Abstract

New Tools for Quantitative Imaging: Camera-Based Flourescence Spectroscopy with Single Molecule Sensitivity

Thorsten Wohland, Associate Professor, National University of Singapore

Humans are very effective in recognizing patterns within images. This is one of the reasons why biology and light microscopy have developed hand-in-hand since the first microscopes were invented at the end of the 16th century. Technical advances, which were made within the last 20-30 years allowed the detection of single molecules even in live samples. However, this capability was mainly restricted to point detectors and most spectroscopic tools are still conducted at single pixels, and thus do not provide a contiguous image of a sample. The recent advent of fast and sensitive electron multiplying charge coupled device (EMCCD) and scientific complementary metal–oxide–semiconductor (sCMOS) cameras makes it now possible to record spectroscopic data in an imaging format. In combination with advanced imaging techniques that have inherent z-sectioning, e.g. total internal reflection (TIR) or single plane illumination microscopy (SPIM) these cameras allow now the recording of fluorescence data over a whole plane or even a volume within a sample. This provides images with novel contrast mechanisms that are not given by fluorescence intensity as in the past but are created by any observable parameter. Here we will concentrate on Imaging Fluorescence Correlation Spectroscopy (Imaging FCS) – a novel technique that was made possible with these new camera technologies – and its application to the determination of contiguous mobility (diffusion or flow), concentration, and interaction maps within biological samples.