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Quantitative description of protein dynamics and interactions in vivo with temporal and spatial resolution is a key step in dissecting molecular mechanisms in cell biology. Fluorescence fluctuation spectroscopy (FFS) has recently emerged as a powerful in vivo tool for assessing molecular concentration and movement and formation of hetero- and homo-oligomeric complexes. This article discusses point FFS-based analysis methods that have proven useful to cell biologists, focusing on the kinds of information they provide, their pros and cons, and the basic instrumentation required. Along the way, we describe briefly a few recent examples where these analyses have helped address important biological questions.

Toward Quantitative “In Vivo Biochemistry” with Fluorescence Fluctuation Spectroscopy