to the tube.

Fig.␣ 60␣ ␣ Bertrand lens engaged␣ 1 Lever for focusing the Bertrand lens, 2 Switch lever for side port, 3 Switch lever for front port

Page 70

Operation of objectives

Immersion objectives OIL: Only use DIN/ISO standard immersion oil.

n. b.:

Observe the safety information on the immersion oil!

W: Water immersion. The special water immersion objectives with ceramic front part can be used for all hydrous solutions. IMM: Universal objective for water, glycerine and oil.

Colour coding of objectives → “Technical data”.

Locking objectives

Some immersion objectives (with knurled grip) can be locked in a shorter position. This prevents any remaining drops of immersion liquid from wetting other objectives or specimens when the nosepiece is turned.

● Press up the front part by about 2 mm.

● Lock the objective in this position by rotating

slightly.

When using the immersion objective again, remember to release the lock, as otherwise the spring mechanism that protects the specimen and objective will not work and the other objectives will no longer be parfocal with the immersion objective.

CORR objectives

These are special objectives which can be adjusted to the thickness of the coverslip.

● Roughly set the correction mount to a medium

or estimated value by turning the knurl.

● Focus the specimen.

● Adjust the correction mount until you obtain

obtimum contrast, fine-tune the focus with the fine drive if necessary. This setting may be very difficult for featureless or low-contrast areas of the specimen.

n. b.:

Page 71

Operation of transmitted light

Brightfield illumination

Illumination techniques where the empty areas of the specimen are the brightest parts are called brightfield. Absorbing specimen structures are required for brightfield imaging, i. e. most specimens will need staining. Alternatives are optical contrasting techniques such as phase or modulation contrast.

Setting the condenser

On the TL illumination column there are height markings – S70, S23 and S1 – (13.3) for setting the correct condenser height. Using the supplied hexagonal screwdriver, slacken the screw (14.1) and adjust the height of the condenser or condenser holder until its upper edge coincides with the corresponding condenser height marking on the illumination column. Retighten the condenser or condenser holder fixing screw.

Setting the aperture diaphragm

The aperture diaphragm determines the lateral resolution, field depth and contrast of the microscope image. The best contrast is obtained when the apertures of the objective and the condenser are roughly the same. When the aperture diaphragm is stopped down to be smaller than the objective aperture, resolving power is reduced, but the contrast is enhanced. A noticeable reduction in the resolving power is observed when the aperture diaphragm is stopped down to less than 0.6x of the objective aperture and should be avoided where possible.

Brightfield illumination with condenser

0.30 S70

Brightfield illumination is possible with objective magnifications of 2.5x to 40x. Turn a 10x objective into the light path and focus the specimen with the coarse and fine drive. Narrow the aperture diaphragm until you obtain the desired image contrast.

Brightfield illumination with condensers

0.53 S23 and 0.90 S1

Brightfield illumination is possible with condenser 0.53 S23 with objective magnifications from 5x to 100x, and with condenser 0.90 S1 from 10x to 100x. A P 1.40 OIL S1 condenser top is available for extremely high resolution.

Page 72

Setting Koehler illumination

Turn a 10x objective into the light path and focus the specimen. – Engage the condenser disc into the “H” =

Hellfeld = brightfield position if necessary. – Close the field diaphragm. – Adjust the height of the condenser until the

edge of the field diaphragm is sharply in focus

and also:

Centre the image of the field diaphragm in the

middle of the field of view with the two cen-

tring screws.

Open the field diaphragm until it just dis-

appears from the field of view.

When objectives are changed, the condenser

centration may have to be slightly adjusted

with the knurled screws and the field dia-

phragm reset.

The field diaphragm protects the specimen

against unnecessary heat and keeps all light

not required for imaging away from the

specimen, thereby enhancing contrast.

Fig.␣ 61␣ ␣ Koehler illumination␣ a Field diaphragm closed, not focused, not centred, b Field

diaphragm focused, but not centred, c Field diaphragm focused and centred, but diameter too small, d Diameter of field diaphragm = diameter of field of view (Koehler illumination)

Therefore it is only opened wide enough to just illuminate the observed or photographed object field. A change in magnification always necessitates adjustment of the field diaphragm.

– Narrow the aperture diaphragm until you

obtain the desired image contrast.

Fig.␣ 62␣ ␣ 1 Binocular observation and phototube, 2 Tube clamp screw, 3 Objective nosepiece, sextuple, 4 Clamp screw for SLR/TV

adapter, front port, 5 Transmitted light lamphousing, 6 Transmitted light illumination column, 7 Condenser holder, 8 Condenser 0.53 S23 with disc, 9 Screw for opening lamphousing 105, 10 Lamphousing 105, 11 Adjustment wheel for tube lens 1x, 1.5x or Bertrand lens (B), 12 Beamsplitter switch rods, 13 Coarse and fine focus

2 3

7 8

9 10

Page 73

Visual comparison of the objective and condenser apertures is done as follows: remove an eyepiece from the eyepiece tube, or engage the Bertrand lens by turning the knurled wheel (62.11), (pos. B) and focus with the lever (62.11). Close or open the aperture diaphragm until the image just shows up in the pupil (= brighter circle) of the objective. This is regarded as the standard setting, i. e. condenser aperture = objective aperture.

