I am reading about "diffraction-limited system" on Wikipedia, which mentioned

"To increase the resolution, shorter wavelengths can be used, such as UV and X-ray microscopes. These techniques offer better resolution but are expensive, suffer from lack of contrast in biological samples and may damage the sample."

without giving a more detailed explanation for why the higher energy EM waves might lead to poor contrast. I have tried to search on Google, but still can't find a satisfying answer. Can anyone here kindly refer me to more detailed discussion on this question? Thank you.

  • $\begingroup$ This source says UV microscopy offers better contrast. I think it all depends on what method you use to visualize the sample after magnification. UV is not visible, so I guess it has to be converted digitally? $\endgroup$ – AliceD Mar 23 '18 at 11:35
  • $\begingroup$ Most biological samples are not electron-dense enough and need to be treated with heavy metals for EM. See wikipedia article for a brief introduction. $\endgroup$ – vkehayas Mar 23 '18 at 15:18
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    $\begingroup$ "EM" in the question referred to "electromagnetic". Heavy metals are used to increase contrast for "electron microscopy". Not the same meaning for "EM". $\endgroup$ – S. McGrew Mar 23 '18 at 17:55
  • $\begingroup$ UV microscopy is not a form of electron microscopy, it seems to be a technique in its own right. $\endgroup$ – AliceD Mar 23 '18 at 18:55

Contrast in optical microscopy typically depends on different substances having different absorption spectra. Practically everything absorbs UV light, so there tends to be less contrast in that range than in the visible spectrum where there is a bit more selectivity. All of this changes when there are contrast-enhancing substances added. For example, molecules that absorb in one wavelength and emit at a different wavelength via fluorescence, attached to antibodies that bind selectively to specific cell components, can provide very high contrast. E.g., see [https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2156041/].

An alternative optical microscopy technique is phase-contrast microscopy which does not use absorption at all. Instead, it measures the effective optical path length through the sample, which depends on local refractive index of the sample.

Yet another alternative is x-ray fluorescence microscopy, which can identify specific elements in the composition of a sample. See, for example,[https://www.americanlaboratory.com/913-Technical-Articles/18833-Three-Dimensional-Elemental-Imaging-Using-a-Confocal-X-Ray-Fluorescence-Microscope/].

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  • $\begingroup$ I suppose I am looking for a more detailed explanation for why "practically everything (in cells) absorb UV light ... (while) there is a bit more selectivity (for the visible spectrum)". But thanks for pointing out to us the alternative optical microscopy, in relation to the fluorescence microscopy. I have found a useful post on chemistry.stackexchange: link, which has nicely given me a chemistry perspective on my problem. $\endgroup$ – FAN FAN Mar 24 '18 at 7:42
  • $\begingroup$ @Fan Fan, if you do a search for "UV absorption spectrum" and proteins, DNA, or lipids, you'll see that a huge number of biomolecules absorb in the UV range (~200nm to ~400 nm). The chemistry of it relates to the energy levels in the types of bonds in the biomolecules, but you'll need a biochemist to explain to a greater depth. $\endgroup$ – S. McGrew Mar 24 '18 at 8:03

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