Multispecies Fluorescence Photoactivation Localization Microscopy by Spectral Measurement

Super resolution microscopy techniques such as fluorescence photo-activation localization microscopy (FPALM), photo-activation localization microscopy (PALM), and stochastic optical reconstruction microscopy (STORM) have revolutionized biological microscopy by allowing an increase in final image resolution by a factor of ten over traditional diffraction limited microscopy. Cells are labeled with photo-activatable/switchable fluorescent molecules which become sparsely activated through a time series of laser illumination and imaging, which records each molecule as a diffraction limited spot. Analysis localizes the position of each molecule using its image. The localized molecule positions are combined to form a final super resolution image. Recent advances with these methods have enabled the determination of the orientation of individual proteins within samples, the tracking of individual proteins in living samples, the imaging of proteins in three dimensions, and the distinction of multiple fluorescent species.Here, we focus on multispecies imaging. Previous super-resolution multicolor techniques have relied upon splitting the fluorescence signal from multiple species into channels of different wavelength bandwidths using dichroic mirrors; the ratio of intensities between these channels provides the means of species distinction. Alternatively, interchanging multiple excitation lasers can excite different fluorescent species at different time periods. Here, we present an alternative method for super-resolution FPALM multispecies imaging. We use a combination of optics to measure the spectral distribution of single molecule fluorescence. In this way we can distinguish multiple distinct fluorescent species. We have applied this technique successfully to obtain super-resolution FPALM images, visualizing multiple photo-activatable fusion proteins simultaneously within NIH-3T3 fibroblast cells.