Talk:Total internal reflection fluorescence microscope

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Comments by Toytoy[edit]

Please simplify the language. -- Toytoy 00:47, Nov 19, 2004 (UTC)

Removed : 200 nm is less than one-third of the wavelength of red light. Not relevant Cnickelfr

It does seem relevant in that normally the axial resolution is limited by the wavelength of illumination. But I don't know whether what you removed specifically was useful or not... -- 18:03, 13 May 2005 (UTC)[reply]

OK then, we could write : ...about 200nm (depending on the wavelegth of illumination). What is unrelevant is that 200nm is 1/3 of red wavelength. It is also 1/2 of green light and 200 nm is waveleght of UV light. so what? Cnickelfr

Actually, this point is critical to the article. The axial resolution of a fluorescence microscope is usually quoted as half of the wavelength of the excitation light according to the Rayleigh criterion. This means that fluorescent molecules closer to each other than this distance cannot be discerned in the axial direction (perpendicular to the image plane). That TIRF microscopy can increase the axial resolution to 1/3 the wavelength used or less is what makes it so useful in molecular biology and biophysics. This fact should be re-emphasized and written back into the article in a clear manner.

--Johnoreo 02:23, 2 October 2006 (UTC)[reply]

added : about 7.5nm Thick

Removed "A popular implementation is marketed as Biacore™". Biacore is not a TIRF microscope. It's an apparatus using Surface Plasmon Resonance of a metal on wich are fixed molecules. Although Surface Plasmon resonnance is caused by an evanescent field like in TIRFM this is quite different. See Plasmon and How does biacore work? TIRFM is a fluorescence microscope, the evanescent field is used to excite fluorescence which is then imaged like in classical fluorescence microscopy. In Biacore resonance of surface plasmon alters the properties of the incident light and reflected light is measured. Biacore is used to measure kinetic constant of molecule interaction (hormone binding on a receptor for exemple)

Epifluorescence microscopy?[edit]

Is this the same as Epifluorescence microscopy? 'cause it sounds simillar. --hydkat 10:07, 14 March 2006 (UTC)[reply]

No, the two techniques are different. They differ in the way that excitation light is directed towards the sample. In epifluorescence microscopy, light is directed parallel to the axis of the objective such that the light transmits straight through the sample. If the sample is large and densly labeled, out of focus blurring can be problematic. TIRF microscopy gets around this problem by directing excitation light towards the sample such that light strikes the substrate-sample interface at an oblique angle (beyond the critical angle given by Snell's law for that interface) either through an optically coupled prism or a high NA microscope objecive (like in the figure). Evanescent waves are created in this case and penetrate very shallow into the sample medium. This causes only the closest fluorophores to the substrate to "light up". Detection of fluorescence emission follows the same path for both epifluorescence and TIRF microscopy. It should be noted that the evanescent waves of fluorescence can be selectively observed as well if the sub-critical angles of detection by the microscope objective are blocked with an obstruction disk placed in the appropriate postion of the microscope detection path - see Axelrod, Journal of Biomedical Optics, 2001 for example.

--Johnoreo 01:35, 2 October 2006 (UTC)[reply]


I think the following comment is technically incorrect:

"Importantly, the fluorescence excitation energy of the evanescent wave is the same as the energy of the wavelength of the light that was totally internally reflected."

The intensity of light that penetrates into the lower index medium is actually a function of the angle of incidence that the illumination light strikes the interface, as well as the distance above the the interface in the lower medium. Is the text trying to stress that the wavelength of the illuminating light does not change upon total internal reflection? I don't see why this is so important, and there is no reason to think that it would be altered in the first place. I suggest this text be removed and new text that goes into some of the finer details of electromagnetic waves at a dielectric interface be added instead.

--Johnoreo 02:07, 2 October 2006 (UTC)[reply]

TIRF is missing[edit]

There is a TIR page and this TIRFM page. But wouldn't it be nice for the overall structure if there was a page entitled TIRF? That one would explain TIRF in general as a spectroscopic tool with some more theoretical detail ,e.g. penetration depth, intensity of the evanescent wave, dependence of incident angle, etc. TIRFM is only one specific TIRF technique, i.e. TIRF imaging. Probably it is the most important one but there are other non-imaging techniques like TIRF sensing or TIRF-FCS.

I agree that TIRF does not always imply imaging. Most notably, TIRF spectroscopy has been going on since the 70's. There is also another related technique, TIR microscopy (not fluorescence) that images the scatter light of particles in the prism geometry. Perhaps they should be distinguished as well. 20:34, 30 November 2006 (UTC)[reply]

The new diagrams[edit]

While the new diagrams are definitely an improvement, there are two things that I would like to see changed:

1. Objective-based TIRF is usually done in the inverted microscope configuration. So the picture should be flipped vertically 2. Fluorescence emission in ray diagrams is usually colored to indicate a redshift. In these diagrams, the illuminating light color and the fluorescence light color should be swapped. 20:34, 30 November 2006 (UTC)[reply]

Diagrams are very good, although the numbering is a bit odd. why first emmision beam (signal) and then excitation beam? better the other way around. maybe for evanescent wave/cover slip the same. --Tcheh (talk) 08:17, 14 October 2010 (UTC)[reply]

Different setups[edit]

The objective can be placed on either side of the specimin Also the laser can be shone from the plane the glass or from the side of the glass —The preceding unsigned comment was added by (talk) 15:58, 9 December 2006 (UTC).[reply]

Proposal for new illustration[edit]

Total internal reflection.

Relevant citation?[edit]

I don't see why the reference to Andre et al. regarding single-molecule imaging is relevant, the paper only has 5 citations according to Google Scholar. I added another reference with 500+ citations which seems to be more relevant. If somebody knows something better, go ahead. (talk) 16:38, 16 January 2012 (UTC)[reply]


I prepared an image File:Tirfm numbers like TIRFM2.svg, which uses the same numbers as TIRFM2.svg to simplify caption of both images together. Just for your information. -- Pemu (talk) 10:33, 4 August 2016 (UTC)[reply]

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