Nikon Eclipse Ti TIFT Ee-1454 Manuel - Page 9

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FRAP on the Fly Calibration

Before you can use FRAP the laser must be calibrated

• Find an empty piece of glass without cells/fluorescence

• Set the exposure time around 100 - 200ms

• Open ILAS2 with icon

• Open calibration tab and put the power of selected laser to 100% (in ILAS2 window)

• Activate the laser [G] and press " Show Live"

• Place laser in upper left corner by moving the red cross, press [H]

• 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

• Calibrate with button [i]

• To check if your calibration was successful open " on the fly" tab

• Change time to 3000

• Increase the power of your laser and press " Show Live"

• Click in the image and check if laser pulse in at the position of your mouse pointer

Troubleshooting - FRAP

FRAP calibration error

• Notification: Calibration canceled (exceeded 15 bits)

Possible errors

• Emitting fluorophore or auto fluorescence

o Ensure that the area of the slide really empty

• Too much laser power or exposure time

o Reduce laser power in the ILAS2 window and/or exposure time in Acquire window

Martijn de Gruiter

Manual: TIRF Microscope

Erasmus OIC

Ee-1454