polarization dependent excitation of fluorescence probes

[Lu Yan]

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​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

[Zdenek Svindrych-2]

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
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*****

Dear Lu,
all fluorescent probes are polarization dependent (the probability of
absorption depends on the mutual orientation of the polarization of the
incoming photon and the dipole moment of the molecule). But the molecules
are usually not well aligned and the excitation light is usually not a pure
linear polarization...

The depletion is also polarization dependent, my guess is that STED works
better when the excitation and the depletion light have the same
polarization. But there are more factors, probably more important: the
traditional donuts are easy to make and are quite robust when it comes to
aberrations and sample inhomogeneities...

Also, small molecules rotate quickly, so the polarization is 'forgotten' in
less than a nanosecond. Fluorescent proteins or well anchored small-molecule
dyes might show the polarization effects better.

Best, zdenek




---------- Původní zpráva ----------
Od: Yan, Lu <[hidden email]>
Komu: [hidden email]
Datum: 19. 6. 2015 18:29:07
Předmět: 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.
*****

​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"

[John Oreopoulos]

*****
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

[Lu Yan]

*****
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 Zdenek,

Thanks for your reply. For the depletion donut, in terms of being robust
when it comes to aberrations, would azimuthally polarized donut be more
robust, i.e. only having azimuthal component which will never give central
intensity under high NA focusing?


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)
[hidden email]
-----------------------------------------------------

On Fri, Jun 19, 2015 at 8:07 PM, Zdenek Svindrych <[hidden email]> 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,
> all fluorescent probes are polarization dependent (the probability of
> absorption depends on the mutual orientation of the polarization of the
> incoming photon and the dipole moment of the molecule). But the molecules
> are usually not well aligned and the excitation light is usually not a pure
> linear polarization...
>
> The depletion is also polarization dependent, my guess is that STED works
> better when the excitation and the depletion light have the same
> polarization. But there are more factors, probably more important: the
> traditional donuts are easy to make and are quite robust when it comes to
> aberrations and sample inhomogeneities...
>
> Also, small molecules rotate quickly, so the polarization is 'forgotten' in
> less than a nanosecond. Fluorescent proteins or well anchored
> small-molecule
> dyes might show the polarization effects better.
>
> Best, zdenek
>
>
>
>
> ---------- Původní zpráva ----------
> Od: Yan, Lu <[hidden email]>
> Komu: [hidden email]
> Datum: 19. 6. 2015 18:29:07
> Předmět: 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.
> *****
>
> ​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"
>

[Lu Yan]

*****
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)
[hidden email]
-----------------------------------------------------

On Fri, Jun 19, 2015 at 8:17 PM, John Oreopoulos <
[hidden email]> 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
>

[John Oreopoulos]

*****
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,

Take a look at this very recent publication:

http://www.nature.com/nmeth/journal/v11/n5/abs/nmeth.2919.html

In regards to making this happen with STED, why not ask Stefan Hell directly?

John Oreopoulos


On 2015-06-22, at 2:39 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.
> *****
>
> 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)
> [hidden email]
> -----------------------------------------------------
>
> On Fri, Jun 19, 2015 at 8:17 PM, John Oreopoulos <
> [hidden email]> 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
>>

[Zdenek Svindrych-2]

*****
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,
I'm not an expert on high-NA focusing of engineered beams. I haven't found
any sound comparison of traditional vs azimuthal donuts, especially
regarding their robustness in scattering media. I believe that due to
symmetry the traditional donut has a central zero even when focused with
high-NA lens (otherwise it would not work quite so well :-).

zdenek


---------- Původní zpráva ----------
Od: Yan, Lu <[hidden email]>
Komu: [hidden email]
Datum: 22. 6. 2015 14:42:35
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.
*****

Hi Zdenek,

Thanks for your reply. For the depletion donut, in terms of being robust
when it comes to aberrations, would azimuthally polarized donut be more
robust, i.e. only having azimuthal component which will never give central
intensity under high NA focusing?


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)
[hidden email]
-----------------------------------------------------

On Fri, Jun 19, 2015 at 8:07 PM, Zdenek Svindrych <[hidden email]> 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,
> all fluorescent probes are polarization dependent (the probability of
> absorption depends on the mutual orientation of the polarization of the
> incoming photon and the dipole moment of the molecule). But the molecules
> are usually not well aligned and the excitation light is usually not a

pure
> linear polarization...
>
> The depletion is also polarization dependent, my guess is that STED works
> better when the excitation and the depletion light have the same
> polarization. But there are more factors, probably more important: the
> traditional donuts are easy to make and are quite robust when it comes to
> aberrations and sample inhomogeneities...
>
> Also, small molecules rotate quickly, so the polarization is 'forgotten'
in

> less than a nanosecond. Fluorescent proteins or well anchored
> small-molecule
> dyes might show the polarization effects better.
>
> Best, zdenek
>
>
>
>
> ---------- Původní zpráva ----------
> Od: Yan, Lu <[hidden email]>
> Komu: [hidden email]
> Datum: 19. 6. 2015 18:29:07
> Předmět: 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.
> *****
>
> ​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"
>"

[Lu Yan]

*****
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 John,

That was a very interesting paper. Thanks much for pointing to me. Stefan
Hell is apparently not an active user in this email list any longer haha...
I will try to ask him directly but I doubt I will get answer soon.

