Posted by
John Oreopoulos on
URL: http://confocal-microscopy-list.275.s1.nabble.com/paper-help-tp4115966p4232499.html
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=1Isolated 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/Polarizedhttp://en.wikipedia.org/wiki/Quarter-wave_plateAs 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
>
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