N3 Laser License Required to operate the microscope
Location: T-lab level 10, Microscope Room
Light SourceWidefield100W
mercury lamp
Lasers
Wavelength (nm)
Maximum power (mW)
Laser type
405
50
Diode
488,515
12
Ar
559
15
Diode
633
10
HeNe
Microscope BodyOlympus 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
CameraPhotometrics
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. 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.
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.
