Fluorescence microscopy methods for measuring the mobility and stability of molecules in 3-D samples

Abstract

In the last decade, confocal laser scanning microscopy (CLSM) has become a very popular tool for biological, medical, pharmaceutical and material research. Because of its optical sectioning ability, a confocal microscope can give insight in the three-dimensional structure of fluorescently labelled samples. Many CLSMs of the latest generation have the additional ability of scanning user-defined regions. This very feature makes them a convenient tool for fluorescence recovery after photobleaching (FRAP) experiments. FRAP is a fluorescence microscopy technique for examining the mobility of fluorescently labelled molecules. In a FRAP experiment, the fluorescent molecules are at first photobleached in a user-defined region of the sample by a powerful laser beam. Immediately after the bleaching process, the fluorescence inside the bleached area will recover due to the inward diffusion of fluorescent molecules from the surrounding unbleached areas. As the rate of fluorescence recovery reflects the local mobility of the fluorescent molecules, analysis of the recovery curve allows to calculate their diffusion coefficient at that particular location in the system. While the principle of FRAP is essentially quite simple, it is not so evident to come to correct quantitative diffusion measurements. Here we present three methods for performing quantitative FRAP measurements on a confocal or multi-photon laser scanning microscope. The first method is based on the bleaching of a large disk bleached by a CLSM with an objective lens of low numerical aperture (NA) [1]. This method is especially suited for performing diffusion measurements in 3-D extended samples, such as solutions, gels or extracellular matrices.