paper help
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Can anyone send me a pdf copy of the following article? Thanks.
Phys. Chem. Chem. Phys., 2008, 10, 3548 - 3560
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Tim zhang <[hidden email]> wrote: >Can anyone send me a pdf copy of the following article? Thanks. > >Phys. Chem. Chem. Phys., 2008, 10, 3548 - 3560 > > |
 
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Shigeo Watanabe |
TIRF and p- s-polarized incident linght
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Dear all, I have questions about TIRF and polarized light. Incident light can be devided into p- and s- polarized light. Then what happen to these two light when evanescent light is induced by these light. Olympus webpage explains these, but I could not understand these phrase.
Olympus web page is here. http://www.olympusmicro.com/primer/java/tirf/evaintensity/index.html I appreciate if there are somebody who can explain these to me. Sincerely Shigeo Watanabe Hamamatsu Photonics KK. |
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John Oreopoulos |
Re: TIRF and p- s-polarized incident linght
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Shigeo, Since the core of my PhD project involves exploiting the polarization of light in the TIRF mode of illumination, I guess I can try to explain this. As you know, light can normally be linearly polarized in any direction that is transverse (perpendicular) to the direction of travel of the light beam using polarization optics. Polarization here refers to the direction of oscillation associated with the electric field vector of the light beam. In the epi-illumination mode for microscopy, the beam of light is directed straight through the sample along the optic axis of the microscope objective - call this direction the z-direction. In this case, this means that the light can be polarized along any direction in the xy plane (the sample/image plane) - the plane that gets projected onto your CCD detector. Now consider what happens when you adjust the beam for TIRF illumination - you set up the beam to impinge on the substrate surface at an oblique angle instead of going straight through. From Snell's law of refraction at an interface, you know that the critical angle for a glass/water interface is ~60 degrees. If you set the beam to impinge the surface at at the critical angle, the refracted beam that emerges from the interface travels horizontally (x-direction) along the surface. You can see this with a bright laser on a TIRF microscope setup and solution of fluorscent dye. Since the laser beam travels along the x-direction in this situation, the same polarization rule applies - now the beam can be polarized in the yz plane depending on the input polarization of the beam before it emerged from the interface. Here is the tricky part: if you increase the angle of incidence beyond the critical angle of the interface, total internal reflection occurs and an evanescent wave/field is created in the lower index medium (water). As a consequence of Maxwell's electromagnetic field equations solved at an interface, the polarization characteristics of the evanescent wave are a bit unusual. These equations have certain boundry conditions that demand that the transition of light (more specifically the light oscillation wavefronts) from one medium to another be continuous for all angles of incidence, and as a consequence, the elecric field vector of the evanescent wave in both mediums (glass and water) must adjust to satisfy this condition. This is a general property that is observed in many physics situations - this idea of continuaty at an interface is the reason that quantum electron tunneling occurs as well. In fact, the evanescent wave is the photon analog of electron tunneling. I would suggest reading Griffeth's textbook Indroductory Electrodynamics for the full details of this dervation about light interactions at an interface. Anyways, it turns out that past the critical angle, light that was originally polarized along the y-axis remains the same ("s-polarization", the s stands for the first letter of the German word for perpendicular to the plane of incidence) but light polarized along the z-direction ("p-polarization, the p stands for the "paralell" to the plane of incidence) for critical angle illumination becomes split into a linear combination of light polarized along z and the x-direction (which is the direction of travel of the evanescent surface wave!). The relative amount of z and x polarization will depend in the refractive indcies and the angle of incidence. This is one of the few examples in nature where light can be setup to partially oscilate along its direction of travel (a longitudinal wave) and this is why the website you mentioned said that the "p-polarized" evenescent electric field vector "cartwheels" along the direction of travel. After reading what I just wrote I see that it still might be unclear. Working with polarization can be tricky because it is the feature of light we as humans are most unfamiliar with since our eyes cannot sense/detect polarization. It's hard to visualize in your mind imaginary field vectors oscillating and traveling in space. If it's still unclear, try reading Dan Axelrod's review papers on TIRF microscopy where he discusses the applications of polarized TIRF illumination. I think there is even a useful Java applet on the Molecular Expressions website that does a bettter job of depecting this. Polarized illumination (epifluorescence or TIRF mode) can be useful for studying the orientations of fluorescent molecules embedded in a sample (linear dichroism, fluorescence anisotropy, etc.) John Oreopoulos
