Leica DM LSP Instructions Manual

Leica DMLSP

Instructions · Bedienungsanleitung

Mode d’emploi

MICROSYSTEMS

UK

This manual is a supplement to the main DMLS
manual, which is also supplied with every
DMLSP polarizing microscope in English,
French and German. The manual DM LS also
contains all the technical data and safety infor­mation.

A special brief instruction manual for the
DMLSP is also available in several languages.

Information about the range of objectives is
given on the separate “OPTICS” data sheet.

D

Zusätzlich ist eine spezielle Kurzanleitung
DMLSP in verschiedenen Sprachen verfügbar.

Über das Objektivprogramm informiert das ge­trennte Datenblatt „OPTIK“.

F

Cette notice est un complément du mode d’em­ploi complet Leica DMLS en français, allemand
et anglais qui est également fourni avec chaque
microscope polarisant Leica DMLSP. Le mode
d’emploi du Leica DMLS contient des données
techniques, des consignes de sécurité et des in­formations générales qui se rapportent au mi­croscope de base.

Diese Anleitung ist eine Ergänzung zur ausführli­chen Anleitung DMLS, die in Deutsch, Englisch
und Französisch jedem Polarisationsmikroskop
DMLSP ebenfalls beigefügt ist. Die Anleitung
DMLS enthält darüber hinaus sämtliche techni­sche Daten, Sicherheitshinweise und Grundla­gen zum Mikroskop.

rd

3

edition, issued in 2000 by/

3. Auflage, herausgegeben 2000 von/
Edtion 1998 par:

Leica Microsystems Wetzlar GmbH

Le mode d’emploi condensé est lui aussi dispo­nible en différentes langues.

Informations sur le programme objectif : voir
feuilles de données séparées «OPTIQUE».

Ernst-Leitz-Strasse
D-35578 Wetzlar (Germany)

Responsible for contents/
Verantwortlich für den Inhalt/
Département responsable du contenu:
Marketing MQM, Product Management

Phone/Tel./Tél. [phone]
Fax [phone]

Leica DMLSP

Instructions

MICROSYSTEMS

4

Contents

Pol components .................................................... 5

Adjustment.............................................................. 7

Operation of objectives (Pol) .............................. 8

Operation of transmitted light polarization ...... 9

Evaluation of conoscopy...................................... 14

Possible errors ...................................................... 15

Incident light techniques .................................... 15

EU conformity declaration .................................. 16

Text Symbols and their meaning:

Caution! Operation errors can dam­age the microscope and/or its acces­sories.

Not part of all configurations/option.

Numbers with an arrow, e.g. → p. 20,
refer to a particular page in this man­ual.

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

*

→ p. 20

(1.2)

5

Pol components

Components

The microscope model DMLSP (Fig. 43) differs in
the following points from the DMLS model
described on pages 5 – 51. However, all points of
the DMLS manual apply to the DM LSP as well
as long as it is equipped with the relevant com­ponents. The following components are the only
ones that cannot be used.

● Filter magazine (Fig. 11)

● Filter holder for 2 filters (Fig. 12). For combina-

tion with IC/P polarizer (43.10) only: the similar
filter holder (43.11), although for one filter only,
should be used instead.

● Heating stages

Pol tubes

Polarization tubes have a groove on the under­side into which a guide pin in the Pol microscope
stand and the Pol module engages, so that the
cross line in the → Pol eyepiece is oriented. For
this purpose, the right-hand eyepiece mount has
a snap-in groove and a mechanical compensa­tion to make sure that the cross line in the eye­piece remains oriented when the interpupillary
distance is adjusted → p. 28. The following tubes
are available, on the outside they are only slight­ly different from the normal tubes illustrated in
Fig. 35 and 37: explanation of abbreviations
→ p. 25.

● LMP--7 Monocular Pol tube

● HC LBP 0/3/4 Binocular Pol tube

● HC L1TP 4/5/7 Trinocular Pol tube with

1 beamsplitter switching position (intensity
ratio of binocular viewing port: monocular
photo/TV exit = 50 %:50 %).

● HC L3TP 4/5/7 Trinocular Pol tube with
3 switching positions (intensity ration bin: ver­tical exit 100% :0 %/50:50%/0 :100%).

