Olympus IX2-ARCEVA User Manual

IX2-ARCEVA

ARC ILLUMINATION TIRF SET

This instruction manual is for the Olympus Arc Illumination TIRF Set. To ensure the safety, obtain
optimum performance and to familiarize yourself fully with the use of this system, we recommend that
you study this manual thoroughly before operating the system.
As this system employs an arc light source (mercury burner or xenon burner), take special care for safety
and please also read the instruction manual for the light source.
Retain this instruction manual in an easily accessible place near the work desk for future reference.

INSTRUCTIONS

A X 7 3 8 7

IX2-ARCEVA

CONTENTS

Correct assembly and adjustments are critical for the system to manifest its full performance. If you are going to assemble
the system by yourself, please read chapter 3, “ASSEMBLY” (pages. 8 to 11). For the assemblies of the modules for which
instruction manuals are available, refer to their instruction manuals.

IMPORTANT — Be sure to read this section for safe use of the equipment. — 1-4

1 NOMENCLATURE 5

2 CONTROLS 6-7

3 ASSEMBLY 8-11

4 OPERATION 12-14

4-1 Preparation

1 Centering the Burner
2 Adjusting the Slit

4-2 Total Reflection Fluorescence Observation

..............................................................................................................................................................................................................

....................................................................................................................

12-13

14

5 TROUBLESHOOTING GUIDE 15

IMPORTANT

This system has been designed to enable total reflection fluorescence observation using an arc light source
(mercury burner or xenon burner).
Total reflection illumination is implemented by limiting the illumination projection area to the area corresponding
to the objective pupil NA of 1.39 (because the reflectance of cytoplasm is 1.38).
}This system offers the following advantages compared to the laser-based total reflection illumination system.

· Higher safety, because the laser light is not used.

· Easy handling without the care of interference stripe, because the light source is not a coherent source.

· Easy switching between multiple wavelengths.
Nevertheless, this system is not applicable to molecular fluorescence observation due to low intensity of the
illumination. Its applications are limited to high-contrast observations of tissues near cell membranes and
physiochemical phenomena.

SAFETY PRECAUTIONS

1. Do not ignite the mercury burner or xenon burner without mounting the lamp housing on the microscope.
The UV light emitted from the burner is hazardous for your eyes. Do not look directly at the light output from the lamp
housing.
For details precautions, also refer to the instruction manual for the light source.

2. When centering the burner or replacing the specimen, be sure to look them through the UV cut plate to protect your eyes.

1

IX2-ARCEVA

Getting Ready

1

1. This manual pertains only to the handling of the total reflection fluorescence illumination system. Please also read the
instruction manuals for the IX2 series (IX81/IX71/IX51) microscope and associated options to understand the comprehen­sive operating instructions of the microscope system.

2. The total reflection fluorescence illumination system is composed of precision instruments. Handle it with care and avoid
subjecting it to sudden or severe impact.

3. Do not use the system where it is subjected to direct sunlight, high temperature and humidity, dust or vibrations.

4. Reserve spaces of 10 cm or more around the lamp housing and power supply to facilitate their ventilation.

5. The fluorescent light illuminator that can be used in combination with this system is only the IX2-RFAL L-shaped reflected
light illuminator.

6. The objectives that can be used with total reflection illumination are only the PlanApoN60XO

7. The illumination field of total reflection fluorescence observation is limited within the area of field number 11.

8. Since the total reflection light is not point-source light, the excitation light incidence angle on the specimen is not
constant and the effusion depth cannot be specified.

9. If an excitation filter is included in the mirror unit during total reflection fluorescence illumination, the repeated reflections
cause the leak of direct light to increase and degrade the contrast considerably. To prevent this, remove the excitation filter
from the mirror unit and mount in the excitation filter slider.

10

. The size of the excitation filter inserted in the filter slot of the excitation slider should be 25 mm in diameter and 6 mm or

less in thickness. Only one ND filter with a diameter of 32 mm and thickness of 1 mm or less can be inserted.

11

. When ordinary fluorescence observation is performed by inserting a 25 mm excitation filter in the excitation filter slider,

the illumination field is limited within the area of field number 20.

. To prevent light leak, be sure to attach the provided 32 mm light shields in the unused positions of the excitation filter

12

slider.

and PlanApo60XO

TIRFM

TIRFM

.

