Saturday, May 21, 2016

Tinier and Tinier- Advances in Electron Microscopy

Phase contrast and phase plates for electrons;  getting to near-atomic resolution.

Taken for granted today in labs around the world, phase contrast light microscopy won a Nobel prize in 1953. It is a fascinating manipulation of light to enhance the visibility of objects that may be colorless, but have a refractive index different from the medium. This allowed biologists especially to see features of cells while they were still alive, rather than having to kill and stain them. But it has been useful for minerology and other fields as well.

Optical phase contrast apparatus. The bottom ring blocks all but that ring of light from coming into the specimen from below, while the upper ring captures that light, dimming it and shifting its phase.

Refraction of light by a sample has very minor effects in normal bright field microscopy, but does two important things for phase contrast microscopy. It bends the light slightly, like a drink bends the image of a straw, and secondly, it alters the wave phase of the light as well, retarding it slightly relative to the unaffected light. Ultimately, these are both effects of slowing light down in a denser material.

The phase contrast microscope takes advantage of both properties. Rings are strategically placed both before and after the sample so that the direct light is channeled in a cone that is then intercepted after hitting the sample with the phase plate. This plate both dims to direct light, so that it does not compete as heavily with the scarcer refracted light, and more importantly, it also phase-retards the direct light by 90 degrees.

Light rotational phase relationships in phase contrast. The phase plate shifts the direct (bright) light from -u- to -u1-. Light that has gone through the sample and been refracted is -p-, which interferes far more effectively with -u1- (or -u2-, an alternate method) than with the original -u-, generating -p1- or -p2-, respectively.

The diagram above shows the phase relationships of light in phase contrast. The direct light is u on the right diagram, and p is the refracted and phase-shifted light from the specimen. d is the radial difference in phasing. Interference between the two light sources, given their slight phase difference, is also slight and gives very little contrast. But if the direct light is phase shifted by 90 degrees, either in the negative (orginal method, left side u1) or positive directions (right, u2), then adding the d vector via interference with the refracted light has much more dramatic effects, resulting in the phase contrast effect. Phase shifting is done with special materials, such as specifically oriented quartz.

Example of the dramatic enhancement possible with optical phase contrast.

A recent paper reviews methods for generating phase contrast for electron microscopy, which, with its far smaller wavelength, is able to resolve much finer details, and also revolutionized biology when it was invented, sixty years ago. But transmission electron microscopy is bedeviled, just as light microscopy was, by poor contrast in many specimens, particularly biological ones, where the atomic composition is all very light-weight: carbons, oxygens, hydrogens, etc, with little difference from the water medium or the various cellular or protein constituents. Elaborate staining procedures using heavy metals have been used, but it would be prefereable to image flash-frozen and sectioned samples more directly. Thus a decades-long quest to develop an electon analogue of phase contrast imaging, and a practical electron phase plate in particular.

Electrons have waves just as light does, but they are far smaller and somewhat harder to manipulate. It turns out that a thin plate of randomly deposited carbon, with a hole in the middle, plus electrodes to bleed off absorbed electrons and even bias the voltage to manipulate them, is enough to do the trick. Why the hole?  This is where the un-shifted electrons come through, (which mostly also do not interact significantly with the specimen), which then interfere with the refracted and shifted ones coming through the carbon plate outside. Which has the effect of emphasizing those electrons phase-shifted by the specimen which escape the destructive interference.
"A cosine-type phase-contrast transfer function emerges when the phase-shifted scattered waves interfere with the non-phase-shifted unscattered waves, which passed through the center hole before incidence onto the specimen."

The upshot is that one can go from the image on the right to the one on the left- an amazing difference.
Transmission electron microscopy of a bacterium. Normal is right, phase contrast is left.

At a more molecular scale, one can see individual proteins better, here the GroEL protein chaperone complex, which is a barrel-shaped structure inside of which other proteins are encouraged to fold properly.
Transmission electron microscopy of individual GroEL complexes, normal on left, phase contrast on right. 

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