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Hi there,
I've got a problem during ROI bleaching with a Zeiss LSM 510. After scanning with the 750nm laser, I draw and ROI and bleach with the same laser. After rescanning, the bleached region is shifted slightly to the left relative to the drawn ROI. This problem is much like the phenomenon described by Karl Garsha on the listserv (
Item #19986 (21 Nov 2003 11:42) - Spot Size, Raster+Shutter, Q-Dots, Spectral Artifacts) (the particularly relavent portion is underlined):
LASER RASTER & SHUTTER. The laser raster is achieved by providing the
beam steering galvos with an electronic signal in the form of a square
wave (at least in our instrument). The top part of the square wave
signal drives the galvo in one direction, and I believe there may be a
second signal in which the current is reversed for the for the
corresponding low part of the square wave to actively drive the galvo
back on the flyback. I think the amplitude of the waves controls the
speed of the raster, and the period controls the distance(zoom or FOV).
So during one line of a scan, the laser beam is moving continuously.
One interesting test is to view a high concentration of rhodamine--if
the concentration is high enough, the rhodamine will quench itself in a
time dependant manner. If the speed of the laser while moving along one
direction is not constant, the pixels in the resultant image will be
darker where the laser moves more slowly. When I discovered this, we
had a very repeatable speed variation across the field of view, so
images of the rhodamine sea were striped, and could even be averaged
over several scans. The pattern changed at different scan speeds and
could have been due to a systematic variation in the top plateau of the
square wave signal or a bad galvo. The shutter is simply a 'garage
door' like affair controled by some sort of solenoid, and it shuts by
gravity. The shutter remains open for the duration of the scan of one
frame (sometimes you can hear it 'click-click-click' with each frame). I
don't think the shutter can open and close any faster than 1msec.
During the scan raster, the laser power is attenuated at the beginning
and/or end of the scan by the AOTF, which can modulate the laser power
much faster than the mechanical shutter. If the offset of the signal
going to the AOTF is miscalibrated, you can get things like a black bar
along one side of an ROI scan, with the ROI being scanned offset from
the one drawn by some distance (dependent on scan speed and zoom). Unfortunately there does not seem to be any software options availble to calibrate the AOTF to the scanning module.
Does anyone know how to calibrate this in the Zeiss LSM 510 software (ver 3.2), or has anyone written a macro that does this?
Thanks,
Dave Anchel,
Toronto
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