http://confocal-microscopy-list.275.s1.nabble.com/paper-help-tp4115966p4232515.html
Sorry, I sent the wrong Youtube link for the MIT video. See this one
> Shigeo,
>
> I'm not sure if the unusual polarization properties of TIRF
> microscopy should be called a "problem". As I said at the end of my
> last posting on this topic, polarized TIRF microscopy/spectroscopy
> can be exploited to assess the orientation or order of fluorescent
> molecules near a surface, and in that case I would consider the
> effect a benefit, not a problem.
>
> On the other hand, if you're talking about single-molecule imaging
> (in vivo or in vitro), the polarization in TIRF is something to be
> aware of and perhaps controlled depending on what information is
> trying to be sought.
>
> Fluorescing molecules behave like tiny electric antenna and the
> absorption of light by a single fluorescing molecule is
> polarization dependent. The origin of this behavior is based on the
> existence of a definite transition dipole moment vector for the
> absorption and emission of light that lies along a specific
> direction within the fluorophore structure. Not surprisingly, these
> transition dipole moments usually lie roughly along the chain of
> conjugated double bonds in the chemical structure which possess
> outer orbital electrons that can easily oscillate along these
> directions. This region of the molecule is called the chromophore.
> The PROBABILITY of light absorption (and subsequent fluorescence
> emission) by a single molecule is maximized when the polarization
> of the incident light (ie: the electric field vector associated
> with the light) is parallel to the transition dipole moment vector
> of the molecule and follows a cosine squared dependence for all
> other angles between the two vectors. If you'd like to see a
> macroscopic example of this with real electric antenna, check out
> the youtube video provided by the physics department at MIT:
>
>
http://www.youtube.com/watch?v=nCAKQQjfOvk&NR=1>
> Isolated fluorescing molecules behave the same way as the
> macroscopic radio antenna, and so the physics is the same.
> Lakowicz's book Principles of Fluorescence Spectroscopy contains a
> full mathematical derivation of the effect at the single-molecule
> level and explains how it is used in fluorescence spectroscopy
> applications. What is truly amazing is that this phenomenon can
> even be observed directly with an epifluorescence microscope
> equipped with a polarizing optic on the illumination side. See this
> paper:
>
> Schutz, G. J., H. Schindler, and T. Schmidt. 1997. Imaging single-
> molecule dichroism. Opt. Lett. 22:651-653.
>
> Again, what is unique about polarized TIRF microscopy compared to
> polarized epifluorescence microscopy is that it is possible to
> preferentially excite single molecules that have their transition
> dipole moment oriented more vertically outside of the xy plane of
> imaging because the "p" polarization of TIRF is directed mostly
> along the z-direction (perpendicular to the imaging plane).
> Therefore, it becomes possible through some clever choices of
> modulated polarized excitation in TIRF to assess the full 3D
> orientation of a fluorescent molecule (on a given time-scale
> determined by the exposure time of imaging) relative to the sample
> substrate (the xy imaging plane again). Truly, the best example of
> this is the work undertaken earlier this decade by the Goldman
> group who used polarized TIRF illumination (and observation of the
> polarized emission) of singly fluorescently labeled Myosin
> molecular motors to assess the protein's structural dynamics on
> actin filaments. I am always astounded by complexity and ingenuity
> of these experiments as well as the information about molecular
> orientation that can be gleaned from them:
>
> Forkey, J. N., M. E. Quinlan, and Y. E. Goldman. 2000. Protein
> structural dynamics by single-molecule fluorescence polarization.
> Prog. Biophys. Mol. Biol. 74:1-35.
>
> Forkey, J. N., M. E. Quinlan, and Y. E. Goldman. 2005. Measurement
> of single macromolecule orientation by total internal reflection
> fluorescence polarization microscopy. Biophys. J. 89:1261-1271.
>
> Note that the equipment used in the above examples is home-built
> and customized.
> So in the cases stated above, I would not call the polarization of
> TIRF a problem, but rather an advantage. If on the other hand you
> are trying to measure some other property other than orientation of
> single molecules, then it might be considered a problem. For
> example, some researchers try observe the diffusion of single
> fluorescent molecules ("single particle tracking") to assess
> structural changes in the environment of the probe. Other
> researchers may try to observe FRET between two single fluorophores
> attached to a protein to assess dynamic changes in protein folding
> conformation at the single protein level ("single molecule
> intramolecular FRET"). In carefully calibrated single molecule
> experiments, it is even possible to count the number of molecules
> in a diffraction limited spot to assess absolute concentrations of
> labeled proteins, DNA, etc. ("number and brightness analysis"). All
> of these types of single-molecule measurements are intensity-based
> just like the single-molecule polarization measurements stated
> above, however. On top of that, single molecules sometimes exhibit
> some complicated photophysics that lead to so-called blinking on
> different time scales. So if you are trying perform one of these
> other types of single molecule measurements, it would be best to
> remove all polarization bias of the excitation of the molecule,
> especially if the molecules under study rapidly rotate or re-orient
> in time. Obviously, one way to do this would be to "depolarize" the
> incident illumination light used in your experiment. Unfortunately
> it is surprisingly difficult to create a perfectly depolarized
> source of light in a microscope since every time the light
> transmits through or reflects from an internal optic (lenses,
> mirrors, etc.) the light becomes polarized a small amount in one
> direction. In addition, lasers usually emit strongly linearly
> polarized light as well. You can check the polarization direction
> of your laser beam by rotating a film polarizer (polaroid) in the
> path of the beam, the direction of polarization being the the angle
> of the polaroid that gives you maximum transmission. Again, it is
> very difficult to depolarize the laser light and so the solution to
> the polarization "problem" in these cases is to create CIRCULARLY
> polarized light using an optic called a quarter-wave plate.
