Manual: TIRF Microscope
Ee-1454
Martijn de Gruiter
Erasmus OIC
9
FRAP on the Fly Calibration
Before you can use FRAP the laser must be calibrated
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Find an empty piece of glass without cells/fluorescence
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Set the exposure time around 100 - 200ms
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Open ILAS2 with icon
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Open calibration tab and put the power of selected laser to 100% (in ILAS2 window)
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Activate the laser [G] and press “ Show Live”
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Place laser in upper left corner by moving the red cross, press [H]
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Repeat for lower right corner
o
If the spot isn’t visible or too big and bright change power and/or exposure time
o
view is rotated, so move laser in other direction
o
keep in mind to move red cross into left and right corners of the calibration screen
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Calibrate with button [i]
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To check if your calibration was successful open “ on the fly” tab
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Change time to 3000
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Increase the power of your laser and press “ Show Live”
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Click in the image and check if laser pulse in at the position of your mouse pointer
Troubleshooting - FRAP
FRAP calibration error
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Notification: Calibration canceled (exceeded 15 bits)
Possible errors
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Emitting fluorophore or auto fluorescence
o
Ensure that the area of the slide really empty
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Too much laser power or exposure time
o
Reduce laser power in the ILAS2 window and/or exposure time in Acquire window
G
i
H
K
J
7
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