Replace the eyepiece or disengage the Bertrand lens. For low-contrast specimens, the aperture diaphragm can be narrowed further for clearer imaging of fainter structures. In polarisation microscopy, narrowing the aperture diaphragm usually results in stronger colours.

n. b.:

The aperture diaphragm in the illumination light path is not for adjusting image intensity.

Only use the brightness adjustment knob or neutral density filters for this.

An aperture diaphragm in the objective is normally opened fully. Narrowing it reduces the intensity and

increases field depth reduces coverslip sensitivity creates a darkfield impression alters contrast

Possible errors

Wrong coverslip thickness or wrong objective. Specimen with coverslip at the top instead of the bottom.

Aperture diaphragm opened too far or closed.

Condenser at wrong height.

Light ring switched in by mistake.

Dirty optics.

Page 74

Operation of phase contrast

Phase contrast observation

Like transmitted light darkfield and transmitted light interference contrast, phase contrast is used to produce high-contrast images of unstained specimens.

Setting phase contrast with condenser 0.30 S70

Phase contrast observation is possible with objective magnifications from 5x to 40x.

Turn a phase contrast objective (engraving e. g. PH2) of the lowest magnification into the light path and focus the specimen. If it is difficult to find the focal plane: temporarily narrow the aperture diaphragm or use a stained specimen and switch the disc to pos. H (= brightfield).

Fig.␣ 63␣ ␣

Centration for phase contrast, viewing with a Bertrand

␣

lens a Condenser in brightfield position (H), b Condenser in PH position, light ring LR not centred, c Light ring and phase ring centred

Set the light ring (64.2) in the condenser disc that corresponds to the objective engraving (PH2). Open the aperture diaphragm (= pos. PH). Move the Bertrand lens into the light path = pos. B by turning the knurled knob and focus the annular structures with the lever (Fig. 64).

Insert the two supplied centring keys into the openings of the disc on the left and right of the label plate (e.g. 3) (Fig. 64.2) and turn them until the dark ring (phase ring in the objective) coincides with the slightly narrower ring (light ring in condenser).

Then repeat the centration process for the other objective/light ring combinations. Disengage the Bertrand lens, pos. 1x.

Fig.␣ 64␣ ␣ Centration process for phase contrast/DF 1 Centring keys in working position, 2 Disc

Page 75

Setting phase contrast with condensers 0.53 S23 and 0.90 S1

Phase contrast observation is possible with condenser 0.53 S23 with objective magnifications from 5x to 100x, with condenser 0.90 S1 from 10x to 100x.

For both condensers, phase contrast is set as described as for the 0.30 S70 condenser. However, before the centration process itself, correct Koehler illumination must be set.

Possible errors

Specimen: too thick, too thin, staining too intense; refractive index of mounting medium and specimen identical, so there is no phase jump.

Specimen slide too thick, so Koehler illumination not possible.

Wedge-shaped coverglass position, so centration of light and phase ring is no longer effective.

Wrong light ring, or light ring has been put in the disc upside down.

Aperture diaphragm not open.

Light ring not centred.

Wrong light ring.

Page 76

Operation of transmitted light darkfield

Darkfield observation

Darkfield observation is not possible with condenser 0.30 S70, with condenser 0.53 S23 it is possible from 5x objective magnification, the max. usable objective aperture is 0.40. With condenser 0.90 S1, DF observation is possible from objective magnification 10x, the max. usable objective aperture is 0.75.

Objectives with higher apertures can be used if it is possible to reduce the aperture with a builtin iris diaphragm. These objectives can be recognised by the fact that the maximum and minimum apertures are given in the objective engraving and in our lists, e. g. 1.30 – 0.60.

Rotate the condenser disc to the H position (= brightfield). Focus the specimen (5x/10x objective). If the specimen plane is difficult to find, temporarily close the aperture diaphragm. Set Koehler illumination, open the aperture diaphragm as far as the stop (= pos. PH) and turn the disc to position D (= darkfield diaphragm).

If the specimen does not appear against a dark background, centre the DF diaphragm with the centring keys. To do this, insert them in the openings in the disc on the left and right of the label plate for the DF diaphragm (D) (64.2) and rotate until a homogeneous dark specimen background is produced.

Page 77

Operation of transmitted light polarisation

DL polarisation

Polarisation contrast for examining birefringent specimens is possible with condenser 0.30 S70 with objective magnifications from 2.5x to 40x, with condensers 0.53 S23 or 0.90 S1 from 5x or 10x to 100x. A P 1.40 OIL S1 condenser top is also available for extremely hgh resolution.

