TOTAL INTERNAL REFLECTION FLUORESCENCE (TIRF

TOTAL INTERNAL REFLECTION FLUORESCENCE (TIRF
TOTAL INTERNAL REFLECTION FLUORESCENCE (TIRF) MICROSCOPY
TURN ON THE FOLLOWING EQUIPMENT
 The power strip for the microscope and accessories
 The fluorescent light
 The CoolSNAP HQ camera on the right (Turn off the QuantEM camera on the left.)
 The lasers (the white toggle switch on the front of the laser unit)
 The computer
START THE NIS ELEMENTS AR SOFTWARE
Choose the TIRF Layout. Right click and choose Layout Manager. Select Import Layouts,
and open the TIRF Layout file found in the Layouts folder on the desktop. Choose Apply.
FIND YOUR FIELD OF INTEREST
Use the 60x/1.49 Apo TIRF oil objective. Optimize the correction collar for either 23° or 37°
and coverglass thickness (should be around 0.17).
Place your cells on the microscope. You need to use a #1.5 glass coverslip.
Turn the black knob on the front of the microscope to Bino.
Choose a filter configuration for the eyes. Open the EPI shutter, and center your cells in the
field of view.
Choose the Magnification – 1x or 1.5x (black knob on the lower right side of the
microscope). Make sure the dropdown list on the top menu matches the magnification so
your images will be calibrated correctly. The 1.5x mag may give a more even field of view.
Close the EPI shutter.
CHOOSE A TIRF OPTICAL CONFIGURATION
Select an optical configuration from the OC Panel. We have single color configurations for
488 TIRF, 561 TIRF and 637 TIRF.
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In the Filters, Shutters, and Switchers window,
the filters should be set as follows:
488 TIRF
Filter Block #5 GFP/mCherry
Filter A #7 FITC (ex)
Filter C #7 FITC (em)
561 TIRF
Filter Block #5 GFP/mCherry
Filter A #8 TRITC (ex)
Filter C #8 TRITC (em)
637 TIRF
Filter Block #1 BF/SEDAT
Filter A #9 Cy5 (ex)
Filter C #9 Cy5 (em)
Select the TIRF switcher button
In the AOTF Pad window, check the laser and
laser power.
CHECK THE CONFIGURATION OF THE MICROSCOPE
Move the DIC analyzer out of the light path.
Pull the mirror switch lever (silver slider on the
TIRF attachment) to the left. (This will block epi
illumination.) Adjust the ND filters as needed.
Turn the black knob on the front of the
microscope to Confocal.
Select Live to open the AOTF shutter and to send
laser light to your sample
Use the laser position adjustment knobs to bring
the beam straight up. (A laser safety lock will
turn off the laser if the microscope head is tilted
back…never look directly into the laser beam.)
The top knob moves the beam forward or
backward, and the front knob moves the beam
left or right.
Focus the beam using the focal point adjustment
TIRF Microscopy (09-15)
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knob. This is a sliver screw behind the filter turret, just in front of the white microscope
base.
SETUP THE CAMERA
In the CoolSNAP HQ Settings window, choose an exposure
time. This camera has a 1392x1040 pixel array, so binning
may be useful to increase signal.
Use 4x calibrated gain.
In the LUTs window, adjust the scale (or use Auto Scale) for
better visualization.
START SCANNING
Turn the front laser position adjustment knob counter-clockwise to bring the laser down to
the right. (Avoid hitting your neighbors with the laser beam.) You will see the beam travel
down the wall, into the dish and then into the objective. Turn the knob until the image goes
black, then come back just a bit. Focus up and down slightly to check if you are in TIRF.
You’ll need to adjust the the laser power, the camera exposure and possibly the camera
gain to get the best image possible while doing the least damage to your cells. You can also
engage ND filters (ND2, ND8 and/or ND16).
SETUP ND ACQUISITION
Set up experiment parameters in the Time, XY Pos, Lambda and Large Image tabs of the ND
Acquisition window.
Because each color requires a slightly different angle, if you choose more than one optical
configuration on the Lambda tab, you may compromise one or both of the settings.
WHEN YOU ARE DONE…
Close Elements, and move files off the C Drive.
Turn off the laser unit, the fluorescent light and the microscope (power strip).
Return the magnification to 1x.
Move the mirror switch lever back to the right.
Move the knob on the front of the microscope back to Bino.
Clean up.
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Laser position adjustment knob (for
forward or backward adjustment)
ND filters
Laser position adjustment knob
(for right or left adjustment)
Mirror switch lever
(pulled out for TIRF)
TIRF Microscopy (09-15)
Focal point adjustment
knob
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