Ordinary tubes can also be used for polarization
microscopy, although the orientation of the cross
line in the eyepiece is not then guaranteed. In
this case the disturbing bearing ring with latch in
the Pol eyepiece can be removed with a very
small screwdriver. If using additional graticules,
e.g. for photomicrography, you should order a
third eyepiece, as 2 different graticules in the left
and right-hand eyepieces are irritating.

Pol eyepieces

On polarized light microscopes like the DMLSP,
the (right-hand) eyepiece has a cross line which
is aligned by a latch and corresponding groove
in the right-hand tube. If aligned in a horizontal/
vertical position (Fig. 41/42) the crossline in­dicates the vibration direction of the polarizer
(east – west) and the analyser (north – south). If
the crossline is set at an angle of 45°, it indicates
the vibration direction in a birefringent specimen
when it is rotated to a diagonal position (= max.
light intensity). There is also a scale for length
measurement → p. 42.

Pol module

The Pol module (intermediate tube, 43.3; 43.4) also
has an orientation (see above). It consists of:

● a switchable analyser (43.4), orientation north
– south.

● a switchable (43.3) and centerable (43.3a)
Bertrand lens for conoscopy → p. 10, with
coupled pinhole diaphragm for conoscopic
isolation of small specimen areas.

6

● Quartz plate. This has a depolarizing effect.
If the tube is mounted directly to the micro­scope, i.e. without the Pol module and there­fore without the quartz plate, anomalous inter­ference colours (pseudodichroism) may occur
when the analyser is disengaged.

Centering nosepiece

All objectives of the quadruple centering nose­piece are centrable to the axis of rotation of the
stage with the two centering keys (1.3; 43.15),
→ p. 5.

Pol objectives

Objectives with the additional engraving P or
POL are manufactured to be particularly low in
strain. However, they may show considerable
signs of strain if subjected to rapid changes in
temperature or mechanical damage.

Pol rotary stage

Together with the centered objectives, the ball­bearing precision stage enables exact angle
measurements.

Pol condensers

The condensers (43.8) also have to be strain-free
for all examinations in polarized light. Therefore,
only the special Pol condensers CLP/PH or UCLP

0.85 (identified by the code letter P and the aper-

ture value 0.85) can be used.

Polarizer

First the switchable polarizer (43.10), rotatable
through 360° and with slot for λ or λ/4 compen­sator above (8.2) or, as a simple solution, the fil­ter holder (28.4) with slot-in polarizer can be
used.

Analyser mount TL L 1/25 (option)

(not illustrated)
In case of alternative outfits without Pol module,

the modular analyser mount TL L 1/25 (11505 121)
can be directly interposed between stand and
tube. This allows the use of fixed and rotatable
analysers (180°) in slider.

Assembly

First read the general assembly instructions on
page 5 – 21. The polarizer (43.10) is screwed onto
the underside of the condenser holder at the left.

Caution!

When assembling the intermediate tube (Pol
module, tubes) and the (right-hand) Pol eye­piece, remember to watch out for the orientation
aids (pin and groove) at the joints.

Operation of transmitted light pol

Basically all information on pages 21 – 52 is valid.
In addition, please observe the following specific
measures for polarization.

7

Method II (Fig. 42)

Move the prominent point on the specimen (42a)
to the centre of the crossline M. Rotate the stage
until the point on the specimen is furthest away
from the centre of the crosslines M (position A,
Fig. 42b). Point A (= maximum distance of the
specimen point from the centre) may even be
outside the field of view. Turning the centering
keys, adjust the image until the specimen point A
is midway (= pos. B) between pos. A and the
centre of the crosslines M (42c). Move point A to
M and check that A stays at M when the stage is
rotated (42d). Repeat the centering process if
necessary.

Each objective must be centered separately. If
an objective is screwed out of the nosepiece,
e.g. for cleaning, and screwed back in the same
place, is centration is more or less retained.

The centering keys are then stored in the two
receptacles on the stage bracket (43.15).

Adjustment

Objective centration

Objectives are centered by adjusting (43.14)
them with two Allen keys (1.3; 43.15) until the
optical axis of the objective (and therefore the
centre of the image) matches the axis of rotation
of the specimen stage (41 and 42). When an
objective is centered correctly, a focused speci­men area does not drift out of the field of view
when the stage is rotated. A specimen point in
the center of the crossline therefore does not
change positon during a complete rotation of the
stage. It is a good idea to use a specimen with
plenty of contrast and detail for the objective
centration.
Disengage the analyser and the Bertrand lens
(43.3; 43.4). Greatly narrow the aperture
diaphragm (43.7). Insert the two objective cen­tering keys above the objective (43.14) you want
to center. Focus the specimen. There are two
similar ways of centering objectives:

Method I (Fig. 41)

Rotate the stage and note the point on the speci­men that remains stationary. This point corre­sponds to the mechanical axis of rotation of the
stage.
Now move this prominent point of the specimen
to the centre of the crosslines with the two cen­tering keys. Rotate the stage and fine-adjust the
centration if necessary.

