Brightness analysis

Brightness analysis provides a powerful tool for the study of protein interactions both in solution and in living cells. We provide a brief survey of some widely used techniques for extracting brightness from fluorescent fluctuation spectroscopy experiments. While all the techniques are equivalent under ideal conditions, we touch upon their relative strengths and discuss in detail a specific scenario wherein the photon-counting histogram (PCH) separates the brightness of rare, bright particles from a dominant background. In a practical vein for ensuring quantitative and unbiased brightness data, we address a number of potential issues stemming from both theoretical assumptions and experimental realities. Two additional issues arising from geometry are examined in greater detail. An oil-immersion objective skews the geometry of the excitation volume as a function of penetration depth. The bias can be characterized and corrected or avoided through the use of a water-immersion objective. Brightness measurements in thin sample geometries, frequently encountered in cells, may be biased. We use z-scan FFS to characterize sample geometry and correct any resulting bias in the brightness.