Olympus Live TIRF

N3 Laser License Required to operate the microscope


Location: T-lab level 10, Microscope Room

Light Source    Widefield           100W mercury lamp

Lasers 

Wavelength (nm)

Maximum power (mW)

Laser type

405

50

Diode

488,515

12

Ar

559

15

Diode

633

10

HeNe

 

Microscope Body    Olympus IX81 Inverted Microscope

Beam splitters and filters

 

DAPI (WU)

FITC (NIB)

TRITC (WIG)

Excitation Filters

360 – 370

470/40

510 – 550

Dichroic Mirrors

400

500

570

Emission Filters

420

515

590

Objectives

Model

Immersion

N.A.

WD (mm)

Remarks

PL FL 10X

Nil

0.30

10

Phase

PL FL 20X

Nil

0.45

6.4-7.6

Phase






 


TIRF 100X

Oil

1.45

 

TIRFM

Camera         Photometrics CoolSNAP K4  2048 x 2048

Operation system and software

WinXP, 4 GB RAM, 3TB system disk, DVD-RW

MetaMorph

Misc. Accessories

Motorized stage with linear encoder (Ludl)

Standard multi-well plate including 96-well automated imaging

Weather Station Incubator, Temperature, Humidity, CO2 control

ZDC: zero focus drift compensator


Zero Drift Control(ZDC unit)


SOP(Olympus TIRF)

1. Switch on switch labelled "Main TIRF Switch" (Try not to shake the laser table when doing so)

2. Switch on Mercury Lamp on top of the rack.

3. Wait until the Light Indicating Burner On is stable. (If it's not stable for 1 min , the lamp need to be replaced. Please contact microscope staff.)

4. Turn on microscope controlling computer.

5. Switch on the K4 camera.

6. Open Metamorph Software and proceed with TIRF imaging.
Standard Operating Procedure

System Start-up

1.       Weather chamber and the air heating unit

The air heater should be left ON ALL THE TIME.
(Please do not touch the plug on the wall.)

Avoid placing electronic units, immersion liquid or sample close to the air heater outlet.

figure 1. air heater outlet


2.      
CO2 and CO2 mixer

Turn on the CO2 and the CO2 mixer ONLY WHEN live cell imaging is required.

Refill the water bottle with MiliQ water if the water level is low.

Open the CO2 wall outlet valve (NEVER adjust the grey flow rate control):

        Red handle parallel to the tube = open

                         perpendicular to the tube = closed.











figure 2. CO2 wall outlet (closed)   











figure 3. CO2 wall outlet (open)

figure 4. Water bottle

Turn on the CO2 mixer using the power switch at the front of the temperature and CO2 mixer control box behind the weather chamber and PC monitor. Wait for a few minutes to confirm that the actual percentage of CO2 is steadily rising towards 5% (in case the percentage does not rise, turn off the mixer using the power switch at the front and turn on again)

figure 5. Temperature control and CO2 mixer

3. Microscope control, stage control, transmitted light and PC

Turn on lamp (when necessary), microscope control and shutters controller.



figure 6. (from left to right)microscope control, lamp, shutters controller

4. Lasers

Turn on the lasers required for the experiment:

 a. 405 nm, switch on power buttom and turn interlock key

 b. 488/514 nm, switch on power buttom and turn interlock key

 c. 559 nm, switch on power buttom, wait until both green light lights up and then turn interlock key.

 d. 633 nm, turn interlock key.

Turn on the shutter control.

DO NOT LOOK AT ANY LASER DIRECTLY.
CLASS 3B Laser License Required to operate the microscope


figure 7. Lasers (wavelength labeled on the power boxes)

    figure 8. Laser shutter controller

Turn on the PC.

5.       Camera


Ensure that the camera is on by checking the power switch 
 figure 9. Camera and its power switch


Basic operation


1.      
CO2 supply

Use the on-stage incubator with CO2 supply and humidity control from the water bottle for live cell imaging.
























figure 10. CO2 supply glass heating cover and protecting frame

Caution: always use the protecting frame together with the heating cover to prevent the condenser from cracking the cover by accident!!!


1.      
Epi-fluorescence and DIC

For epi-fluorescence observation, use the fluorescence filter sets WU (for DAPI), NIB (for FITC) and WIG (for TRITC).For DIC, avoid driving the condenser too low to prevent from cracking the heating cover. The relay lens below the weather chamber offers 1.6x more magnification, but sacrifices the contrast of DIC. Clean the DIC light path with a clean piece of tissue paper only if necessary.


figure 11. filter list, relay lens, glass cover for DIC and field aperture

3.      
Laser output power

Adjust the laser output power using the wheels on the front board of the control box.


figure 12. laser output power control

4.      
Microscope

Switch the light path between the eyepiece and the camera by pressing the button on the front board of the microscope.

Turn on/off & up/down the transmitted light from the buttons on the front board of the microscope. The relative light intensity is displayed in a line of green lights.

Change the axial position of objective turret with the buttons on the front board of the microscope or with the focusing knob on the side of the microscope. Fine/course turning can be chosen from the button beside the focusing knob. ‘Escape’ the objective every time the objective is changed.




















figure 13. front panel of the microscope, control pad and focusing knob


5.      
TIRF

For TIRF imaging, push the laser safety slider in GENTLY.

Pull out the switch bar at the back of the microscope to switch from mercury lamp to laser mode.

Turn the laser emission key to position ‘on’ to let through the laser.

Turn the TIRF illumination angle control knob (behind the weather chamber) outwards to reach the critical angle (until only the surface of the sample is illuminated). Try not to turn it too far out after the critical angle is reached.


figure 14. TIRF laser safety slider position out/in, laser emission key, TIRF illumination angle control

Cautious: do not touch the laser fibre or change its position under any circumstance. Please contact the microscopy core staff if you need alignment of the light path.

Finishing experiments

1. Clean the objective if an immersion lens has been used

figure 17. Lens cleaning Tools
2. Lower the objective turret all the way down using the focusing knob3. 

3. Close the software

4. Shut down the PC

5. Check that the CO2 outlet valve is close

6. Turn off the CO2 mixer






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