Abstract
Multi-color stochastic optical reconstruction microscopy (STORM) is routinely performed; however, the various approaches for achieving multiple colors have important caveats. Color cross-talk, limited availability of spectrally distinct fluorophores with optimal brightness and duty cycle, incompatibility of imaging buffers for different fluorophores, and chromatic aberrations impact the spatial resolution and ultimately the number of colors that can be achieved. We overcome these complexities and develop a simple approach for multi-color STORM imaging using a single fluorophore and sequential labelling. In addition, we present a simple and versatile method to locate the same region of interest on different days and even on different microscopes. In combination, these approaches enable cross-talk-free multi-color imaging of sub-cellular structures.
Highlights
Stochastic optical reconstruction microscopy (STORM) [1] and similar methods [2,3] enable fluorescence imaging beyond the diffraction limit, extending the spatial resolution of optical microscopy to nanometer length scales
We demonstrate a strategy for multi-color STORM imaging based on correlative microscopy, in which the same sample is repeatedly imaged using different modalities –instead of varying the modality, we vary the color, and perform sequential STORM imaging
Our simple strategy eliminates a large number of technical problems and enables cross-talk free, multi-color STORM with the best performing fluorophore
Summary
Stochastic optical reconstruction microscopy (STORM) [1] and similar methods (including photoactivated localization microscopy, PALM, and fluorescence photoactivated localization microscopy, fPALM) [2,3] enable fluorescence imaging beyond the diffraction limit, extending the spatial resolution of optical microscopy to nanometer length scales. The position of a single fluorescent molecule can be precisely determined if its image is isolated in space [4,5]. Photoswitchable fluorophores [6,7,8,9] can be used to overcome the problem that when multiple fluorescent molecules overlap in a diffraction limited volume, their images merge, making it difficult to determine their positions. By switching most of the fluorescent molecules into a dark state and photoactivating only a sparse subset of them, it is possible to obtain isolated images of single molecules and localize their positions precisely. By repeating the photoactivation, imaging and localization, a high resolution image of the underlying structure can be reconstructed from fluorophore positions
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