2

13

. The aperture iris diaphragm for use in ordinary fluorescence observation is not provided. If it is required to use the aperture

iris diaphragm, replace the slit iris diaphragm unit of this system with the aperture iris diaphragm unit of the IX2-RFAL.

14

. When the IX2-RFAC, IX2-RFACA and a standard mirror unit are used in combination, it is required to readjust the centering

of the slit iris diaphragm unit because, otherwise, the contrast of the total reflection fluorescent light would fluctuate due
to variation in the illumination light projection positions between turret positions.

15

. The IX2-SFR right-hand knob stage is not recommended because the stage knobs will degrade the operability of the slit

iris diaphragm unit considerably. Instead, it is recommended to use the IX-SVL-2.

16

. When the IX2-RSPC right-side port adapter is used, some positions of the excitation filter slider would be unusable due

to interference with the camera. The following table shows the relationship between the camera size and unusable filter
positions.

3

Height from the center of
C-mount

Less than 27 mm

27 mm to 45 mm exclusive

45 mm or more

}The camera interference can be reduced by using the U-FWR filter wheel or a commercially available motorized filter

wheel.

Depth from the center of
C-mount

Less than 34 mm None

34 mm to 70 mm exclusive Deepest (vacant) position is unusable.

70 mm or more The two deepest positions are unusable.

Less than 20 mm None

20 mm to 56 mm exclusive Deepest (vacant) position is unusable.

56 mm or more The two deepest positions are unusable.

Interference condition

None

IX2-ARCEVA

Maintenance and Storage

2

1. Clean all glass components by wiping gently with gauze. To remove fingerprints or oil smudges, wipe with gauze slightly
moistened with a mixture of ether (70%) and alcohol (30%).

Since solvents such as ether and alcohol are highly flammable, they must be handled carefully. Be sure to keep
these chemicals away from open flames or potential sources of electrical sparks –– for example, electrical
equipment that is being switched on or off. Also remember to always use these chemicals only in a well-venti­lated room.

2. Do not attempt to use organic solvents to clean the non-optical components. To clean them, use a lint-free, soft cloth
slightly moistened with a diluted neutral detergent.

3. Never attempt to disassemble any part of the system, for this may degrade the performance.

Caution

3

If the system is used in a manner not specified by this manual, the safety of the user may be imperiled. In addition, the
equipment may also be damaged. Always use the equipment as outlined in this instruction manual.

The following symbols are used to set off text in this instruction manual.

: Indicates that failure to follow the instructions in the warning could result in bodily harm to the

user and/or damage to equipment (including objects in the vicinity of the equipment).
#: Indicates that failure to follow the instructions could result in damage to equipment.
}: Indicates commentary (for ease of operation and maintenance).

4

NOMENCLATURE

}The modules enclosed in belong to the Arc Illumination TIRF Set.

· The illumination field in fluorescence observation is limited to the area of FN 20.

· The illumination field in total reflection fluorescence observation is limited in the area of FN 11.

5

Recommended stage IX-SVL-2
Features high operability of iris

diaphragm unit.

UV cut plate

Objective
(Note)Only the PlanApoN60XO

and PlanApo60XO
be used with total reflection
fluorescence observation.

Fluorescent mirror unit cassette

· IX2-RFACEVA

· IX2-RFACA

· IX2-RFAC

TIRFM

can

TIRFM

Microscope frame

(IX2 series)

· IX81

· IX71

· IX51

L-shaped reflected light illuminator

IX2-RFAL

Arc Illumination

TIRF Set

IX2-ARCEVA

Lamp housing

· U-LH100HG

· U-LH100HGAPO

· U-LH75XEAPO

Power supply

CONTROLS

IX2-ARCEVA

Arc Illumination TIRF Set

IX2-ARCEVA

Field iris diaphragm lever

L-shaped reflected light illuminator
IX2-RFAL

(Note)The two filter sliders and aperture

iris diaphragm unit are not used
with this system.

Slit selector lever
Pushed in: IN

Pulled out: OUT

Attaching screws

Field iris diaphragm
centering screws

Slit iris diaphragm unit

Wedge prism slider
See detailed view (page 7).

