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Good evening,

Kevin and George have already provided good answers. What I write in the
following lines is more of a concept than a practical solution in case of
camera detection based systems.

Two very interesting - to my mind - approaches have been described and
successfully used by Åslund, Carlsson, Liljeborg and others in the 1990s
on a conventional CLSM. The approaches, both called "IMS Technique", are
based on lock-in detection principles:

In brief:

Provided, one has n e |N >1 dye substances, which can be excited at
available laser wavelengths without any cross excitation, the resp. laser
beams each exciting one, and one only, out of the n dyes can be modulated
using, e.g., n electro optic modulators at n individual frequencies and
phases.

First approach:
If the periods of the n modulation frequencies are long compared to the
decay times of the dye substances, the resulting n fluorescences will then
also be modulated at the same frequencies and phases (at which, in this
case, phase shifts related to the different decay times of the dyes can be
neglected). Irrespectively of any spectral overlap of the fluorscence
spectra of the dyes, the resulting fluorescence signals as detected by one
photomultiplier can be read out via n lock-in amplifiers and be recorded
at negligibly large cross talk to n channels.

Second approach (basically a life time method):
If the periods of the modulation frequencies are short scompared to the
decay times of the lasers, the resulting fluorescences will then also be
modulated at
the same frequencies but different phases (in this case, other then in the
first approach, phase differences as induced by the different decay times
of the different dyes cannot be neglected but are the detection
magnitude). If the n dyes have n different decay times, at which any Delta
T between any two dyes must be large enough to cause a specific phase
shift of the fluorescences, n lock-in amplifiers can be used to read out
the resp. signals from one photomultiplier - with sufficiently small
transition time spread - at negligible cross talk into n channels.

A task as far as I know not done on a regular basis and possibly un-done -
besides the financial challenge, of course - for this method to be applied
to spinning disk systems including camera detection would be to read out
the signals as detected by the camera via lock-in amplifiers.
Alternatively, one might try to use PMT arrays.

References:

E.g.

K. Carlsson, N. Åslund, K. Mossberg & J. Philip, "Simultaneous confocal
recording of multiple fluorescent labels with improved channel separation"
J. Microsc. 176, 287-299 (1994).

K. Carlsson & B. Ulfhake, "Improved fluorophore separation with IMS
confocal microscopy" NeuroReport, 6(8), 1169-1173 (1995).

K. Carlsson, "Signal-to-noise ratio for confocal microscopy when using the
IMS technique" Micron 26, 317-322 (1995)

P.J. Helm, O. Franksson & K. Carlsson, "A confocal scanning laser
microscope for quantitative ratiometric 3D measurements of [Ca2+] and Ca2+
diffusion in living cells stained with Fura-2" Pfluegers Arch - Eur J
Physiol, 429, 672-681 (1995).

K. Carlsson, A. Liljeborg, "Confocal fluorescence microscopy using
spectral and lifetime information to simultaneuosly record four
fluorophores with high channel separation", J. Microsc. 185, 37-46 (1997).

PJ Helm, A Patwardhan, and EMM Manders (1997),
A study of the precision of confocal, ratiometric, Fura-2 based measurements,
Cell Calcium 22(4):287-298

K. Carlsson & A. Liljeborg, "Simultaneous confocal lifetime imaging of
multiple fluorophores using the Intensity-modulated Multiple-wavelength
Scanning (IMS) technique," J. Microsc. 191, 119-127 (1998).

as well as

Åslund, N.; Carlsson, K.S. Apparatus for Quantitative Imaging of Multiple
Fluorophores.
U.S. Patent 5,294,799, 15 March 1994.

Åslund, N.; Carlsson, K.S. Apparatus for Quantitative Imaging of Multiple
Fluorophores Using Dual Detectors. U.S. Patent 5,418,371, 23 May 1995.



Best wishes,

Johannes


-- 
P. Johannes Helm, M.Sc. PhD
Seniorengineer
CMBN
University of Oslo
Institute of Basic Medical Science
Department of Anatomy
Postboks 1105 - Blindern
NO-0317 Oslo

Voice:	+47 228 51159
Fax:	+47 228 51499

WWW:	folk.uio.no/jhelm

> *****
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http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Dear Everyone:
>
> I have been trying to visualize a macromolecular structure with 4 color
imaging. I have been doing this with spinning disc microscopy as I need
complete sections of cells also to know the distribution of the
structure
> inside the cells in 3D. So far things are working properly BUT ...
>
> We now would like to upgrade to 5 color imaging as we want to visualize
additional components of the structure. I checked the spinning disc
setting
> myself and I asked several senior users of spinning disc and got the
opinion
> that "5 color imaging with spinning disc without bleed-through is hardly
possible, if possible at all". The main reason being that it is not very
likely to set up emission filter combinations for 5 color imaging
without
> bleed-through. Spectral mixing is technically possible but not very
promising.
>
> I also learned that 5 color imaging is possible with laser scanning
microscopy with which one can specifically define the emission band pass
range. But with laser scanning microscope it will cost me much more time
to
> collect enough data for statistical analysis.
>
> So my questions would be: can anyone of you share your
> experience/opinion/literatures on 5 color imaging? I also tried to look for
> literatures describing multi-color (> 4 color) imaging (I think there
must
> be decent papers about it) but so far I have not found any. Maybe some
of
> you have some nice papers about this?
>
> Thank you very much for your input.
>
> Nice weekend!
>
> Aromis
>