PHYC500/2007F:Interactions

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Living cells in an electron microscope?

Electron microscopy requires that cells be "fixed" (chemically cross-linked). Why? Why can't we look at living cells in an electron microscope? - JLT

• I think the reason why we can't look at living things is the fact that we have to put the samples in a vacuum. The EM uses an electron beam to illuminate and create an image of a cell. Hence, molecules in air would scatter the electrons :( -KS user page

• Great answer, Kathrin! This reminded me of a talk I saw a few years ago (a scientist from IBM) where they developed a very thin sample chamber that allowed them to do transmission electron microscopy (TEM) on a sample in solution (I think this was a gold nanowire growing). So, the majority of the TEM was in vacuum, except for a thin slice of liquid-phase sample (vacuum tight). So, I think in situ TEM has been demonstrated in proof of principle. I would guess it's pretty difficult to think of doing something similar w/ living cells, but maybe not impossible? (I suppose also the cells would die quickly while being zapped with electrons.)--Steven J. Koch 00:04, 29 August 2007 (EDT)

Controls for cross-linking experiments?

If you use a chemical that can cross-link biological components together (when prepping samples for electron microscopy), what controls could you do to see whether that cross-linking changes the spatial distribution of the components? - JLT

• First, I would tag the biological component of interest with a gold particle. Then I would take an image of the sample with no cross-linker. I would then compare an image made from an identically prepared sample with cross-linker added. If there exists regions where the gold particles are in higher concentration, as compared to the sample with no cross-linker, I would say that the cross-linker worked and the biological components are now fused together. - Andy_

I'm not sure I made my question clear. Electron microscopists HAVE to use crosslinkers (fixatives) to look at their cells. They also usually assume that the fixative does not change the spatial distribution of the components. How can they be sure that the fixative does not? You can't do EM without the fixative. - JLT

• Can we use optics microscope to measure the flourecent tagged particle to see whether there's a difference of concentration before and after crosslink? I mean we can make a sample and measure the distribution of particles of interests with optics microscope and then do the crosslink thing and measure it again with optics microscope to make true nothing changed during the crosslinking. -Fang

• I would tag the components of interest with quantum dots at very low labeling density so that individual emitters could be localized in an optical microscope with nanometer accuracy. I would then image the cell while fixing it. If the relative positions of well localized emitters changes during the fixing process, then you've got a problem. Otherwise, you can conclude that the crosslinker has not affected the spatial distribution of components. Additionally, since QDs are electron dense, they can be used in the electron microscope. Is there a technique that does not require the use of an optical microscope? -Paul