Crossing the polarisers

First: Set Koehler illumination. Remove the specimen from the light path; remove the Bertrand lens and fluorescence filter cube if necessary; turn the condenser disc and turret for objectiveside IC prisms to pos. H. Insert the polariser into the filter holder with the engraving facing upwards. Turn the filter holder to the right into the light path.

Push the analyser into the 2nd clickstop position in the microscope with the engraving ICT facing upwards.

Set the optimum extinction position by rotating the polariser and watching the empty field of view. Put a specimen on the stage.

For Pol colour contrast, the ICT analyser can be turned over, with the lambda engraving facing upwards, to activate a whole-wave compensator.

Page 78

Operation of transmitted light interference contrast

TL interference contrast

TL interference contrast observation is possible with condenser 0.30 S70 with objective magnifications from 10x to 40x, with condensers

0.53 S23 or 0.90 S1 from 10x to 100x. For objective 100x there is also a condenser top P 1.40 OIL S1 for extremely high resolution.

Crossing the polarisers

Remove the Bertrand lens and fluorescence filter cube from the light path if necessary; turn the condenser disc and turret for objective-side IC prisms to pos. H. Focus the specimen (20x objective). Set Koehler illumination exactly (not needed for condenser 0.30 S70). Remove the specimen from the light path.

Insert a polariser into the filter holder with the engraving facing upwards. Turn the filter holder to the right into the light path. Push the analyser into the 2nd clickstop position in the microscope with the engraving ICT facing upwards.

Set the optimum extinction position by rotating the polariser and watching the empty field of view.

Page 79

Centration of the condenser prisms

Objectives for ICT

If you have ordered a complete microscope, this adjustment will already have been made at the factory. However, it is advisable to check the centration from time to time, particularly after transport: disengage the objective-side IC prisms (pos. H).

Remove an eyepiece from the eyepiece tube. Engage the condenser-side IC prisms one after the other (the whole-wave compensator must not be active, i. e. the lambda engraving is on the bottom side of the analyser). When the centration is correct, the dark stripe must be in the centre of the pupil (= brighter circle) of the objective (Fig. 65).

If not, proceed as follows: Put one of the supplied centring keys in the disc opening on the

left of the label plate for the IC prism (e. g. 64.2) (Fig. 65) and turn it to centre the stripe.

Transmitted light interference contrast is possible with the brightfield and phase contrast objectives which have the code letter of the pupil position in the first line of engraving, e.g. A (see separate objective chart). An IC condenser prism, e.g. K6, must also be available for the objective. An up-to-date table of possible prism combinations (objective chart) is enclosed separately with each configuration.

Fig.␣ 65

Objective pupil with correctly centred compensation stripe

Fig.␣ 66 Centration for interference contrast 1 Centring keys in working position

Page 80

Choice of prisms

Choose the objective-side prism with the letter indicated in the top line of the objective engraving, e. g. C for pupil position C, by rotating the turret.

Choose the condenser-side prism that corresponds to the magnification of the objective used, e. g. pos. 40 for objective 40x, by rotating the disc.

Setting ICT contrast

Turn the objective-side prism turret to the left and right (Fig. 67). Also adjust the contrast with the aperture diaphragm. Optimum contrast for specimens with parallel structures can be obtained by rotating the specimen. Colour contrast: Turn over the analyser, so that the lambda sign can be seen on the top.

Sources of error if ICT image quality is unsatisfactory

Embedding medium, specimen slide (petri dish) or specimens (e. g. crystals, fibres) are of birefringent material. The phase shifts caused by birefringence disturb the interference contrast image. This can sometimes be remedied by rotating the specimen.

Fig.␣ 67␣

Setting ICT contrast

Page 81

Operation of incident light fluorescence

n. b.:

Only with microscope with integrated incident light fluorescence axis.

Fluorescence observation

Focus the specimen in transmitted light first, if possible (perhaps Phaco or ICT).

Select a filter cube to suit the excitation and emission spectrum of the specimen and move into the light path by rotating the turret. Open the iris diaphragm of the objective. Switch the magnification changer* (on the DM IRB-SLR and DM IRBE versions) by turning the knurl to pos. 1x. Switch off the transmitted light illumination. Open the incident light path. The switch rod should be pulled out fully. If the background is too red, engage a BG 38 filter. Push the switch rod in halfway.

The BG 38 filter should always be used for photography. When not looking through the microscope, always block the incident light path to prevent specimens fading. Push the switch rod in all the way.

The 3 clickstop positions of the switch rod mean:

Switch rod

Stop v Incident light path blocked

(light stop)

BG 38 BG filter engaged

Incident light path open

Page 82

Centration of the 12 V 100 W, Hg, Xe lamps

Lamphousing 107/2 for 12 V 100 W halogen lamp

This lamphousing is permanently set and does not require centration. However, it is essential that the lamp is aligned straight in its mount.

