Re: Ratiometric measurements for multiphoton microscopy

Posted by Johannes Helm on
URL: http://confocal-microscopy-list.275.s1.nabble.com/Ratiometric-measurements-for-multiphoton-microscopy-tp7585091p7585093.html

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Dear Konstantin,

we had done something like the thing you describe for a CLSM in the 90s.
Please, check these papers:

A)
P.J. Helm, O. Franksson, and K. Carlsson (1995),
A confocal scanning laser microscope for quantitative ratiometric 3D
measurements of [Ca2+] and Ca2+ diffusions in living cells stained
with Fura-2,
Pflügers Archiv - European Journal of Physiology, Vol. 429, pp. 672-681

B)
PJ Helm, A Patwardhan, and EMM Manders (1997),
A study of the precision of confocal, ratiometric, Fura-2 based [Ca2+]
measurements,
Cell Calcium 22(4):287-298


These systems might work even in the multi photon case.

HOWEVER:
Concerning A)
1)
Using excitation ratiometric dyes will be troublesome in the multi
photon case:
a)
It is difficult, often impossible, to avoid cross excitation in
non-linear excitation. An exception is the CFP-YFP-FRET based Chameleont
dye. However, you would need TWO lasers for this dye, which would be
rather expensive and space consuming ("stapling" lasers is certainy not
recommended, however, you could install them on a sub-table-top shelf,
which some Companies selling and producing Optical tables offer).
b)
Whe using the Chameleont dye, you would need a FRET detection. You might
check another paper with a proposal on how to measure FRET:

Helm P.J. (2012),
Proposal of a New Method for Measuring Foerster Resonance Energy
Transfer (FRET) Rapidly, Quantitatively and Non-Destructively,
International Journal of Molecular Sciences 13(10):12367-12382

c)
Using an emission ratiometic dye might be technically easier, although I
know that one needs "this dye" and cannot use "that one" for some
reasons.

2)
You would have to carefully select your EOMs (or AOMs)!

Concerning B)
WARNING:
One needs a really large number of detected photons in order to be able
to use the raw data as basis for a fully quantitative ratiometric
analysis.


Please, note that Kjell Carlsson and Anders Liljeborg had continued to
work on the  IMS method, which is a beautiful technology, indeed. It can
be used - and has been so, although not for ratiometric imaging, for
which it nevertheless would be suitable - for suppressing cross talk and
bleed through between different imaging channels. I also allow myself to
suggest this, in my opinion outstanding, paper,

Carlsson K1, Liljeborg A.
Simultaneous confocal lifetime imaging of multiple fluorophores using
the intensity-modulated multiple-wavelength scanning (IMS) technique.
J Microsc. 1998 Aug;191(2):119-127.

Best wishes,

Johannes

--
P. Johannes Helm, M.Sc. PhD
Seniorengineer
University of Oslo
Institute of Basic Medical Sciences
Department of Molecular Medicine
Division of Physiology
P. O. Box 1103 - Blindern
NO-0317 Oslo
Norway

Voice: [phone]
Fax: [phone]



On 2016-04-25 11:32, Microscopia-IBIS wrote:

> *****
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> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> Post images on http://www.imgur.com and include the link in your
> posting.
> *****
>
> Dear all.
>
>
>
> One of our groups is interested in doing fluorescence ratiometric
> measurements (e.g., FURA-2, ROS) using a multiphoton microscopy system.
> From
> what we understand the laser tuning speeds won't be fast enough to
> switch
> between two different excitation wavelengths, and we were wondering if
> there
> could be ways to maintain these ratiometric attributes while still
> performing a laser two-photon excitation. Any ideas or suggestions will
> be
> very much appreciated.
>
>
>
> Thanks and regard,
>
> Dr. Konstantín Levitskiy
>
> Servicio de Microscopía
>
> InstitutodeBiomedicinadeSevilla - IBiS
>
> Campus del Hospital Universitario Virgen del Rocío
>
> Avda. Manuel Siurot s/nº
>
> 41013 Sevilla
>
> Tlfno: 955 92 3030
>
> Email: [hidden email]
>
> Web: www.ibis-sevilla.es
>
>
>
>
>
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