Talk:Confocal laser scanning microscopy

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Explained the image formation and the resolution enhancement as good as I understood it. However, I could not find the explanation of the resolution enhancement in the literature, i.e. I did not verify its correctness by consulting a specialist on this topic. I drew the figure of the confocal principle with Xfig.

To my knowledge, the enhancement of lateral resolution in CLSM is due to :
  • Coherence of the laser light
  • Small size of the illuminated field (point scanning)
see : "foundations of confocal scanned imaging in light microscopy" by Shinya Inoue in "Handbook of biological confocal microscopy" JB Pawley.
It is wrong that "the effective volume of light generation is usually smaller than the volume of illumination" both are diffraction limited, the volume of light generation might be a bit bigger because of the longer wavelength of the emission, but basically they are the same. The only increase in resolution comes from the fact that you have to multiply the illumination and collection efficiency to get the signal intensity and because of this you get a factor of squareroot two better resolution. The main effect is the out of focus fluorescence which in the case of non confocal fluorescence microscopy can flood your signal totally so that you have in fact very very poor resolution. Grmf 09:48, 31 August 2007 (UTC)

[edit] Article Title

This article should probably be retitled to "confocal microscopy" and some text about non-laser confocal microscopy added (the above book is a good reference). For instance, confocal microscopes are still being made today that use a mercury lamp instead of a laser. These are mainly spinning-disk confocal microscopes (also need to be covered in this article), although some models of spinning-disk confocal microscope (at least one recent model made by Perkin-Elmer) do use a laser.

[edit] Schematic representation

A very good article!

There are, nevertheless, some things wrong with the schematic representation of the Confocal Microscope.

  • there should not be a reflector
  • the red lines should go no further than where they meet inside the sample
  • the blue lines should originate in that point and be the same as the red lines (although in opposite direction) till they hit the mirror
  • the aperture for the blue lines should (in the case as presented) be located higher, cutting of a part of the area between the blue lines, reducing it to where the red lines would have been if they where reflected in the mirror

Or, to sum it all up in other words: the reason that confocal microscopes generate sharp images is the fact that it uses information from the confocal plane and the confocal plane only - but in the picture used, the image would be blurred because it contains a lot of noise: the out-of-focus blue area is larger than the focussed red one.

These remarks only affect the picture, not the explanation in the article. The explanation is fine.

For an example, see Leica Micro-systems (producers of confocal microscopes) - in this image the yellow area is the complete area inside the red and blue lines.

Done. danh 16:59, 15 September 2006 (UTC)

The image is still incorrect, it is usually the laser light (one or several lines of different wavelength) which is reflected by the beam splitter and the fluorescence light passes through. At least all dichroic mirrors for fluorescnece microscopes work this way. Furthermore if the color for the beams should be an indication of the wavelength it has to be the other way round, the laser excitation is with short wavelength e.g. blue and the emission with longer wavelength green or red, this is how fluorescence works. A good shematics should show how the out of focus light does not pass the pinhole. Look at the pages from Olympus, Nikon ect. which all have much better information than in this wikipedia article. Grmf 09:48, 31 August 2007 (UTC)

[edit] Phonograph?

Maybe it's just me, but I would never confuse the CLSM with a phonograph album, as linked in the article. Perhaps something else was intended?

I think the point that the author was trying to make was that a phonograph works on a similar principle to AFM i.e. involves a stylus scanning a surface, though I could be mistaken.

  • Agreed. I re-worded the sentence and removed the phonograph link (which, in this case, should be reached via the AFM page if necessary. MarcoTolo 17:57, 22 August 2006 (UTC)

An AFM does not work like a phonograph, this is a misconception which is even presented at scientific conferences, a stylus profiler comes close to a phonograph, but in an AFM you have a piezo which moves the tip ap and down and a feedback loop which controls this movement, this is much different from a phonograph! Grmf 09:48, 31 August 2007 (UTC)

[edit] Citations?

I really miss citations in this arcticle! I think this is a prerequisite for a reputable article. Seth2007 13:32, 19 April 2007 (UTC)

[edit] free 3D reconstruction?

It'd be great someone reading this would donate to the public a still from a 3D reconstruction, or can someone troll through the PLoS articles and find a nice one? I'll look there a little, myself. That's one of the great beauties of the technology, and it'd be cool to show it off. — eitch 16:13, 2 April 2008 (UTC)