Lamphousing 107 L for 12 V 100 W halogen lamp

(Fig. 68) 3 alternative centration methods:

Method 1: Centration with a centring aid

On the right side of the microscope there is an adjustment window showing an image of the light source. The reflector for lamp adjustment is inserted in the filter turret instead of a filter cube and turned into the light path.

Centre the lamp as described for method 2 while watching the light source in the adjustment window.

Method 2: Centration in the rear focal plane of the objective

1. Turn a low-power objective into the light path and, using the BF reflector, focus on a strongly reflecting specimen (e. g. surface mirror) with the coarse and fine drive. Open the field and aperture diaphragm (72.1 + 72.3).

2. Remove the eyepiece from the right or left tube and look into the empty eyepiece tube.

3. Slightly reduce the light intensity until the back objective pupil (back lens surface of the objective) can be clearly seen.

4. Adjust the lamp collector (68.4) until you see the structure of the lamp filament. The filament image is divided into two with a pale stripe in the middle (Fig. 69). Please note that only the central area of the filament can be seen and that the image is very low in contrast.

Fig.␣ 68␣ ␣ Lamphousing 107 L␣ 1 Cover fixing screws, 2 Screw for horizontal adjustment, 3 Screw for vertical adjustment, 4 Collector focusing

Fig.␣ 69␣ ␣ Lamphousing 107/2 and 107L Reflection of the lamp filament (greatly schematized): the reflection is actually very low in contrast, the pale overlap area is wider and more blurred. For lamphousing 106 z the reflection is rotated by 90°.

Page 83

5. Using an Allen key, adjust the screw for horizontal adjustment (68.3) until the pale stripe of the filament image is in the centre of the pupil.

6. Then adjust the screw for vertical adjustment (68.2) to align the filament image vertically in the centre of the pupil.

5. Using the centring screws, slide the image of the filament into the middle of the centration area marked with a dot or cross, as described in Method 2.

Lamphousing 106 z L with halogen lamp, Xe and Hg lamps

Method 3: Centration in the plane of the specimen stage

1. Put a piece of paper or non-shiny piece of Leica packaging on the specimen stage and roughly focus the surface with a low-magnification objective.

2. Set the field and aperture diaphragms at the middle position.

3. Make a dot or cross on the centration area with a felt or ball point pen and slide it into the centre of the spot of light. Fix with the specimen clip if necessary.

4. Screw out the objective or turn an empty nosepiece position into the light path.

Fig.␣ 70␣ ␣ Lamphousing 106z with Hg 100W lamp␣ 1 Lamp adjustment, vertical, 2 Reflector adjustment, vertical, 3 Focusing of the reflector image, 4 Reflector adjustment, ho- rizontal, 5 Lamp adjustment, horizontal, 6 Collector focusing, 7 Cover fixing screw

(switch gas discharge lamps on and off at separate power units)

For lamphousing 106z the direct lamp image and the reflection of the reflector are focused separately and aligned to each other. Either of the above methods can be used for imaging the lamp filament or arc.

Centration of 12 V 100 W halogen lamp

Move the reflection of the filament to the side or entirely out of the light path by adjusting the centring screws on the back of the lamphousing (70.5, 71). Focus the direct image of the filament with the collector adjustment (70.1) Then, using the centring buttons, adjust the image of the filament until the centration area or rear focal plane of the objective is half filled (Fig. 71b). Then focus the reflection of the filament with the centring buttons for the reflector adjustment and align symmetrically to the direct image (Fig. 71c).

2 3 4

Risk of glare with gas discharge lamps! Use neutral density filter (see p. 56).

Page 84

Hg 100 W and Xe 75 W lamps

n. b.:

Never look straight into the light path! Remember the risk of glare when switching to the BF or Smith reflector!

Centration of Xe or Hg gas discharge lamps

Move the reflection of the discharge arc to the side or entirely out of the light path by adjusting the centring screws on the back of the lamphousing (70.2, 70.3, 70.4). Focus the direct image of the arc with the collector adjustment (70.6).

Caution:

Use the neutral density filter to reduce the intensity of the discharge arc image on the centration areas due to the risk of glare damaging the eyes.

Centre the arc images as follows:

Hg 50 W mercury lamp

Using the centring buttons (70.1, 70.5) move the direct image of the arc to the right or left of an imaginary line through the middle of the centration area. Then focus the reflection (70.3) and, using the centring buttons of the mirror adjustment (70.2, 70.4), move the reflection until it is symmetrical with the direct image (Fig. 71c).

