Olympus 1X71 Manuel utilisateur

Revised December 2014

UCL Institute of Child Health

User guide

Olympus 1X71

Dr Bertrand Vernay

Light Microscopy Facility Manager

Wellcome building, Office W2.09

Tel Office: 42224

Tel Mobile: 07582249337

Email: b.vernay@ucl.ac.uk

Table of contents

page 3.........Ownership

page 3........ Access Rules

page 3.........Olympus Customer Support Contact

page 4 ........ General Specifications

Microscopy Techniques Available

Objectives

Filter Cubes

Camera

Fluorescence Illumination

Pixels to Microns Calibration

Consumables List

page 5......... Quick User Guides

Transmitted Light

Epifluorescence

Image Capture

page 6......... Halogen Lamp Operation

page 7 .... Kohler Illumination

page 8........ Adjusting the Objective Correction Collar

page 9........ Prior Lumen200 Metal Halide Lamp Operation

page 10 .. Image Capture with HClmage

page 11 ... Selecting the Right Fluorochrome/Filter Set

page 11 DAPI

page 12 Endow GFP/EGFP Bandpass

page 13 DsRed(TRITC/Cy3)

page 14 Cy5

page 15 Cy7

page 16 ....Prior Lumen200 Spectral Output

page 17 ... Hamamatsu ORCA-R

Spectral Response

Ownership

Prof. Jane Sowden, Developmental Biology Unit (Purchased in 2011)

Access Rules



No access without prior training by the Light Microscopy Facility Staff



Free of hourly charge for Sowden and Ferretti groups, £1 hourly charge for

all other users towards the cost of the consumables is expected



Prof. Sowden team has priority over other users.



Users must always record their activity in the Log book



Problem(s) with the microscope should be reported as soon as they are

noticed

Olympus customer support service: http://www.olympus.co.uk/microscopy

The system is not covered by a maintenance contract

Olympus requires a PO number before sending an engineer

General specifications

Microscopy techniques available



Brightfield



Phase contrast



Epitluorescence

Objectives

O l y mp u s U P l an F L N 1 0 x P h 1 NA 0 . 3 WD 1 0 . 0 m m

Olympus LUCPlanFLN 20x Ph1 NA 0.45 WD 6.6 -7.8 with correction collar

Olympus LUCPlanFLN 40x Ph2 NA 0.6 WD 3.0-4.2 with correction collar

Z e i s s o b j e c t iv e s c a n a l s o b e u s e d

Filter cubes (see p11-15)

Position

Filter set name

Exciter

Beamsplitter

Emitter

DAPI

350/50x

400LP

ET460/50m

GFP

ET470/40x

495LP

ET 525/50m

DsRed (TRITC/Cy3)

ET545/30x

T570LPXR

ET620/60m

Cy5

ET620/60x

T660LPXR

ET700/75m

Cy7

ET710/75x

T760LPXR

ET810/90m

empty (brightfield)

Camera (see p16)

Hamamatsu ORCA R2CCD Camera with HClmage Capture Software

Fluorescence illumination (see p16)

Prior Lumen 200 Metal Halide Light Source (2000 hours/bulb)

Pixels to Microns calibration

5x objective binning 1x1 1 pixel = 1.88 um

10x objective binning 1x1 1 pixel = 1.03 um

20x objective binning 1x1 1 pixel = 0.514 um

40x objective binning 1x1 1 pixel = 0.256 um

Calibration with a micrometer under transmitted white ligh t

Consumables list

Price correct as of November 2013

Prior Lumen 200 bulb LM375 (£550, Prior Scientific Instruments Ltd)

Prior Lumen 200 light guide LM587 (£400, Prior Scientific Instruments Ltd)

Halogen bulb 12V/100W (£1.8, Technical Lamp Supplies UK)

Quick user guides

Users must always record their activity in the Log book

Transmitted light

1. Halogen Lamp Pow er Su pply Unit TH4 "O N"

2. Light Path se lector on "Ocular"

3. Koh l e r i l lumin a tion ad j u s te d

4. Correct phase ring in position (10x & 20x Ph1, 40x Ph2)

5. Fil t e r c u be on p o sit ion #6

Epifluorescence

Warnings:

Do not shut the unit down within 30 minutes of powering up the unit.



After shutting down the unit allow 30 minutes before re-powering up



After shutting down the unit allow 30 minutes before changing the bulb.

Failure to do so is likely to result in damage to the bulb.

