Single Plane Illumination Fluorescence Correlation Spectroscopy (SPIM-FCS) Using a Position-Sensitive Detector

Abstract

Measuring the time- and position-dependent intensity distribution of fluorescent probes is a promising approach to characterize the mobility of molecules inside living cells and the accessibility of cellular compartments. Typically Fluorescence Correlation Spectroscopy (FCS) based on confocal microscopy is used for such measurements, which implies high time-resolution but only for a single-position measurement. In order to achieve a high time-resolution and parallel multi-position measurements, we built a Single Plane Illumination Microscope (SPIM) equipped with a high-speed image acquisition device and high-NA detection optics. The image sensor is based on a 32x32 pixel array of avalanche photodiodes and its read-out time is on the order of 10µs for a complete 32x32 pixel frame. This is fast enough to resolve the typical motion time-scale of small molecules (like eGFP) moving inside living cells. As an option a fast CCD camera can be used, which trades a larger field of view for a lower time-resolution. The performance of the system is being characterized by diffusion measurements of water-soluble quantum dots and fluorescent beads. The data acquisition system that we developed uses programmable hardware for some tasks and is fast enough to allow real-time correlation of few pixels. We can also use the complete experimental dataset for higher-level evaluations using regularized global fits.