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Hi Ohkyung-
It seems like this could be one of two things- either the first dichroic
that is available on the Zeiss META channel ((UV/488/543/633) or the
pins that are put in place for the Zeiss scope to prevent reflected
light from hitting the META PMT array. The initial dichroic for the META
channel will reflect the indicated wavelengths of light onto the sample
and block those wavelengths from reflecting back off the sample and
transmitting through the detection beampath. So in the setup you are
using, they are set up to block light at whatever the indicated UV
wavelength is, the 488 wavelength, the 543 wavelength, etc. That can
create the partial "dips" in your spectra. Are the dips at these
approximate wavelengths? If you are exciting with 350 nm light, you
certaintly don't need a dichroic that includes the wavelengths of 488,
543, and 633- do you have another dichroic available in the turret? The
other possibility involves the pins that are put in place for the META
detector when multiple laser lines are on at the same time. I believe
(and I know this list has discussed this before) that when any given
laser line (e.g. 514 nm) is turned on, a blocking pin is put in place
after the prism dispersion element of the META channel that diminishes
the amount of 514 light that reaches the detector. This is generally a
quite helpful feature since reflected light from the laser will
overwhelm your PMT, even with appropriate other filters in place (the
META detector is the one instance where one may be using an excitation
wavelength that is NOT below the wavelengths are you detecting in
emission, so reflected laser light is a big concern). But it also causes
these dips in spectra that make them not line up with published spectra.
This isn't a problem if your "control" spectra for spectral unmixing was
also taken with the same "track" and settings.
-Jason
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-----Original Message-----
From: Confocal Microscopy List [mailto:[log in to unmask]] On
Behalf Of Oky
Sent: Thursday, November 08, 2007 7:41 PM
To: [log in to unmask]
Subject: Beam splitter: UV/488/543/630
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Dear all,
* What is the difference of the detector of CLSM (Zeiss LSM 510 META)
and fluorescence spectrometer (microplate reader, Molecular Devices
spectraMax)? Or excitation source? For the microplate reader, I used the
excitation wavelength at 350 nm and collected fluorescence intensity
between 360 - 720 nm. The emission wavelength interval was 2 nm. For the
META detector, UV laser at 364 nm was used for excitation and used all
32 channels for collection of emission. The emission wavelength interval
was given as 10.7 nm.
I have scanned some wood cell wall utilizing both instrument. I expected
very similar spectra from both. The microplate reader seems to generate
similar spectra to those in literatures, but the spectra from the META
detector were very different from those of the microplate reader. The
spectrum from the META detector consists of four major peaks with
successively decreased intensity. If I connect only the peaks, it would
be similar to the spectrum from the microplate reader.
I have been trying to figure out what causes the difference. I want to
make sure whether the characteristics of the instrument or something I
have done incorrectly cause the differences in the spectra from both
instrument. Here are a few possible reasons I could draw. 1. differences
in light source and optical setup in each instrument 2. beam splitter
(UV/488/543/630; the number may not be exact, but
close) in Zeiss LSM 510 META
3. reflection between cover glass and sample surface
Thanks.
--
Ohkyung Kwon, Ph D
http://www.meso.or.kr/portfolio
http://www.wpskorea.org
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