1. Prior Lumen 200 module on

2. Prior Lumen 200 intensity knob >0%

3. Light Path selector lever on "Ocular"

4. Correct filter cube in position

5. Fluorescence shutter open

Image Capture

1. Start Camera controller (press until LED turns green)

2. Compu te r on (Logi n: Jane / Password: Ja*e)

3. HClmage software open

4. Light Path se lector lever on Camera

5. Correc t transmitted ligh t/e pifluores cence set -up

6. " L i v e " m o d e

7. Adjust exposure time accordingly. Make use of the Histogram and the Saturatio n

options

8. " A b o r t "

9. " C a p t u r e l "

1 0 . S a v e a s i n M y D o c u m e n t s > U s e r N a m e _ U n i t > F i l e N a m e . t i f

1 1 . Shut-down: exit HClmage, log out windows session, camera on stand -by (press

until LED turns orange)

Halogen lamp operation:

Turning on the lamp

1. Make sure the light intensity control knob

(5)

is in the MIN (minimum intensity)

position on the microscope frame.

2. Make sure the light intensity control knob

(1)

is in the MIN (minimum intensity)

position on the TH4 module.

3. Set the main switch

(2)

"I"

(ON) on the TH4 module.

4. On the microscope front, press the transmitted light ON-OFF button

(6)

so that

the button is illuminated.

Adjust the brightness with the light intensity control knob

(5).

6. To turn OFF, set the transmitted light ON-OFF button

(6)

to OFF

Halogen lamp operation:

Turning off the lamp

1. Set the light intensity control knob (5) to the MIN (minimum intensity) position on

the microscope frame.

2. Set the light intensity control knob (1) to the MIN (minimum intensity) position on

the TH4 module.

3. Set the main switch (2) to "0" (OFF) on the TH4 module.

Kohler illumination:

1. Rotate the turret (1) to the "BF" position. (Any of positions 3,4 or 5, position 1=Ph1, 2 = Ph2)

2. Sli de t he ape rture iris diap hr agm lever ( 2) t o fully open the d iaphragm .

3. Sli de th e field iris diaph r agm le ve r (3) to the fully open posi ti on .

4. En ga ge th e 1 0x obj ective and br ing t h e spe cim en in to foc u s.

5. Using the field iris diaphragm lever (3) , completly close the field iris diaphragm.

6. Rotate the condensor height adjustment knob (4) to bring the field iris diaphragm

image into focus.

7. Cen ter the fie ld iris diaphr agm ( 3) using the conden ser centerin g kno bs (5).

8. Open the field iris diaphragm (3) until its image reach the limits of the field of view,

adjust the centering if necessary.

9. Ope n the f ie l d ir is d iaphr a gm (3) u n t il n o t v isible .

Step 6 Step 7 Step 8 Step 9

Adjusting the objective correction collar

Correction is possible according to the vessel bottom thickness.

1. When the thickness of the vessel bottom is known, match the scale reading of the

correction collar to the thickness of the vessel in use.

2. If the thickness of the vessel is unknown or diverge from the manufacturer

specifications, the optimum position for the correction collar can be obtained by

judging the image resolution and contrast. When a satisfactory image is not obtain

after focusing:

1. Rotate the correction collar to the left and right, refocus each time and

compare the images.

2 Then rotate the collar in the direction yielding a better image, rotate the

correction collar to the left and right, refocus each time and compare the

images.

3 Repeat this cycle until the position with the optimum image is found.

20x Correction Collar 40x Correction Collar

Correction Collar Scale

0 mm

0.17 mm (glass coverslip #1.5)

0.5 mm

1 mm (most tissue culture plates)

1.5 mm

2 mm

Prior Lumen200 Metal Halide Lamp Operation:

Starting Up the Lumen

1. Switch the Lumen power switch on.

2. Make sure the ventilation vent on the left hand side is unobstructed or the lamp

will overheat resulting in automatic shutdown and damage to the module.

3. Allow 1-5 minutes for light to reach 70% of output.

4. Allow 30 minutes for the Lumen to reach operational temperature.

Warning:

Do not power down the unit within 30 mins of power up. This may re-

duce the effective lifetime of the bulb.

Shutting down the Lumen

The following warnings apply as damage to the bulb may result if instructions not

followed:

1. Warning: Do not shut the unit down within 30 minutes of powering up the unit.

2. Warning: After shutting down the unit allow 30 minutes before re-powering up or

changing the bulb. Failure to do so is likely to result in damage to the bulb.

Warning: the air

outlet for heat

ventilation must

not but obstructed

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