4-position excitation filter slider
See detailed view (page 7).

Slit focus adjustment screw

Adjustment range: ± 1.5 mm

Slit centering screws

Adjustment range: ± 1.5 mm

Allen wrench for use in centering

Column

Adjuster

Click additional parts
Balls, springs, screws (x 2 each),

and Allen wrench

Width across flats: 3 mm

Width across
flats: 2 mm

6

4-position excitation filter slider

ND filter insertion slots
Filter diameter: 32 mm.

Thickness: 1 mm or less.

Light source side

Vacant position

Wedge prism slider

32 mm light shields (x 4)
To be inserted in the ND

filter insertion slots of the
unused positions.

Objective side

ND filter insertion slot
Filter diameter: 32 mm.

Thickness: 1 mm or less.

Slider knob

Sticker attaching positions

x 6

Slider knob

Light leak prevention plate

Excitation filter insertion slots
Filter diameter: 25 mm.

Thickness: 6 mm or less.

Vacant position

Wedge prism

Shutter

Push ring driver

Handwritten indication stickers

Target

7

IX2-ARCEVA

ASSEMBLY

The filters and sliders are very hot during and immediately after use. Confirm that they are cooled down enough
before replacing them.

#When a commercially available, non-Olympus low reflection ND filter is used, the filter may sometimes be cracked

or burnt.

Attaching the Click Additional Parts (Fig. 1)

1

1. Using the provided Allen wrench (width across flats 2 mm), remove the
screw, which functions as the cap, on the center of the bottom side of the
filter insertion slot of the IX2-RFAL.

2. Put the ball @ and spring ² into the center of the bottom side of the filter
insertion slot and fix them with the screw ³. Use the provided Allen wrench
for use in centering (width across flats: 3 mm) for clamping the screw.

@
²
³

Fig. 1

8

³

Fig. 2

@

²

Mounting the Excitation Filter (Figs. 2 to 4)

2

}When the IX2-RFACEVA mirror unit cassette is used, the operation in step

1 below is not necessary because no excitation filter is provided with it.

1. Using the push ring driver ², turn the excitation filter push ring @ of the
mirror unit to be mounted in the fluorescence mirror unit counterclockwise
and remove the excitation filter ³.

2. Using the push ring driver ², turn the push ring ƒ of the excitation filter
slider | counterclockwise to remove it.

9

³

Fig. 3

²

ƒ

#The excitation filter should be inserted in the specified orientation.

Insert it so that the indication arrow on the filter frame points the
objective. Ensure the orientation of insertion of the excitation filter
slider into the illuminator before assembly.

3. Drop the excitation filter ³ into the excitation filter slider so that the indica­tion arrow on the filter frame faces upward (if the up surface comes on the
objective side) and fix with the push ring ƒ.

|

…

Fig. 4

³

IX2-ARCEVA

4. Write the name (or abbreviation) of the mounted filter on one of the
provided handwritten indication stickers and attach it to one of the sticker
attaching positions … (position on the left or right of the position in which
the filter is mounted).
In the above case, attach the sticker to the position on the left of the filter
position because the left side comes near the slider inlet when the slider
is set on the illuminator.

Replacing the Modules Provided with the IX2-RFAL

3

}Replace the following modules as shown in the illustration on page 6.

· Aperture iris diaphragm: Remove the attaching screw and replace with
the slit iris diaphragm unit.

· Filter slider (aperture iris diaphragm side):Remove the slider knob and

replace with the wedge prism
slider.

· Filter slide (lamp housing side): Remove the slider knob and replace

with the excitation filter slider.

10

Mounting the IX2-RFAL

4

Mounting the UV Cut Plate

5

}Refer to the instruction manual for the IX2 reflected fluorescence system.

Mounting the ND Filter

6

}The ND filter attenuates the excitation light and helps delaying color

fading of the specimen.
Insert the ND filter gently into the ND filter insertion slot of the slider.

11

OPERATION

4-1 Preparation

@

²

³

@

²

|

Fig. 5

³

Fig. 6

IX2-ARCEVA

Centering the Burner (Fig. 5)

1

}The following settings are the original settings required for this system.

