*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
Post images on http://www.imgur.com and include the link in your posting.
*****

Hi Lu,
of course, STED *is* a nonlinear technique in a sense that the intensity of 
fluorescence is a nonlinear function of the intensity of incoming light (in 
this case the intensity of depletion). The peak depletion intensity has to 
be well above saturation threshold of this process to allow for significant 
increase in resolution (see the famous Hell-modified Abbe formula).

On the other hand, the theory of linear super-resolution imaging (such as 
SIM, image scanning microscopy aka AiryScan, etc) states that only a factor-
of-two resolution improvement is possible, compared to widefield 
fluorescence.

Different polarization states have been used as a 'contrast' in structured 
illumination (not sure about superresolution, but Rainer Heintzmann has 
demonstrated it in optical sectioning SIM, see "picoSIM"), anyway, you are 
still limited by 2x resolution increase. If you aim at 'higher' 
superresolution, you need to exploit some non-linear behavior of 
fluorescence intensity...

Best, zdenek


---------- Původní zpráva ----------
Od: Yan, Lu <[log in to unmask]>
Komu: [log in to unmask]
Datum: 22. 6. 2015 15:20:36
Předmět: Re: polarization dependent excitation of fluorescence probes

"*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
Post images on http://www.imgur.com and include the link in your posting.
*****

Wow... John, thanks so much for this comprehensive list of literature. The
main reason I had this question was that, the other day when I was looking
at some Hell's presentation, he talks about how STED is not a nonlinear
tech., and how its key is all about being able to manipulating probes in
two distinguishable states, since we are a fiber group, I was wondering if
the probe states can be distinguished by two polarizations of excitation
beams, i.e. azimuthal polarized and radially polarized, therefore form some
imaging contrast.

So in your opinion, would that be doable?

cheers,
lu

-----------------------------------------------------
Lu Yan
Nanostructured Fibers and Nonlinear Optics Laboratory
Electrical and Computer Engineering
Boston University
8 St. Mary St., Boston, MA, 02215
617.353.0286 (office) | 617.358.5917 (lab)
[log in to unmask]
-----------------------------------------------------

