New Microscopy Techniques Reveals Inner Workings of Cells As Never Before [II]

 Microscopy  

Researchers have developed a suite of new optical techniques that smash through the diffraction barrier in microscopy and allow us to see, in detail, the inner workings of cells. New fluorescent tags are also used that light up structures within the dense darkness inside cells. These new optical approaches are making what was once an invisible part of ourselves, visible. Continued from Part I.

The essential activity of cell biology happens on scales too small to see through a conventional light microscope.  Now, with new super-resolution microscopy techniques scientists are able to look where they haven't been able to before.

The methods include optic and dye techniques:

SIM (~100 nm)

Structured illumination microscopy (SIM) shines a striped pattern of light onto a sample. That light interacts with light from fluorescent tags on cellular material and generates a pattern of interference called a moiré fringe. Using a series of moiré fringes it’s possible to mathematically extract and reconstruct a super-resolution image. SIM is ideal for looking at entire cells in 3-D, ensembles of cells or multiple cellular structures at once.
SIM: Lothar Schermelleh, Univ. of Oxford

STED (~30–70 nm)

When a focused light beam hits a fluorescent-tagged specimen, it generates a blurry halo. With stimulated emission depletion microscopy (STED), a second laser shines a doughnut-shaped beam of light that turns off the excited molecules in the halo. This provides a sharper view that, when scanned across the sample, produces a super-resolution image.
STED: R. Medda, D. Wildanger, L. Kastrup and S.W. Hell/Max Planck Institute

PALM (~10–55 nm)

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Photoactivated localization microscopy incorporates into a sample special fluorescent proteins that can be toggled between on and off states when hit with a particular wavelength of light. This allows researchers to illuminate a subset of molecules in a sample and eliminate overlapping fluorescence that would blur details if everything in the sample was lit up at once. iPALM (interferometric PALM) provides images in 3-D.
PALM: J.A. Galbraith, G. Shtengel, H.F. Hess and C.G. Galbraith/NINDS/NIH, Janelia Farm Research Campus/HHMI and NICHD/NIH

STORM (~20–55 nm)

Stochastic optical reconstruction microscopy, developed around the same time as PALM, also relies on fluorescent tags that can be switched on and off. In STORM’s case, the tags can be dyes or proteins. Using dyes may require an extra step, but they can be switched on and off more quickly and don’t burn out as fast as fluorescent proteins. Dyes can also be attached to genetic material.
STORM: M. Bates et al/Science 2007

Light sheet (~100 nm)

Imaging cells can be a violent process. The heat from light can cook cells, and the tinier the object the more light scientists need to see it (or to “interrogate” it). Light sheet microscopy hits a sample with a thin sheet of light that excites only the molecules in a single plane, minimizing damage. Bessel beam imaging uses superthin sheets of light to image live cells — and their innards — in action (see images, “A FLY IS BORN” and “SIGNED, SEALED, DELIVERED,” in slideshow above). Simultaneous multiview light-sheet microscopy (see image, “PACKING IT IN,” in slideshow above) uses thicker sheets to track ensembles of cells.


SOURCE  Science News, Top Image K. deLuca/Colorado State Univ.

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