Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
Hi Carol,
A question from soneone who ran a Leica SP1 for five years:
Is the silicon surface sending only reflection?
That is, if you get rid of the cells, culuture media, etc, and do a
spectral scan from say 450 to 650 nm with 5 nm bandpass and 5 nm step size,
do you just see reflections at the laser wavelength(s), i.e. 488 and 568?
This should be done after seeting the detector offset to be above zero (at
zero laser power) and gain at less than saturation when the detection band
is centered on 488 nm.
If that spectral scan works (reflections only at laser line or lines), you
can repeat with more experiment components.
FYI - if you have a mirror slide, the reflections from it had better be
only at the laser lines. If you get the same problem with a mirror slide as
you described with your sample, I'd suspect the instrument settings (or the
instrument) being messed up.
George
p.s. if you do not have a reflection slide, you can try to get the Leica
service engineer to come over with their's, or buy one for a few dollars at
http://www.nanofilm.com <http://www.nanofilm.com> ... if you have any
interference filters, any that reflect at the riight wavelength will get the
job done.
George McNamara, Ph.D.
Support Scientist
CITI
City of Hope National Medical Center
1500 E Duarte Rd
Duarte, CA 91010
626-359-8111x60035 voice
[log in to unmask]
George McNamara, Ph.D.
Consultant
Congressman Julian Dixon Image Core
The Saban Research Institute of Childrens Hospital Los Angeles
4650 Sunset Blvd., MS 133, SRT 1016
Los Angeles, CA 90027
323-669-2548 voice
[log in to unmask]
-----Original Message-----
From: Carol Bayles [ mailto:[log in to unmask]
<mailto:[log in to unmask]> ]
Sent: Tuesday, August 16, 2005 12:10 PM
To: [log in to unmask]
Subject: Reflected light
Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
<http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal>
I run a core facility and several of the users grow their cells on
silicon chips, which are very reflective. If they try to see red and
green fluorescence in the cells with our Leica SP2 confocal system,
the reflected light is so bright that the fluorescence is
overwhelmed. We tried closing the pinhole to .65 airy disk and that
helped, but the background is still high and signal is lost. It also
helped to switch to the triple dichroic, relative to the double.
Interestingly, with our old Bio-Rad MRC 600 and our 1024, the
reflected light is not a problem. However, the signal is low and the
noise is much higher on these systems.
Is there anything I can do to reduce the reflected light on the
Leica? Why the difference between the systems?
Thanks,
Carol
--
<><><><><><><><><><><><><><><><><><><><><><>
Carol Bayles, Manager
Microscopy, Imaging & Fluorimetry (MIF)
Biotechnology Resource Center
160a Biotech Bldg
607-254-4860
www.brc.cornell.edu
Confocal and Multiphoton Microscopy
Nanobiotechnology Center
www.nbtc.cornell.edu
Cornell University
Ithaca NY 14853
|
