Beam splitter: UV/488/543/630

[Oky]

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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

[Miller, Jason]

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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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University of California- San Francisco
 
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-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On
Behalf Of Oky
Sent: Thursday, November 08, 2007 7:41 PM
To: [hidden email]
Subject: Beam splitter: UV/488/543/630


Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal

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

[Oky]

Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal

Thank you for your prompt reply.

But I am still wondering the effect of light source in both instrument.
In the case of CLSM is clear. It uses laser. Then how about the
microplate reader? When I specify an excitation wavelength, does the
microplate reader generate laser or use a filter? If it uses a filter
for certain excitation wavelength, how do I know the performance or
the spectral characteristics of the filter?

Again thanks for the answers in advance.


--
Ohkyung Kwon, Ph D

http://www.meso.or.kr/portfolio
http://www.wpskorea.org

[Julio Vazquez]

Search the CONFOCAL archive at http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal =

Oky, 

the spectramax uses a monochromator, while the LSM uses a set of fixed laser lines for excitation. When you do a spectral scan on the LSM, the detection bands that match the excitation have to be removed to prevent ugly reflection artifacts. If you have a four-band beam splitter, these four bands will be suppressed from your final image.  You can circumvent this by using a single band beamsplitter (primary dichroic). For instance, if you excite with a 488 laser line and a 488 dichroic, your emission spectrum above 500 nm should show no gaps... 

As for the spectramax, I am not sure how they separate excitation from emission, but they show polarizers in their illustrations.  I can imagine that if you use  polarized light for the excitation, and then use a polarizer to filter the emission, you could remove the excitation light at any given wavelength, without need for any fixed-band dichroics or other blocking optics. This allows you to measure the spectrum over the entire range, without any gaps... just a guess...  Such a system would eat out a lot of the emission light, but for a plate reader, this is probably not as big an issue as for a confocal, where light is more limited. I am just speculating here... I don't really know the details of their optical path. 

Also, another difference might be that the emission may depend to some extent on the excitation. On the LSM, you use one (or a few) fixed excitation lines, while with the spectramax and its monochromator, you have more flexibility on the excitation side...  In this situation, the emission at let's say 600 nm may be different depending on whether you are exciting at 488nm or at 560 nm, for example.

you can find technical info on the spectramax at this site:

http://www.moleculardevices.com/pages/instruments/spectramax_m5.html

--

Julio Vazquez

Fred Hutchinson Cancer Research Center

Seattle, WA 98109-1024

http://www.fhcrc.org/

==

On Nov 8, 2007, at 10:07 PM, Oky wrote:

Search the CONFOCAL archive at

http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal

Thank you for your prompt reply.

But I am still wondering the effect of light source in both instrument.

In the case of CLSM is clear. It uses laser. Then how about the

microplate reader? When I specify an excitation wavelength, does the

microplate reader generate laser or use a filter? If it uses a filter

for certain excitation wavelength, how do I know the performance or

the spectral characteristics of the filter?

Again thanks for the answers in advance.

-- 

Ohkyung Kwon, Ph D

http://www.meso.or.kr/portfolio

http://www.wpskorea.org

[Grant MacGregor]

Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal

Has your META been calibrated ?


On Nov 8, 2007, at 7:40 PM, Oky wrote:

> Search the CONFOCAL archive at
> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>
> 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