Using the centring buttons (70.1, 70.5) move the direct image of the arc to the middle of the centration area, with the bright tip of the arc, the focal spot of the cathode, just off centre. Then focus the reflection (70.3) and, using the centring buttons of the reflector adjustment, move the reflection until it is symmetrical with the direct image (Fig. 71a, b, c). The V-shaped emissions of the arcs of the direct image and the reflection can be superimposed.

Caution:

The bright tip of the light arcs, the focal spots of the cathode, must never be projected on top of one another, as there is then a risk of explosion due to overheating. Replace spent burners in good time and dispose of in an environmentally compatible way. Open lamphousing only after cooling and disconnection from the mains. Wear gloves and mask if using Xe lamps. Hg lamps will reach their full intensity only after a few minutes, they do not ignite when hot.

Page 85

Fig. 71

Schematic diagram of the lamp centration in lamphousing 106 z (in reality the lamp images are not as sharp)

a direct lamp image, focused, but decentred b direct lamp image in correct position c indirect and direct lamp image in correct position

Halogen lamp

Hg 50 lamp

Hg 100 / Xe 75 lamp

Page 86

Centring the aperture diaphragm

Centring the field diaphragm

Turn a low to medium objective magnification 10x/20x into the light path and focus a specimen with the coarse and fine drive.

Remove an eyepiece from one of the two eyepiece tubes and look into the empty tube or move the Bertrand lens into the light path. Regulate the light intensity so that the rear objective pupil (rear lens surface of the objective) can be clearly seen.

Using the adjustment button (72.1), open the aperture diaphragm nearly to the edge of the pupil. Centre the aperture diaphragm to the edge of the pupil with the centring screws (72.2).

The aperture diaphragm influences the resolution, contrast and field depth of the microscope image. Image quality greatly depends on how carefully it is set. It may not be used for regulating the image intensity.

Turn a low to medium objective magnification 10x/20x into the light path and focus a specimen with the coarse and fine drive. Open the field diaphragm almost as far as the edge of the field of view. Using the centring buttons (72.4), centre the field diaphragm to the edge of the field of view.

The field diaphragm is imaged on the surface of the specimen, framing the illuminated field. Normally, the field diaphragm is opened until it just disappears out of the field of view. When imaging reduced picture diagonals such as in photomicrography or TV microscopy, the field diaphragm can be narrowed to frame the picture format, enhancing the image contrast. The aperture diameter of the field diaphragm remains the same for all objective magnifications.

Fig.␣ 72␣ ␣ Aperture and field diaphragm␣ 1 Aperture diaphragm adjustment, 2 Aperture diaphragm

centring screws, 3 Field diaphragm adjustment, 4 Field diaphragm centring screws

Page 87

Possible errors

Low-contrast image due to:

Weak fluorescence, insufficient brightness:

Wrongly stored, overaged or faded specimens.

Fast fading of the specimens (e. g. for FITC).

Unspecified filter combination.

Numerical aperture of the objective too low.

Eyepiece magnification too high.

Spent lamp.

Room too bright.

Trinocular tube: wrong beamsplitter setting.

Stray light due to reflections at the condenser.

Excitation bandwidth too wide.

Inspecific staining.

Fluorescing mounting medium.

Autofluorescence of objective or immersion oil.

Glass surfaces dirty.

Page 88

Operation of filters

Light filters

Up to max. 3 light filters can be inserted in the filter holder (1.16). They can be switched in and out the light path as required.

Filter Use

Grey filter Grey filters (neutral density filters) are used to

attenuate the light without influencing the colour temperature. The engraved value, e. g. N16, indicates the attentuation value. So N16 means reduction to 1/16 = 6.3 % transmission.

Green filter, panchromatic for general enhancement of contrast in black-

and-white photography.

DLF Conversion filter for colour photography with

daylight film.

ALF Enhances contrast for colour photography with

artificial light film.

VG9 (green filter ) Contrast enhancement for chromosome photo-

graphy.

Page 89

Operation of the slide overlay device

Slide overlay device

The slide overlay device is used for reflecting measurement and comparison patterns, µm marks, marker arrow, company logo and quality data etc. into the microscope image so they appear on the photograph.

Slides with the following line patterns are available:

Marker arrow Measurement scale 10 mm = 100 divisions µm marks for 2.5x – 100x objectives 10 x 10 mm grid division

You can make your own masks with any measurement and comparison line patterns, quality data, company logos, etc.

The original master has to be copied on a 35 mm negative, i. e. white line patterns on a dark background, preferably using fine-grain document film, and then framed in a customary 50 x 50 mm slide frame.

The original is imaged 2␣ :␣ 1 in the intermediate image plane of the microscope. A distance of e. g. 5 mm in the slide overlay is enlarged to 10 mm in the intermediate image plane of the microscope.

The overlay is only possible in beamsplitter position 50/50 (switch rod) in the middle position of the tube (FSA 25 PE).

The framed slide is inserted in the fitted slide holder (74.6), with the lettering on the white side of the slide facing the lamp.