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John Oreopoulos |
Re: TIRF and p- s-polarized incident linght
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I just remembered another physical analogy that can help explain the reasons why certain polarizations of light reflect or transmit through a refractive index interface. Imagine the polarization of light is represented by a long wooden stick. Now imagine throwing this stick into a large pool of water at an oblique angle such that its long axis is oriented paralell to the surface of the water. The stick will enter the water easily in this case. Now imagine throwing the stick towards the water at an oblique angle with the long axis perpendicular to the water surface. I think you can imagine in your mind that as the lower part of the stick strikes the surface first and begins to move slower in the water, the top part of the stick still outside the water will begin to rotate and "cartwheel" towards the surface because of its momentum. It's a very crude mechanical analogy and there are several things wrong with it, but I think you get the general similarity with the situation encountered with light waves. Perhaps someone else out there knows of a better analogy. I think I remember reading a version of this story with bouncing sticks on an interface in Richard Feynman's books or in Eugene Hecht's textbook on optics. John Oreopoulos
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Shigeo Watanabe |
Re: TIRF and p- s-polarized incident linght
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John, Thank you so much for explaining in a way for me to understand. Now I am getting to understand better. I just could not believe the cartwheeling polarized light still, though. Sincerely, Shigeo Watanabe
I just remembered another physical analogy that can help explain the reasons why certain polarizations of light reflect or transmit through a refractive index interface. Imagine the polarization of light is represented by a long wooden stick. Now imagine throwing this stick into a large pool of water at an oblique angle such that its long axis is oriented paralell to the surface of the water. The stick will enter the water easily in this case. Now imagine throwing the stick towards the water at an oblique angle with the long axis perpendicular to the water surface. I think you can imagine in your mind that as the lower part of the stick strikes the surface first and begins to move slower in the water, the top part of the stick still outside the water will begin to rotate and "cartwheel" towards the surface because of its momentum. It's a very crude mechanical analogy and there are several things wrong with it, but I think you get the general similarity with the situation encountered with light waves. Perhaps someone else out there knows of a better analogy. I think I remember reading a version of this story with bouncing sticks on an interface in Richard Feynman's books or in Eugene Hecht's textbook on optics. John Oreopoulos On 2009-12-07, at 11:07 PM, John Oreopoulos <john.oreopoulos@...> wrote: Shigeo, Since the core of my PhD project involves exploiting the polarization of light in the TIRF mode of illumination, I guess I can try to explain this. As you know, light can normally be linearly polarized in any direction that is transverse (perpendicular) to the direction of travel of the light beam using polarization optics. Polarization here refers to the direction of oscillation associated with the electric field vector of the light beam. In the epi-illumination mode for microscopy, the beam of light is directed straight through the sample along the optic axis of the microscope objective - call this direction the z-direction. In this case, this means that the light can be polarized along any direction in the xy plane (the sample/image plane) - the plane that gets projected onto your CCD detector. Now consider what happens when you adjust the beam for TIRF illumination - you set up the beam to impinge on the substrate surface at an oblique angle instead of going straight through. From Snell's law of refraction at an interface, you know that the critical angle for a glass/water interface is ~60 degrees. If you set the beam to impinge the surface at at the critical angle, the refracted beam that emerges from the interface travels horizontally (x-direction) along the surface. You can see this with a bright laser on a TIRF microscope setup and solution of fluorscent dye. Since the laser beam travels along the x-direction in this situation, the same polarization rule applies - now the beam can be polarized in the yz plane depending on the input polarization of the beam before it emerged from the interface. Here is the tricky part: if you increase the angle of incidence beyond the critical angle of the interface, total internal reflection occurs and an evanescent wave/field is created in the lower index medium (water). As a consequence of Maxwell's electromagnetic field equations solved at an interface, the polarization characteristics of the evanescent wave are a bit unusual. These equations have certain boundry conditions that demand