Fluorescent Speckle Microscopy

Extract

Whereas too much of a good thing can be bad, too little of a good tiling can be good. In this case, the accidental dilution of X-rhodamine tubulin injected into living cells resulted in a heterogeneous labeling of micro tubules, rather than visualization of continuous structures. This heterogeneous labeling rendered a speckled image, hence the term fluorescent speckle microscopy (FSM) was introduced. It turns out that FSM yields particularly useful information on dynamic events within living cells, for example the assembly and disassembly of microtubules.

There are two recent reviews of FSM by Clare Waterman-Storer and Gaudenz Danuser, one emphasizing the biologic applications of the technique, the other emphasizing the quantitative aspects. For their purposes, they defined a “speckle” as a diffraction-limited region of the image that is significantly brighter than its immediate environment. It can be calculated from the point-spread function (which is determined by the numerical aperture of the lens) that optimally a diffraction-limited region of about 250 nm can be imaged.