The slide holder can be adjusted on all sides, so that the overlay can be positioned anywhere in the microscope image. Remember that when you move the slide, the overlay in the image will move in the opposite direction. This takes a bit of getting used to.

The white line pattern can be given a coloured background by inserting 32 mm colour filters in the filter slot (74.7).

Page 90

Operation of the macro device

Like the slide overlay device, the macro overlay (Fig. 73) only works in the 50/50 beamsplitter position (switch rod in middle position) of the FSA 25 PE tube. The microscope illumination is left switched off to avoid disturbing image brightening. The object is placed on the stage under the mirror housing of the macrodual zoom (73.11) and illuminated.

Fig.␣ 73␣ ␣ Macro device on FSA 25 PE tube with tube adapter 1 Tube flange, 2 Coupling ring, 3 Reflection optics, 4 Coupling ring, 5 Macro adapter, 6 Screw ring, 7 Zoom setting ring 1: 4, 8 Scale of zoom factor, 9 Scale of magnification factor of the

working distance, 10 Scale of object distance from the bottom edge of the mirror housing, 11 Mirror housing

Stand lamps, cold-light illuminators and fibreoptic lamps, etc. are suitable sources for microscopy. The image is observed in the microscope tube and focused by turning the knurled ring (73.10). The magnification can be changed continuously in a range of 1 : 4 by adjusting the zoom ring (73.7). When changing the magnification with the zoom control the image has to be slightly refocused with the knurled ring (73.10). The zoom magnification factors can be read on the scale (73.8). The magnification also changes when the distance between the object and the macro attachment is varied.

1 3 5

7 9

1110

642

Page 91

The total magnification in the microscope, the reproduction ratio on the photograph or TV image can be quickly and easily measured with a scale and calculated.

n.b.: For normal viewing without the macro mirrorhousing or macrodual zoom, put on the cover to avoid disturbing overlay effects.

The mirror housing (73.11) can be rotated through 360°, for example to alter the angle at which the photograph is taken. This is done by loosening the Allen screw.

The intermediate image magnification M

of the

macro object can be worked out from the eyepiece field of view and the diameter of the object field (measured with a graduated ruler) as follows:

M1 =

field of view Ø

M1 = –––––––––––– e. g. –––––––––––––––––– M =0.1

object field Ø

M1 =

z. B.

10x/20 eyepiece

object field = 200 mm

M1 =

Viewed with a 10x eyepiece, this intermediate image of 0.1x gives a total magnification of 1x in the microscope eyepiece (0.1 x 10 = 1x).

The total magnification of the film plane of a camera is derived from multiplying the intermediate image magnification M

by the

magnifications of the photo eyepiece and camera attachment, e. g.: intermediate image magnification 0.1x photo projection lens 10x camera factor 35 mm 0.32x

0.1 x10x1.32=0.32x The total magnification at the 35 mm camera of

the ORTHOMAT

E is therefore 0.32x.

Fig.␣ 74␣

Slide overlay on the FSA 25 PE tube (with tube adapter) 1 Tube flange, 2 Coupling ring of reflection optics, 3 Reflection optics, 4 Coupling ring of slide overlay device, 5 Knurled focusing ring, 6 5 x 5 cm slide holder, 7 Filter slot, 8 Illumination adapter of lamphousing

2 4 6

1 3 8

Fig.␣ 75␣ ␣ Transformer

Page 92

The total magnification can be roughly calculated with the scale divisions on the macrodual zoom:

The following factors have to be multiplied for this: – Magnification factor of the working distance

(scale (73.9), e. g. 0.11x) – Zoom factor (scale (73.8), e. g. 1x) – Correction factor of the reflection optics

(without engraving 1.17x) – Eyepiece magnification (e. g. 10x)

e. g. 0.11 x 1 x 1.17 x 10 = 1.29

The total magnification in the eyepiece would

therefore be 1.29x.

Use of the macrodual zoom as a drawing device Drawing microstructures under the microscope has the advantage over photomicrography that significant details can be highlighted and that structures can be depicted in three dimensions. This is not possible with photomicrography. Apart from this, drawing with the superimposed image method is a valuable didactic exercise. It is done by superimposing the drawing area (the area of the stage under the mirror housing of the macrodual zoom) onto the microscope image. The drawing area or sheet of paper is homogeneously illuminated with a stand lamp or table lamp. The microscope illumination and illumination of the drawing area are matched providing the lamps are adjustable; otherwise the brightness of the drawing area can be varied by altering the proximity of the lamp.

Page 93

Length measurements

The following components are required for length measurements: – Graticule with scale in eyepiece (Fig. 76) or in

the slide overlay device (Fig. 74).

– Transmitted light stage micrometer for cali-

bration.

