in reply to Dave:
>I can see the advantage of this for each line being closer in time, but
>does it take significantly longer to do the entire field?  If you had a
>cell at high mag, how different in time is the beginning of the scan >(top of cell) to the end of scan (bottom of cell). Dave


The total time for a field varies with the image quality you like to
have at a given signal strength. In some of our experiments we do one
frame in around 500 msec (which corresponds to top and bottonm of a cell
at high mag). This can be a long time for a sharp rising Ca2+ transient.
When using fura-2 or BCECF in the confocal it is therefore a big
advantage doing a bidirectional scan with 351 in one direction and 364
in the flyback or 457 and 488 for BCECF. Getting a line ratio within
msec. I see the same advantage when doing faster scans in small ROIs
where you scan between 10 to 25 frames per second for recording fast
Ca2+ changes. For the dual excitation dyes where you record the two
emission signals with the same PMT you have to use the AOTF to fine tune
the intensities as the PMT sensitivity can not be changed on a line
basis. We have used these features also for double volume measurements
in the nucleus and the cytosol (labelled by two dyes) to monitor if cell
volume changes simultaneously in both compartments.

In our system, a LSM 510, we could not measure any delay in image
acquisation by using multitracking in the line scan mode compared to
unidirectional scanning. On other brand confocals I have measured some
extra delay just when doing only wavelength switching but not filter
switching between frames. For all physiology people this is quite
important but not for recording labelled specimens.

Roland





Dr. Roland Nitschke
University of Freiburg
Institue of Physiology
Hermann-Herder-Str. 7
D-79104 Freiburg

phone ++49 761 203 5195
fax       ++49 761 203 5191
E-mail  [log in to unmask]