Super-Resolution Microscopy

STORM is the most widely used SRM method, and it uses the sequential activation and time-resolved localization of photoswitchable fluorophores to generate high-resolution images. To achieve a high-quality multicolor image, specific labeling is required using direct labeling, protein conjugates, or antibody staining. Ideal fluorophores for STORM should be very bright and have a high rate of photoswitching cycles. They should also exhibit minimal photobleaching in thiol-containing buffers. With appropriate dye and buffer combinations, an optimized STORM system can generate images with 5 nm resolution. Numerous publications show combinations of Molecular Probes® dyes with a variety of targeting and labeling specificities used to generate multiplex STORM images.

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Structured illumination is used to enhance spatial resolution and involves illuminating the sample with patterned light and using software to analyze the information in Moiré fringes outside the normal range of observation. Reconstruction software deciphers the images at about 2-fold higher resolution than the diffraction limit, or ~100 nm. SIM has advantages over other SRM methods in that it can be used for imaging thicker sections, for 3D imaging, and for live-cell imaging. Image quality increases with bright and photostable dyes as well as precise targeting. Fluorescent proteins are commonly used for SIM investigations of live cells in addition to multiplexing with organic dyes and Qdot® probes.

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STED microscopy uses two laser pulses to localize fluorescence at each focal spot. The first pulse is used to excite a fluorophore to its fluorescent state, and the second pulse is a modified beam used to de-excite any fluorophores surrounding the excitation focal spot. The focal spot is raster scanned across the sample to generate an image, so the acquisition speed is relatively slow for large fields of view. A major advantage of this technique is the large depth of field (up to 10–15 µm deep) that can be imaged with high resolution.

With appropriate dyes, a resolution of 30 nm can be achieved in x and y, and the z resolution is comparable to conventional confocal microscopy. For optimal performance in STED, fluorophore properties should match the excitation and depletion beam wavelengths of the instrument system. Numerous publications show Molecular Probes® dyes used in STED microscopy.

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TPE is an optical process that uses multiphoton absorption to image live samples, and this technique results in less phototoxicity than conventional confocal microscopy. Two-photon microscopes use infrared excitation light to penetrate further into tissues and with minimal scatter compared to exciting at longer wavelengths. For each excitation of the fluorophore, two photons of  infrared light are absorbed, and excitation is limited to a tiny focus volume, so there is less contribution from out-of-focus fluorescence.

Two-photon imaging has been applied to a myriad of live-cell applications including imaging of ion concentrations, cytoskeletal organization, reactive oxygen detection, and vesicle recycling. Publications show Molecular Probes® reagents used in all of these applications and provide guidelines on probe selection.

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