M

MM

AA

A

B

M

B

abc

d

Fig. 41

Centration method I

Fig. 42

Centration method II

8

Crossing the polarizers

Focus on an empty area of the specimen or
remove the specimen from the light path.
Remove compensators (43.10; 43.20; 28.2), Ber­trand lens (43.3) from the light path.
Turn the condenser disc of the UCLP* condens­er. In case of condenser CLP/PH* (43.8) pull out
the light ring slider to the BF = brightfield posi­tion.
Engage the analyser (43.4).

Watching the empty field of view, rotate the
polarizer (43.10) until you obtain the optimum
extinction position. The setting may be inaccu­rate if the specimen, condenser lenses or polar­izers are dirty, so clean them beforehand if nec­essary!

Fig. 43 Controls of the polarizing microscope Leica DMLSP
1 Eyepiece, with adjustable eyelens* and anti-glare protec-

tion, removable, 2 Interpupillary distance setting, 3 Bertrand
lens, off/on, 3a Centration

+

of Bertrand lens, 4 Analyser
off/on, 5 Objective, objective nosepiece, 6 Object guide* and
specimen, 7 Aperture diaphragm, 8 CLP/PH* condenser,
9 Condenser centration

+

, on left and right and condenser
fixing screw on the right, 10 Polarizer with clamp screw for
rotation, hinged, with slot for

λ or λ/4 compensator, 11 Filter

holder* with filter*, 12 Field diaphragm, 13 Brightness adjust­ment for transmitted light

+

(left, hidden in the illustration),
14 Objective centration, 15 Holes for keeping centering keys
when not in use, 16 Condenser height adjustment

+

,

17 Condenser height stop, 18 Focusing

+

, fine and coarse,

19 Mains switch, 20 Slot for compensators

* not part of all configurations

+

control also situated on right of microscope

Operation of objectives (Pol)

1

19

2

3
4

5
6

7
8

9

11
12

13

10

3a

+

20
14

15

16

17

18

9

A particularly exact way of crossing the polar­izers is to use the built-in Bertrand lens (43.3)
or the auxiliary telescope (25.1) as follows:
Use an objective with a fairly high magnification,
e.g. 40x, 50x, 63x.
Open the aperture diaphragm (43.7) (PH position).
For the auxiliary telescope: foxus until the some­what brighter circle in the middle of the field of
view is sharply defined.
When the polarizer is not quite adjusted, 2 dark
stripes are visible which close to form a cross
when the polarizers are exactly crossed (44a).
This cross usually does not close completely in
the case of objectives and condensers without
the P engraving.

The following section is only intended to give a
rough survey of the examination methods.
Further details can be found in textbooks on
polarization microscopy.

Examinations: One polarizer only

If specimens are to be examined with other
transmitted light methods such as brightfield,
phase contrast and darkfield instead of with
crossed polarizers, it is usually sufficient to dis­engage either the analyser or the polarizer. If the
image is not bright enough, both the polarizer
and the analyser should be disengaged.

Birefringent specimens with inherent colours
may exhibit changes in brightness and/or colour
when the stage or polarizer is rotated (with
analyser disengaged). This so-called dichroism
or pleochroism is a key indication in crystal
examinations. However, this effect can be simu­lated on non-polarizing microscopes which have
no depolarizing quartz plate, or also if an inci­dent light reflector has been left in the light path
when transmitted light is switched on.

Fig. 44 Crossing the polarizers, viewing with a Bertrand lens
and a high-aperture objective, without a specimen
a exactly crossed, b not exactly crossed
Often, Pos. a cannot be set at all if there is strain in the con­denser or objective

Operation of transmitted light polarization

a

b

10

Crossed polarizers

The DIN and ISO standard vibration directions
are indicated on the microscope (sticker).

