Nanometer imaging by differential confocal microscopy and its applications in biology

By utilizing the sharp slopes of the confocal response function, the authors developed a novel optical technique-differential confocal microscopy-for nanometer surface imaging. With a simple set-up, a 2 nm depth resolution has been demonstrated. Neither servo feedback loop nor lock-in detection are used, hence the technique can be used for high-speed surface profiling at long working distance. Because the technique uses no interferometric effects, it offers large open-loop dynamic range and is compatible with fluorescence microscopy. These features make it ideal for real-time monitoring cell membrane motion in natural environments. Using this technique the authors measured the dynamic viscoelastic and biochemical responses of living cells to sub-micrometer laser-driven deformation. Cytoplasmic gel-to-sol transition was detected with high sensitivity, and its influence on the viscoelastic response and Ca²⁺ release was studied