> Circularly polarized light can be considered light composed of an
> equal amount "s" and "p" polarized light with a 90 degree phase
> shift between these two electric field vectors. To learn more about
> this, see these links:
>
>
http://en.wikipedia.org/wiki/Polarized>
http://en.wikipedia.org/wiki/Quarter-wave_plate>
> As you know now, "p" polarization in TIRF contains light partially
> polarized along the x-direction, but mostly along the z-direction.
> "s" polarized light in TIRF is purely along the y-direction. So
> circularly polarized light used in TIRF microscopy gives you
> polarized illumination that is somewhat isotropic or unbiased, but
> not perfectly so. You will sometimes (but not always) see mentioned
> in the methods sections of single-molecule study publications the
> addition of a quarter-wave plate in the laser illumination beam
> path. For one example, see the final paragraph of the following paper:
>
> Toprak, E., J. Enderlein, S. Syed, S. A. McKinney, R. G. Petschek,
> T. Ha, Y. E. Goldman, and P. R. Selvin. 2006. Defocused orientation
> and position imaging (dopi) of myosin v. Proc. Natl. Acad. Sci. U.
> S. A. 103:6495-6499.
>
> Now I think I am in a good position to properly answer your two
> questions:
>
> 1. Most commercial TIRF microscope systems utilize laser
> illumination (with the exception of the so-called "white-light"
> TIRF systems that use a Mercury lamp), and so the incident light
> will be linearly polarized along a certain direction depending on
> the rotation angle of the laser cavity relative to the microscope
> entry port. As far as I know, the current commercial systems make
> no attempt to control this, so yes, in a single-molecule experiment
> (and depending on the orientational dynamics of the molecules under
> study) you might be preferentially selecting/exciting a sub-
> population of molecules that eventually emit fluorescence.
>
> 2. I believe that current commercial TIRF microscope systems only
> utilize a single incident laser beam directed through a single
> point on the periphery of a high NA microscope objective and not a
> ring-shaped illumination pattern that encompasses the entire
> periphery of the objective aperture. I am aware of a few examples
> of these "ring-beam" TIRF systems in the literature, but they are
> all home-built setups that "solve" this polarization problem. I
> have also seen examples of "flying-spot" TIRF illumination where
> the single beam is forced to rotate rapidly along the periphery
> leading to the same unbiased polarization effect that a ring-beam
> provides. These types of illumination are difficult to align and
> would only be of interest to those who are concerned with very
> precise single-molecule measurements where polarization bias
> matters. It is for these reasons that I suspect the commercial
> vendors choose to work with systems that utilize only a single
> illumination beam, but even here you have no control over the
> illumination polarization unless you insert your own polarization
> optics into the optical train. This not easy to do on a commercial
> system and I have had experience doing this on a home-built TIRF
> system which is much more forgiving when it comes to inserting
> additional optics.
>
> I think the key point here is that polarization bias in
> fluorescence imaging (epifluorescence or TIRF or even confocal) is
> a concern to only a select group of researchers, mostly in the
> single-molecule/biophysics community. In most cell biology
> applications, the researcher concentrates on imaging the location
> of a labeled structure in a cell, tracking it in time, and perhaps
> looking for colocalization in two different channels. In most
> cases, the structure of interest will be labeled at a concentration
> such that hundreds or thousands of molecules are attached to it
> with a RANDOM orientation and the polarization of illumination
> light won't matter (no linear dichroism will be observed). But even
> here I should stress the phrase "most cases" since there have been
> reports on the listserver about polarization effects, especially
> for membrane probes. Sometimes, a fluorescence image can look quite
> different depending on the polarization of illumination. Dan
> Axelrod has already shown how membrane blebbing or invagination can
> be imaged using polarized TIRF illumination:
>
> Sund, S. E., J. A. Swanson, and D. Axelrod. 1999. Cell membrane
> orientation visualized by polarized total internal reflection
> fluorescence. Biophys. J. 77:2266-2283.
>
> John Oreopoulos
>
>
> On 24-Dec-09, at 2:12 AM, Shigeo Watanabe wrote:
>
>>
>> Dear All,
>>
>> I have previouly asked how polarized the P- and S-polarized light
>> are at TIRF illumination.
>> Thanks to John, now I get to know that P-polarized light is
>> converted to "cartwheel" polarized light while S-polized light is
>> intact.
>> As he suggested I read the review of Dan Axelrod about the effect
>> (or problem) of this cartwheel polized light when observing the
>> sample.
>> What I understand in his review is that cartwheel light excites
>> only molecules which is parallel to z-axis.
>>
>> Researchers who I talked with about TIRF problem also mentioned
>> that normal TIRF system which use single incident light from one
>> entering direction excite only a fraction of molecules which is
>> paralle to poloized evanescent light direction and then they
>> prefer to use the ring-like illumination for TIRF to excite every
>> single molecules.
>>
>> Now I am confused about the actual TIRF problem.
>> Quesitons I have are these.
>> 1)Does evanescent light excite the only molecules which are
>> paralle to polarization of evanescent light because of cartwheel
>> polarized light even when incident light is non-polarized light???
>> I am wondering what the actual polarization of evanescent light
>> produced by non-polarized incident light, which is mixture of P-
>> and S- polarized light.
>>
>> 2)Do commercial TIRF systems have this polarized problem? Do they
>> use single incident light or ring-like incident light?
>>
>>
>> I appreciate if anyone help to answer these questions.
>>
>> Sincerely
>> Shigeo Watanabe
>> Hamamatsu Photonics KK
>>
>>
>>
>>
>>
>>
>>
>>
>>