Thanks,
Lu

On Mon, Jun 22, 2015 at 3:33 PM, John Oreopoulos <
[hidden email]> 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,
>
> Take a look at this very recent publication:
>
> http://www.nature.com/nmeth/journal/v11/n5/abs/nmeth.2919.html
>
> In regards to making this happen with STED, why not ask Stefan Hell
> directly?
>
> John Oreopoulos
>
>
> On 2015-06-22, at 2:39 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.
> > *****
> >
> > 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)
> > [hidden email]
> > -----------------------------------------------------
> >
> > On Fri, Jun 19, 2015 at 8:17 PM, John Oreopoulos <
> > [hidden email]> 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
> >>
>

[Lu Yan]

*****
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 Zdenek,

I have done some high NA focusing studies in the past. The azimuthal donut
actually remains the central intensity null under the tight focusing. As
for the robustness in scattering media, I don't know how it will behave
comparing with the circularly polarized donut.

Cheers,
Lu

On Wed, Jun 24, 2015 at 5:31 PM, Zdenek Svindrych <[hidden email]> 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.
> *****
>
> Hi Lu,
> I'm not an expert on high-NA focusing of engineered beams. I haven't found
> any sound comparison of traditional vs azimuthal donuts, especially
> regarding their robustness in scattering media. I believe that due to
> symmetry the traditional donut has a central zero even when focused with
> high-NA lens (otherwise it would not work quite so well :-).
>
> zdenek
>
>
> ---------- Původní zpráva ----------
> Od: Yan, Lu <[hidden email]>
> Komu: [hidden email]
> Datum: 22. 6. 2015 14:42:35
> 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.
> *****
>
> Hi Zdenek,
>
> Thanks for your reply. For the depletion donut, in terms of being robust
> when it comes to aberrations, would azimuthally polarized donut be more
> robust, i.e. only having azimuthal component which will never give central
> intensity under high NA focusing?
>
>
> 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)
> [hidden email]
> -----------------------------------------------------
>
> On Fri, Jun 19, 2015 at 8:07 PM, Zdenek Svindrych <[hidden email]>
> 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,
> > all fluorescent probes are polarization dependent (the probability of
> > absorption depends on the mutual orientation of the polarization of the
> > incoming photon and the dipole moment of the molecule). But the molecules
> > are usually not well aligned and the excitation light is usually not a
> pure
> > linear polarization...
> >
> > The depletion is also polarization dependent, my guess is that STED works
> > better when the excitation and the depletion light have the same
> > polarization. But there are more factors, probably more important: the
> > traditional donuts are easy to make and are quite robust when it comes to
> > aberrations and sample inhomogeneities...
> >
> > Also, small molecules rotate quickly, so the polarization is 'forgotten'
> in
> > less than a nanosecond. Fluorescent proteins or well anchored
> > small-molecule
> > dyes might show the polarization effects better.
> >
> > Best, zdenek
> >
> >
> >
> >
> > ---------- Původní zpráva ----------
> > Od: Yan, Lu <[hidden email]>
> > Komu: [hidden email]
> > Datum: 19. 6. 2015 18:29:07
> > Předmět: 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.
> > *****
> >
> > ​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"
> >"
>

[Zdenek Svindrych-2]

*****
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 <[hidden email]>
Komu: [hidden email]
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)
[hidden email]
-----------------------------------------------------

On Fri, Jun 19, 2015 at 8:17 PM, John Oreopoulos <
[hidden email]> 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
>"

[Guy Cox-2]

Yes, but, there are all sorts of non-linear processes you can exploit.  In SIM you can use saturation to get again theoretically unlimited resolution.  The late Mats Gustafsson took it up to third-harmonic level.   Before I 'finally finally' retired I was looking at the fact that photo-conversion of FPs is also non-linear and could be used for super-resolution.  (See FOM proceedings from a few years ago).  There really are many interesting avenues to explore.

                                                  Guy

Guy Cox, Honorary Associate Professor
School of Medical Sciences

Australian Centre for Microscopy and Microanalysis,
Madsen, F09, University of Sydney, NSW 2006

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Zdenek Svindrych
Sent: Saturday, 27 June 2015 4:42 PM
To: [hidden email]
Subject: 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.
*****

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 <[hidden email]>
Komu: [hidden email]
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) [hidden email]
-----------------------------------------------------

On Fri, Jun 19, 2015 at 8:17 PM, John Oreopoulos < [hidden email]> 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
>"