On Fri, Jun 19, 2015 at 8:17 PM, John Oreopoulos <
[log in to unmask]> wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> Post images on http://www.imgur.com and include the link in your posting.
> *****
>
> Dear Lu,
>
> I can't say much about question #2 you raised since I have no experience
> with STED, but in regards to question #1...
>
> Pretty much all fluorescent probes can undergo photoselection
> (preferential absorption of linearly polarized light, aka linear 
dichroism)
> since all fluorescent probes usually possess an linear absorption
> transition dipole moment that runs parallel to some part of the probe's
> chemical structure. What varies for these fluorescent probes is to what
> degree the emission light (ie: the fluorescence) is polarized along the
> same direction of the input polarization, and this in turn depends on how
> mobile the fluorescent probe is in its local molecular environment as well
> as how long the probe has to move around before emitting fluorescence (ie:
> the length of the fluorescence lifetime). Generally, large fluorescent
> probes, such as the fluorescent proteins like GFP and all its spectral
> variants don't move very much during their relatively short fluorescence
> lifetime and emit mostly polarized light if excited with polarized light.
>
> Other photochemical processes such as FRET can also lead to depolarization
> (in fact, this is one way to detect FRET).
>
> Here are some references of interest that demonstrate the many examples in
> the literature where researchers have exploited this effect to learn some
> new science:
>
>
>
> Lakowicz, J., Principles of fluorescence spectroscopy. 3rd ed. 2006, New
> York: Springer.
>
> Axelrod, D., Fluorescence polarization microscopy, in Methods in cell
> biology, T. Langsing and Y. Wang, Editors. 1989, Academic Press: San 
Diego.
> p. 333-352.
>
> Schutz, G.J., H. Schindler, and T. Schmidt, Imaging single-molecule
> dichroism. Optics Letters, 1997. 22(9): p. 651-653.
>
> Harms, G.S., et al., Single-molecule anisotropy imaging. Biophysical
> Journal, 1999. 77(5): p. 2864-2870.
>
> Sund, S.E., J.A. Swanson, and D. Axelrod, Cell membrane orientation
> visualized by polarized total internal reflection fluorescence. 
Biophysical
> Journal, 1999. 77(4): p. 2266-2283.
>
> Forkey, J.N., M.E. Quinlan, and Y.E. Goldman, Protein structural dynamics
> by single-molecule fluorescence polarization. Progress in Biophysics &
> Molecular Biology, 2000. 74(1-2): p. 1-35.
>
> Gidwani, A., D. Holowka, and B. Baird, Fluorescence anisotropy
> measurements of lipid order in plasma membranes and lipid rafts from
> RBL-2H3 mast cells. Biochemistry, 2001. 40(41): p. 12422-12429.
>
> Inoue, S., et al., Fluorescence polarization of green fluorescence
> protein. Proceedings of the National Academy of Sciences of the United
> States of America, 2002. 99(7): p. 4272-4277.
>
> Jameson, D.M. and J.C. Croney, Fluorescence polarization: Past, present
> and future. Combinatorial Chemistry & High Throughput Screening, 2003.
> 6(3): p. 167-176.
>
> Borejdo, J., et al., Changes in orientation of actin during contraction of
> muscle. Biophysical Journal, 2004. 86(4): p. 2308-2317.
>
> Lopes, S. and M. Castanho, Overview of common spectroscopic methods to
> determine the orientation/alignment of membrane probes and drugs in 
lipidic
> bilayers. Current Organic Chemistry, 2005. 9(9): p. 889-898.
>
> Vrabioiu, A.M. and T.J. Mitchison, Structural insights into yeast septin
> organization from polarized fluorescence microscopy. Nature, 2006.
> 443(7110): p. 466-469.
>
> Greeson, J.N. and R.M. Raphael, Application of fluorescence polarization
> microscopy to measure fluorophore orientation in the outer hair cell 
plasma
> membrane. Journal of Biomedical Optics, 2007. 12(2).
>
> Piston, D.W. and M.A. Rizzo, FRET by fluorescence polarization microscopy,
> in Fluorescent proteins, second edition. 2008, Elsevier Academic Press 
Inc:
> San Diego. p. 415-430.
>
> Jameson, D.M. and J.A. Ross, Fluorescence polarization/anisotropy in
> diagnostics and imaging. Chemical Reviews, 2010. 110(5): p. 2685-2708.
>
> Ghosh, S., et al., Chapter sixteen - dynamic imaging of homo-FRET in live
> cells by fluorescence anisotropy microscopy, in Methods in enzymology, P.
M.
> Conn, Editor. 2012, Academic Press. p. 291-327.
>
>
>
> Cheers,
>
> John Oreopoulos
> Staff Scientist
> Spectral Applied Research Inc.
> A Division of Andor Technology
> Richmond Hill, Ontario
> Canada
> www.spectral.ca
>
>
>
> On 2015-06-19, at 5:36 PM, Yan, Lu wrote:
>
> > *****
> > To join, leave or search the confocal microscopy listserv, go to:
> > 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,
> >
> > I am hopeful I could get some help here. I figured a lot of times 
getting
> > answers here is much quicker than looking into literature.​
> >
> > So my questions are:
> >
> > 1. Are there any fluorescence probes that would be excited by only a
> > certain polarization of the light, i.e. the excitation of the probe is
> > polarization dependent?
> >
> > 2. Why are the azimuthally polarized donut beams not being widely used 
in
> > the STED area? I only saw a few papers talking about using it in STED. 
Is
> > it because the polarization is not ideal for the depletion or just
> because
> > the beam is harder to make than the circularly polarized donut beams?
> >
> > Thanks much,
> > Lu
>"