Important: If using a magnification changer* (DM IRB-SLR and DM IRB/E stands): Remember to take the additional magnification value into consideration separately instead of extrapolating the micrometer values of the other objectives from the calibration of one objective. Measurement errors may occur if the eyepiece is not pushed into the tube as far as the stop.

Before measurement, the

micrometer value of the objective/eyepiece combination must be known, i. e. the distance in the specimen that corresponds to a scale interval in the graticule you are using.

Calibration: Align the stage micrometer and the graticule parallel to one another by rotating the eyepiece and adjust the zero marks of the two scales to exactly the same height (Fig. 76).

Read how many scale divisions of the stage micrometer correspond to how many on the microscope scale (graticule) and divide the two values.

Example: If 1.220 mm of the stage micrometer corresponds to 100 divisions of the measurement scale, the micrometer value is = 1.220 : 100 =

0.0122 mm = 12.2 µm. For extremely low objective magnifications it may be that only part of the measurement scale can be used for calibration.

Connections for TV cameras and photomicro equipment

All the variants of the Leica DM IRB stand have a photo/TV exit on the left side. There are also photo/TV exits in the trinocular tubes for vertical adaption of camera systems.

Fig. 76 Graticule scale in the eyepiece (left) and image of the stage micrometer (right)

Page 94

Various adapters are available for connecting TV cameras with c-mount or B-mount objective thread:

Recorded picture diagonal in mm with

1 inch

/3 inch

camera camera camera camera

Without zoom magnification, for 1 chip cameras

c-mount adapter 1x HC 16 11 ␣ 8 ␣ ␣6.5 541 510 c-mount adapter 0.63x HC

␣ – 17.5 12.7 ␣ 9.5 541 537 c-mount adapter 0.5x HC ␣ – ␣ ␣ – 16 12.5 541 511 c-mount adapter 0.35x HC ␣ – ␣ ␣ – – 17.1 541 512 c-mount adapter 4x HC+)␣ ␣ 4 ␣ 2.8 ␣ 2 ␣ ␣ 1.5 _

Without zoom magnification, for 1␣ –␣ 3 chip cameras

each in connection with 0.5x HC TV optics (screw connection

) c-mount adapter 1x – ␣ ␣ – 16 12.5 541706 B-mount adapter 1x ␣ – ␣ ␣ – 16 12.5 541 702 B-mount adapter 1.25x ␣ – 17.5 – –.5 541 539 F-mount adapter 1x ␣ – ␣ ␣ – 16 12.5 541540 F-mount adapter 1.25x ␣ – 17.5 – –.5 541 541 required for each: TV adapter 0.5x HC

/2 inch

/3 inch Order no.

541 706

With zoom magnification (Vario TV adapter)

c-mount, 0.32 –1.6x HC ␣ – ␣ ␣ – 19 B-mount, 0.5 –2.4x HC (SONY)

in preparation

Fig.␣ 77␣ ␣ C-mount adapter on side port 1 TV camera, 2 Adapter with c-mount thread (or B-mount

bayonet), 3 Clamp screw, 4 Photo adapter tube

++)

from zoom factor 0.42x only!

␣– ␣␣– 16–3.3 –.5 541518

124

++)

–5 18–3.8 541517

Page 95

Calculation of the magnification on the monitor

For all TV exits the magnification on the monitor can be calculated with the following formula:

DM IRB with side photo port and front port

DM IRB-SLR) and correspondingly two beam-

( splitter switch rods (81.1 and 81.2).

= objective magnification x tube factor x

x ––––––––––––––––––––

adapter magnification

monitor diameter

chip diameter of camera

Beamsplitting for photomicrography or TV microscopy

DM IRB with side photo port only and correspondingly with one beamsplitter switch rod (81.1).

– Image recording via phototube:

Switch rod pushed in = 100 % light to the tube (82.1).

– Image recording via side photo port:

Switch rod pulled out = 80 % light to the side port and 20 % light to the tube (82.2).

– Image recording via phototube or side port:

Use the upper switch rod (81.1) as for the version with

side photo port only.

Push the lower switch rod (81.2) in (82.3 and

82.4).

– Image recording via front port* (SLR/TV) or

phototube: Upper switch rod (81.1) pushed in, lower switch rod (81.2) pulled out = 50 % light to the front port and 50 % to the tube (82.5).

Fig.␣ 78␣ ␣ Adaption of the front port for the SLR camera 1 SLR adapter, 2 T2 connector ring, 3 SLR camera

Fig.␣ 79␣ ␣ Adaption of the front port for TV camera 1 TV adapter 0.63x, 2 TV camera with c-mount thread

Page 96

n. b.:

Long-term video microscopy The solid and therefore stable basic body of the stand takes a while to stabilise thermally after the microscope is switched on. For investigations taking over > 30 min. therefore, the microscope should be switched on about 1␣ – 2 hours beforehand.

