Optimal Conditions for Live Cell Microscopy and Raster Image Correlation Spectroscopy

Abstract

Live cell microscopy is now commonplace across all fi elds of the life sciences, as well as, many of the physical sciences. In order to properly study physi- ological processing within living cells, tissues, or organisms it is crucial that viabil- ity of the sample takes the forefront as the most important aspect of the experiments. If samples are subject to high levels of light, phototoxicity can alter the very physi- ological processes under investigation. In order to minimize damage to the sample it is crucial to have as sensitive a microscope platform as possible so that light impact on the sample will be minimized. In order to minimize this impact, many aspects have to be kept in mind to maintain the sample and protect it from phototox- icity such as (1) keeping the cells in a favorable environment; (2) using transmitted light techniques when possible and carefully selecting fl uorescent dyes; (3) using low light densities of optimal wavelengths to image; (4) optimizing the light path for maximal effi ciency; and (5) using sensitive detectors. These aspects are dis- cussed in detail with suggestions how to maximize your sample viability while per- forming live cell microscopy. Many researchers want to measure submicroscopic molecular dynamics in living samples. One novel technique that has been recently developed for this purpose is raster image correlation spectroscopy (RICS). RICS was developed to measure molecular dynamics, concentrations, and intermo- lecular interactions. It has the advantage over other dynamic fl uorescence measure- ments in that it only requires very low laser intensities to measure molecular dynamics. Measuring dynamics using other techniques often requires the use of a high intensity of laser light to bleach, activate, or photo-switch fl uorescent mole- cules. Therefore, RICS is ideally suited for live cell microscopy. Two color cross-correlation RICS, ccRICS, is even more powerful determining if two pro- teins are moving together and determining the concentration and dynamics of the protein complex.