If the specimen contains many non-birefringent
or opaque particles, the polarizer is frequently
turned out of the crossed position by a few
degrees so that these particles show up at least
faintly (they remain dark when the polarizers are
exactly crossed). It is not customary to examine
specimens with the polarizers parallel, as this
method of identifying birefringence is not sensi­tive enough.

Change in brightness when birefringent objects
are rotated

When the stage is rotated, the brightness of
birefringent (anisotropic) objects changes peri­odically. During a full rotation the object dis­appears four times after each 90° interval. The
four dark positions are called extinction or nor-
mal positions. Exactly between each of these
extinction positions the object can be observed
with maximum light intensity. These are the four
diagonal or 45° positions. In the extinction posi­tions the object vibration directions run parallel
to the transmission directions of the polarizers,
at maximum intensity the object vibration direc­tions represent the angle bisectors of the polar­izer directions. The crosslines in the (right-hand)
eyepiece of polarized light microscopes can
either be aligned at N – S/E – W, i.e. in the polar­izer directions, or at 45° angles, i. e. correspond­ing to the object vibration directions in the
diagonal position.

λ and λ/4 compensator

Depending on the microscope model, the quar­ter- and whole-wave compensators are either
slotted in the compensator slot (43.20) or above
the polarizer (43.10; 28.2) or in the light ring slot
(CLP/PH) or integrated in the condenser disc
(UCLP) (9.6). When a compensator is engaged,
the phase difference is increased or decreased
(see Fig. 45). The vibration direction γ (i.e. corre­sponding to the greater refractive index n

γ

) can

be determined from the colour changes.

Fig. 45 Interference colours in relation to phase difference, or
to thickness and colour change for the addition and subtraction
position of a whole-wave and a quarter-wave compensator

Black
Lavender gray

200

1st order

400

600

800

2nd order

1000

1200

Phase difference

1400

3rd order

1600

– λ

+ λ

Gray blue
Yellowish-white

Vivid yellow

Red-orange
Deep red

Indigo
Sky blue
Greenish blue

Light green
Pure yellow

Orange red
Dark violet red

Indigo
Greenish blue

Sea green
Greenish yellow

Flesh color
Crimson

Matt purple

λ

– –
4

λ

+ –
4

11

Quartz wedge

The quartz wedge (46.6) is inserted into the com­pensator slot (43.20). It allows phase shifts from
0 to about 4 λ (orders).

Circular polarization

Birefringent objects exhibit four extinction posi­tions for one stage rotation. Particularly when
scanning a large area of the specimen, some of
the birefringent objects will always happen to be
at the extinction position. Circular polarization is
used for simultaneous observation of the inter­ference colours of all objects:
Remove the specimen from the light path or find
an empty area of the specimen. Cross the polar­izers exactly – they must also be exactly at the
N – S/E – W positions.
Insert quarter-wave compensator (46.5) in the
compensator slot (43.20). Push the quarter-wave
compensator (46.1) into the slot above the po­larizer (43.10) and rotate until the empty field of
view appears at its darkest position (first cross
polarizers exactly!). The quarter wave compen­sator that can be integrated in the condenser
disc (9.6) is not suitable for circular polarization.

Compensators for quantitative measurements

Only in conjunction with polarized light micro­scopes in transmitted light. Adjustable compen­sators are used for exact measurements of
phase differences. For a known specimen thick­ness d and the measured phase difference
gamma (Γ) the birefringence ∆n’ can be worked
out using the following formula:

d

Γ = d x ∆n’ [nm] or ∆n =

Γ

Fig. 46 Compensators
1, 2

λ/4 and λ compensator in holder Ø 32 mm. Only for polarized light microscopes: 3 λ/4 and

λ compensator for condenser disk, 4, 5 λ/4 and λ compensators for compensator slot (43.20),

6 Quartz wedge, 7 Tilting compensator, 8 Brace-Koehler compensator

1

2

3

4

5

6

78

12

To perform the measurement, the compensator
is introduced into the tube slot and adjusted until
the object to be measured is in its maximum
extinction position. For this purpose the object
has to be moved into a certain diagonal position.
Further details are given in the instructions for
the use of the compensators.
The following compensators are available:

Elliptical Brace-Koehler compensator (46.8)

Rotary compensator with compensator plate of
about λ/10 phase difference. Measurement is
carried out in white or in monochromatic light.
Measurement range up to approx. 50 nm.