that the transition of light (more specifically the light oscillation wavefronts) from one medium to another be continuous for all angles of incidence, and as a consequence, the elecric field vector of the evanescent wave in both mediums (glass and water) must adjust to satisfy this condition. This is a general property that is observed in many physics situations - this idea of continuaty at an interface is the reason that quantum electron tunneling occurs as well. In fact, the evanescent wave is the photon analog of electron tunneling. I would suggest reading Griffeth's textbook Indroductory Electrodynamics for the full details of this dervation about light interactions at an interface. Anyways, it turns out that past the critical angle, light that was originally polarized along the y-axis remains the same ("s-polarization", the s stands for the first letter of the German word for perpendicular to the plane of incidence) but light polarized along the z-direction ("p-polarization, the p stands for the "paralell" to the plane of incidence) for critical angle illumination becomes split into a linear combination of light polarized along z and the x-direction (which is the direction of travel of the evanescent surface wave!). The relative amount of z and x polarization will depend in the refractive indcies and the angle of incidence. This is one of the few examples in nature where light can be setup to partially oscilate along its direction of travel (a longitudinal wave) and this is why the website you mentioned said that the "p-polarized" evenescent electric field vector "cartwheels" along the direction of travel. After reading what I just wrote I see that it still might be unclear. Working with polarization can be tricky because it is the feature of light we as humans are most unfamiliar with since our eyes cannot sense/detect polarization. It's hard to visualize in your mind imaginary field vectors oscillating and traveling in space. If it's still unclear, try reading Dan Axelrod's review papers on TIRF microscopy where he discusses the applications of polarized TIRF illumination. I think there is even a useful Java applet on the Molecular Expressions website that does a bettter job of depecting this. Polarized illumination (epifluorescence or TIRF mode) can be useful for studying the orientations of fluorescent molecules embedded in a sample (linear dichroism, fluorescence anisotropy, etc.) John Oreopoulos On 2009-12-07, at 5:44 PM, Shigeo Watanabe <[hidden email][hidden email]> wrote: Dear all, I have questions about TIRF and polarized light. Incident light can be devided into p- and s- polarized light. Then what happen to these two light when evanescent light is induced by these light. Olympus webpage explains these, but I could not understand these phrase.
Olympus web page is here. http://www.olympusmicro.com/primer/java/tirf/evaintensity/index.html I appreciate if there are somebody who can explain these to me. Sincerely Shigeo Watanabe Hamamatsu Photonics KK. |
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Tim Feinstein-2 |
Re: TIRF and p- s-polarized incident linght
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In reply to this post by John Oreopoulos
Dear John,
Thank you for your frequent contributions to the confocal list. I have found them consistently useful and insightful. I am particularly interested in your last message concerning polarization and TIRF. Our lab is working with various product reps to find out how easily our lab can adapt our epifluorescence imaging to detect polarization-based anisotropy (we plan to try it and see what happens). Given the usual concern about high-NA lenses and polarization it never occurred to us to try TIRF, yet your Biophys. J. article of this year indicates that it might work for us. If I tried reproducing your setup, would you mind answering a few questions if/when they come up? I have rebuilt confocals but would hardly qualify as a specialist in optical physics. Thanks again and all the best, Tim Timothy Feinstein, PhD University of Pittsburgh Dept. of Pharmacology Pittsburgh, PA On Dec 8, 2009, at 12:46 PM, John Oreopoulos wrote:
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Tim Feinstein-2 |
Re: TIRF and p- s-polarized incident linght
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In reply to this post by John Oreopoulos
The last email was sent to all by mistake. Apologies.
Tim Feinstein |
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Shigeo Watanabe |
Re: TIRF and p- s-polarized incident linght
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In reply to this post by John Oreopoulos
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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John Oreopoulos |
Re: TIRF and p- s-polarized incident linght
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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 > > > > > > > > > |
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John Oreopoulos |
Re: TIRF and p- s-polarized incident linght
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Sorry, I sent the wrong Youtube link for the MIT video. See this one
instead: http://www.youtube.com/watch?v=4xF1Fq2wB1I&feature=channel John On 30-Dec-09, at 9:46 AM, John Oreopoulos wrote: > 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 >> >> >> >> >> >> >> >> >> |
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