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Hi,
I haven't used Zeiss Meta system and am not sure about  spectral 
response curve of the system.

But as far as I know, most of these spectral detection  systems are 
calibrated only for
 wavelength accuracy. Not for spectral response. 
We were  using a mixture of  UV excited  dyes to make a broad band 
spectra sample 
and measuring the spectra using a  spectrofluorimeter with spectral 
correction. Then use the same
dye solution under your microscope and measure the spectral profile.  
But important thing is to remember that when you use a *mixture of 
dyes*, depending on excitation
wavelength and whether single photon or two photon, the emission spectra 
can be different. So one
has to use the same excitation wavelength for both systems.  Since we 
have a 405 nm diode laser in
the microscope, we were able to use 405nm excitation in both microscope 
and fluorimeter. We
were using the ratio  of the obtained spectra after normalization as a 
response curve to measure the
unknown spectra. 
But one problem is that,  if you use 405nm excitation for estimating 
correction, the 405 reflection
mirror's transmission is included in the correction.  When you switch to 
another excitation wavelength,
it has a different dichoric mirror in path. So still, the whole 
instrument response curve will be slightly
different for each mirror in path. 
Haridas

PS. Now, many of the spectrofluorimeters are also not corrected and you 
may have to make sure
that they have a response correction factor incorporated in the 
software. If not there are
some protocols available from the manufactures for using Rhodamin B or 
such dye solution to
estimate the correction factor.




Magnus Lilledahl wrote:
> Search the CONFOCAL archive at 
> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal Hi
>
> System: Zeiss LSM 510 META with Coherent MIRA Ti:Sapphire
>
> I wanted to measure the two photon excited fluorescence spectra of 
> various types of tissues (intact tissues in reflection mode). To 
> achieve this I planned on using the lambda mode configuration with the 
> metadetector the find the spectral respons.
>
> My question is whether the system is calibrated for variation in 
> respons at different wavelengths (due to variation in transmission in 
> optical system, grating efficency and detector sensitivity) so that it 
> represents what is actually emitted from the sample and can be 
> compared to spectra aquired with different equipment. If this is not 
> the case, is there a way to do a reference measurement to calibrated 
> for these effects?
>
> Best regards,
>
> Magnus Lilledahl
> NTNU, norway


-- 
Haridas Pudavar, Ph.D. 
Inst. for Lasers, Photonics & BioPhotonics,
458, NSC, Dept. of Chemistry,
SUNY at Buffalo, Buffalo, NY-14260
Ph: (716) 645 6800 x  2220
Fax: (716) 645 6945