Fig.␣ 81␣ ␣ Switch rods for beamsplitting 1 Upper beamsplitter switch rod (SIDE), 2 Lower beamsplitter

Fig.␣ 80␣ ␣ Leica DM IRB, equipped with three TV cameras

Fig.␣ 82␣ ␣ Beamsplitting 1 100 % light to the tube, 2 80 % light to the side photo port, 20 % to the tube, 3 100 % light to the tube, 4 80 % light to the side

photo port, 20 % to the tube, 5 50 % light to the front port, 50 % to the tube

switch rod (FRONT)

SIDE OFF

FRONT OFF

SIDE ON

FRONT OFF

SIDE OFF

FRONT ON

Page 97

Operation of LMC

Leica modulation contrast (LMC) is a special form of oblique illumination based on the principle of Hoffmann modulation contrast.

In this technique, the phase gradients of an unstained specimen are converted into differences in amplitude with the aid of a modulator. This gives a three-dimensional impression similar to an interference contrast image. Unlike interference contrast, however, the specimen can be observed through birefringent plastic materials such as petri dishes.

Further advantages of this technique are:

– high contrast – high resolution – halo-free, high-contrast relief image – long free working distance of the condenser – easy assembly and adjustment

use for both stained and unstained specimens.

–

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Principle of LMC

The principle

Leica modulation contrast (LMC) is based on the principle of Hoffmann modulation contrast. This imaging technique is particularly suitable for unstained, colourless objects with little image contrast. Such objects change the phase of the light when it passes through them. The conversion of these phase gradients into differences in amplitude results in a three-dimensional image similar to that of differential interference contrast.

To realise this technique, a light slit diaphragm and an objective with integrated modulator are required. The modulator is a filter built into the rear focal plane which divides it into three zones, a dark zone, a grey zone and a light zone.

The light coming from the light slit diaphragm is diffracted at the object into different directions, depending on the object’s refractive index gradient, so that some of the rays have to pass through the light zone of the modulator and some through the dark zone. The non-diffracted direct light passes through the grey zone and produces the grey background of the entire field of view. Most of the rays diffracted at the object pass through the light zone and produce the image.

If the condenser is set at the “brightfield” position and the specimen is removed, the dark and the grey zone can be seen at the edge of the field of view. The image of the slit diaphragm is in the light zone. To adjust, the light slit diaphragm is rotated until the bright stripe of the slit image covers the grey stripe of the modulator.

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Components

The components

LMC consists of the following components:

S40/0.50 LMC condenser

The condenser (order no. 521 225) is supplied with a condenser disc to accommodate 3 LMC diaphragms, plus two phase contrast light rings and a brightfield position (3x LMC, PH1, PH2, brightfield). Adhesive labels are enclosed for labelling the individual positions. In the front focal plane of the condenser a light slit diaphragm has already been assembled in each of the LMC positions, corresponding to the supplied objectives.

LMC objectives

The following objectives are available:

C PLAN 10x/0.22 LMC C PLAN L 20x/0.30 LMC C PLAN L 40x/0.50 LMC N PLAN L 20x/0.40 CORR LMC N PLAN L 40x/0.55 CORR LMC PL Fluotar L 63 x/0.70 CORR LMC

In the rear focal plane of the objectives a special modulator, similar to the phase contrast rings, has been fitted.

When unpacking, check that you have all the components.

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Assembly/adjustment

Assembly

When taking the following steps, consult the manual for the Leica DM IRB/E manual.

Before installing the LMC components, remove the field diaphragm. Also remove any filters, prisms and interference contrast components.

Screw the LMC objectives into the objective nosepiece.

Replace the condenser on the microscope with the S40/0.50 LMC condenser. First check that the inserted light slit diaphragms match the objectives in the nosepiece. The light slit diaphragms are labelled, for example, LMC 10, LMC 20 or LMC 40.

Example: LMC 10 diaphragms belong to the C PLAN 10x/0.22 LMC objective.

The light diaphragms are usually assembled at the factory to match the supplied objectives. If the light slit diaphragms are supplied separately, they must be inserted in the positions in the condenser disc to match the objectives used.

n. b.:

Adjustment

Open the aperture diaphragm on the condenser fully.

Switch on the light. Select a medium brightness setting.

Set the condenser to the brightfield position and turn the first LMC objective into the light path (usually the objective with the smallest magnification).

Engage the Bertrand lens using the adjustment wheel on the right side of the microscope.

You will now see the modulator built into the objective as a grey rectangle at the edge of the field of view. Its position (top, bottom, left, right) is not fixed and may vary for different objectives. Focus the image of the modulator using the Bertrand lens.

Turn the condenser to switch to the light slit diaphragm whose name corresponds to the engraving on the objective (e. g. LMV 10 for the C PLAN 10x objective). You will now see a bright rectangle.

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Leica DM IRB User Manual

Specifications and Main Features

Frequently Asked Questions

User Manual