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Dear Everyone:
Thank you all for your input. Just back from my weekend so I need some time
to digest these info especially with the suggested papers. Also I'll have an
appointment with the head of our imaging center to discuss about it so I
guess I might get a feasible plan.
What confuses me a bit is that it seems that 5 color imaging is really
doable (even routine) but the people around me ,including those who started
to use confocal microscopy from the 1st generation, are not very positive
about this. As a person coming from conventional molecular biology
background I'll try to understand the discrepancy.
After collecting/understanding enough info I'll try to compose a 'protocol'
of 5/6 color imaging as I presume more people might be interested in it.
More questions from me can be expected.
Thanks again.
Best wishes,
Aro
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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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>
> 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
>
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