Hi all, I am the confocal learner.

When I used indo-1(Molecular Probe product) to measure calcium
concentration, I got the poor fluorecence signal, even I used the calcium
calibration Kit( Molecular Probe), the fluorecence density of both channel
are only about 80( including dark currents 40, the fluorecence range from 0-
250), normally it should be 150 or more.

The following are some phenomenones.

1. I use LSM410 laser scanning confocal microscope(Zeiss, Thornwood, NY, USA).
2. Increasing UV laser density, the fluorecence density increase.
3. Increasing indo-1 concentration from 5 to 10 micromolar, the fluorecence
increase.
4.  Different objective can lead to different fluorecence density, I don't
know which kind of objective(40*, numeber apture is 0.75) is the most
suitable for my case?  the excitation laser is 364 nm uv laser, and 400-435
and 450-505 nm bandpass filters used for indo-1 emission.


Any suggestion are appreciated.

Xi-Qing

Xi-Qing Wang

Assmann Lab
Biology Department
208 Mueller Laboratory
Penn State University
University Park, PA  16802
phone: (814) 863-9578(o)