Tilting compensator B (Berek compensator)
measuring up to 5 orders

Compensator (46.7) with MgF

2

plate for meas­urements in monochromatic or white light of up
to about 5 orders phase difference. The phase
difference can be read directly from the sum of
the two angles of compensation produced when
the compensator plate is tilted in both directions,
from a supplied calibration chart.

Tilting compensator K,
measuring up to 30 orders (like 46.7)

For the measurement of phase differences in
white or monochromatic light up to the maximum
phase difference mentioned above. The com­pensator plate is made of calcite; evaluation is
based on simple calculation by means of
enclosed tables and the stated calibration con­stant. A programmed computer can be used for
evaluation of measurements taken with tilting
compensators. The necessary formulae and
parameters are given in:
Kornder, F. and W. J. Patzelt: The use of mini­computers to evaluate polarization-optic com­pensator measurements. – Leitz Scientific and
Technical Information IX/1, 30 – 32, 1986.

13

Conoscopy of crystals

Birefringent crystals cause interference patterns
(Fig. 47) in the exit pupil of the objective (i.e.
inside the objective). These are also called
conoscopic images. The shape of these interfer­ence patterns and the way they change when
compensators are used supply information on
the number of crystal axes (uniaxial or biaxial
crystals), the orientation of these axes and the
plus or minus sign of the birefringence (positive
or negative birefringent crystal).

As these interference patterns occur in the
pupil, they are not normally visible during normal
microscopic observation (orthoscopy). Their ob­servation can be improvised by removing one of
the eyepieces and looking into the tube with one
eye from a distance of a few centimetres.
Observation is better with the auxiliary telescope
for phase contrast (Fig. 25.1). However, other
crystals in the field of view disturb the interfer­ence patterns of a crystal in the centre, so that it
needs to be isolated. This can only be done with
the Bertrand lens (43.3) in the Pol module, as
here isolation is performed by a fitted dia­phragm. The diameter of the isolated object field
is about 55 µm for a 40x objective, about
36 µm for a 63x objective and 23 µm for a 100x
objective.

Setting the microscope for conoscopy

The most suitable object areas for conoscopy
are those that show the lowest possible phase
differences (chart in Fig. 45). Exact centration
of the strain-free Pol objectives and exactly
crossed polarizers are essential for perfect
conoscopic observation.
Turn an objective with as high an aperture as
possible (e.g. 40x, 50x or 63x) into the light path.
Open the aperture diaphragm (43.7). Move the
crystal you want to examine as near to the cen­tre of the field of view as possible.
Narrow the field diaphragm (43.12) as well, if
necessary.
Push in the Bertrand lens (43.3). To improve the
quality of the image, especially for small crystals,
lower the stage by about 0.2 mm.

The insert the centering keys (1.3) into the two
openings (43.3a) one after the other and adjust
until the circular area (objective pupil) is aligned
to the centre of the crosslines.

14

Fig. 47 Determination of the optical character of uniaxial structures.
Positive and negative uniaxial crystal, cut vertically to the optical axis.
Biaxial positive and negative crystal, cut vertically to the acute bisectrix.

Determination of optical character

Uniaxial crystals (Fig. 47)

Uniaxial crystals observed in the conoscopic
(divergent) beam show a dark cross, whose cen­tre indicates the position of the optical axis. The
cross is surrounded by coloured interference
fringes*.

For the determination of the optical character,
cutting directions where the optical axis of the
crystal is slightly inclined to the direction of
observation are also suitable. The optical char­acter can mostly be determined even when the
centre of the cross is outside the field of view.

Biaxial crystals (Fig. 47)

Cutting directions where the bisectrix of the two
optical axes is parallel to the viewing direction
(section vertical to the acute bisectrix) are par­ticularly suitable for determining the optical
character.

In the divergent beam a dark cross will be seen
which opens up into the two brances of a hyper­bola, the so-called isogyres, when the stage is
being rotated. The cross and the branches of the
hyperbola are surrounded by interference
fringes. According to Fig. 47 or the rule men­tioned below the optical character can be deter­mined from the displacement direction of these
fringes after operation of the compensator. The
symmetry plane of the isogyres (axial plane)
must be vertical to the γ direction of the compen-
sator.

Evaluation of conoscopy

Uniaxial

Without
compensator

With λ compensator*
vibration
direction λ

Displacement of
the stripes with
compensators

With thin specimens or specimens with low birefringence, only the cross is visible.
* with the λ/4 compensator, black dots will occur instead of the black arcs

↔

Biaxial