http://confocal-microscopy-list.275.s1.nabble.com/5-color-imaging-6-color-would-be-even-better-tp7579662p7579678.html
right lasers in the system. Not many are equiped with five lasers. If
C3.5/Texas Red and Cy5. We did this with 405, 488, 561, 594 and 633. You
pretty strongly in the orange channel when excited simultaneously. See
Cytogenet Genome Res. 2006;114(3-4):367-78. Review.
been used to get 8-color labeling. The key to success was to use very
identify all 24 human chromosomes by a unique combination of colors. See
PLoS Biol. 2005 May;3(5):e157. Epub 2005 Apr 26.
Nat Genet. 1996 Apr;12(4):368-75.
> *****
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>
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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
>
>> *****
>> To join, leave or search the confocal microscopy listserv, go to:
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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
>>
>
Steffen Dietzel, PD Dr. rer. nat
Marchioninistr. 15, D-81377 München
Marchioninistr. 27, München-Großhadern