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Are there any microscopy modalities or techniques for in vivo imaging at higher resolutions than the diffraction limit?

I was looking at this list: Super resolution microscopy, but they don't talk about in vivo imaging.

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3 Answers

Any superresolution techniques relying on stochastic sampling (e.g. PALM, STORM) is extremely difficult to accomplish in vivo, as the tissue would be moving (e.g. due to respiration) and this would hinder a lot the reconstruction of the image.

Apparently it may be possible to do it with STED but I could not find the related paper*, although the same authors recently published some interesting work on living brain slices.

* Note that the images in that article come from this paper, which is not done in vivo.

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I beg to differ on the PALM/STORM point. While I'm a little biased as a co-author of Live-cell dSTORM with SNAP-tag fusion proteins, we had very good experience with live cells and dSTORM. After all, a dSTORM image can be taken in ten seconds, and many biological processes are slower. –  thiton Feb 14 '13 at 18:02
    
@thiton: again, I reiterate that when speaking about in vivo (at least in physiology) one considers a living animal, not a cell culture, which is still considered in vitro (or, for living tissue I ex vivo). I doubt you had many problems of respiration with your cells... –  nico Feb 14 '13 at 18:57
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Yes. A decade ago I've been working on a software for microscope that does what you want.

Here's how the product looks after 12 years of development.

It's optical, however it produces Z map instead if image, and has resolution is order of magnitudes below the diffraction limit: http://www.amphoralabs.com/production/10404/10708

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That does not work in vivo, does it (cell culture does not count as in vivo)? –  nico Jan 24 '13 at 7:34
    
Wikipedia thinks you're wrong. "In microbiology in vivo is often used to refer to experimentation done in live isolated cells" –  Soonts Jan 24 '13 at 18:21
    
well, when speaking about physiology, in vivo means on living animals, isolated cells are considered ex vivo. –  nico Jan 24 '13 at 19:45
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There have been experiments with Live-cell dSTORM with SNAP-tag fusion proteins in Markus Sauer's lab in Würzburg. If you are using the physiology definition of in-vivo, I'm pretty sure some of my co-workers have measured on live organisms. However, this is work in progress and not fully published; if you are interested, you should ask Markus Sauer directly.

You can use the dSTORM-like techniques (STORM, dSTORM, GSDIM, RPM, etc.) in live cells because the chemical environment in the cell is favourable and the redox system of the cell works for you. The key problem is to get the fluorophores into the cell, but SNAP-tags can help you to do it.

As one of the co-authors, I can tell you that live cell was some pain, but considerably less than getting 3D localization microscopy to work.

PALM is a different story because you generally have longer acquisition times. STED is difficult: If the cell wasn't dead before, the STED beam will ensure it is, either through the intensity or by unbalancing the chemical environment sufficiently. You might have your image before the cell dies, or maybe not.

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