The details may be variable according to the instruction manual for each
lamp housing.
@ Field iris diaphragm:Pulled-out position (Closed)
² Slit: Pulled-out position (Slit OUT)
³ Wedge prism: Nearest position (Vacant position)
| Excitation filter: B or IB excitation filter engaged. Also the B or IB

mirror unit should be engaged in the light path.

Adjusting the Slit (Figs. 6 to 8)

2

}Open the field iris diaphragm @, set the slit selector lever ² to the OUT

position and set the wedge prism ³ to the vacant position before the
following adjustment. (Fig. 6)

1. Engage the PlanApoN60XO
quires immersion oil) in the light path.

2. Place a fluorescent specimen on the stage and bring the specimen into
focus using general fluorescent light of B or IB.

3. Remove the specimen, rotate the revolving nosepiece and screw the
target into an objective position.

or PlanApo60XO

TIRFM

objective (which re-

TIRFM

12

²

|

Fig. 7

Fig. 8

ƒ

³

4. Push in the slit selector lever ² (IN position) and set the wedge prism
slider ³ to the intermediate click position to engage the slit and wedge
prism in the light path. (Fig. 7)

5. Using the Allen wrench (width across flats 3 mm), turn the two centering
screws | alternately so that a crescent slit is visible on the target.
Adjust so that the slit is located as close as possible to the perimeter of
the target.

6. If the slit is not sharply visible, adjust the focus adjustment screw ƒ using
the Allen wrench (width across flats 3 mm).

13

4-2 Total Reflection Fluorescence Observation

IX2-ARCEVA

· The only objective that can be used in total reflection fluorescence observation is the PlanApoN60XO

· Since the illumination intensity is lower than with the laser light source, this system is not applicable to molecular
fluorescence observation. Its applications are limited to high-contrast observations of tissues near cell membranes and
physiochemical phenomena.

1. Set the slit to the OUT position and the wedge prism to the pulled out vacant position.

2. Using an objective other than the PlanApoN60XO
fluorescence observation.

3. Push in the field iris diaphragm lever to the open condition and engage PlanApoN60XO
tive in the light path.

4. Focus on the lowest position of the specimen (position closest to the cover glass).

5. Set the wedge prism in the intermediate click position and push in the slit to the IN position.
Now the microscope is ready for total reflection fluorescence observation.
The effusion depth can be fine adjusted by slightly rotating the lower slit centering screw as described below.

· Clockwise: This moves the slit inward, increasing the effusion depth.

· Counterclockwise: This moves the slit outward, decreasing the effusion depth.

or PlanApo60XO

TIRFM

, set up the microscope condition for general

TIRFM

TIRFM

or PlanApo60XO

TIRFM

or PlanApo60XO

TIRFM

TIRFM

objec-

.

14

TROUBLESHOOTING GUIDE

Under certain conditions, performance of the system may be adversely affected by factors other than defects. If problems
occur, please review the following list and take remedial action as needed.
If you cannot solve the problem after checking the entire list, contact Olympus.

15

Phenomenon

a. Total reflection fluorescence cannot

be obtained.

b. The field of view is irregular during

total reflection fluorescence obser­vation.

Cause Treatment Page

The PlanApoN60XO
PlanApo60XO
engaged in the light path.

The wedge prism and slit are not
engaged in the light path.

The slit positioning is incorrect. Adjust using the target.

An excitation filter is left in the mirror
cassette unit.

The lamp housing is not centered
properly.

TIRFM

or

TIRFM

objective is not

Engage them in the light path.

Remove it.

Center it correctly.
If the field is still irregular after adjust­ment, the irregularities may sometimes
be eliminated by decentering the lamp
housing slightly.

14

13

9

12

EC REP

Shinjuku Monolith, 3-1, Nishi Shinjuku 2-chome, Shinjuku-ku, Tokyo, Japan

Wendenstraße 14-18, 20097 Hamburg, Germany

3500 Corporate Parkway, P.O. Box 610, Center Valley, PA 18034-0610, U.S.A.

One Corporate Drive, Orangeburg, NY 10962, U.S.A.

491B River Valley Road, #12-01/04 Valley Point Office Tower, Singapore 248373

31 Gilby Road, Mount Waverley, VIC., 3149, Australia

5301

Blue Lagoon Drive, Suite 290 Miami, FL 33126, U.S.A.

01/10