Super-resolution microscopy (SRM) describes any optical technique used to resolve structures beyond the diffraction-limited resolution of conventional light microscopy. The fine mapping of cellular structures such as neural synapses, golgi apparatus, and nuclear membranes reveals more biological information when components are resolved beyond the diffraction limit and features can be elucidated at the sub-organelle level.
In conventional microscopy, due to interactions between visible light and the surrounding medium, a single fluorophore—which is less than a few nanometers in diameter—can only be resolved as a point spread function that is roughly half the light wavelength for lateral resolution and roughly twice that in depth. This means that if two or more fluorophores are within a few hundred nanometers of one another, their images become blurred together, limiting resolution.
SRM offers techniques to approach the resolution historically reserved for electron microscopy with all the benefits of targeting and multiplexing for biological context. To achieve optimum performance in the various SRM techniques and more traditional two-photon microscopy, different fluorophore requirements must be met for each (details below).
We are a leader in fluorescence technology, and Molecular Probes® products are widely published in SRM applications. The Molecular Probes® product range offers multiple dye combinations that can be selected for multiplexing in any of the SRM techniques. Individual SRM techniques are highlighted below. Select the technique you're interested in, and the linked pages will help you identify the dyes that work best for that application. Check back frequently; this is a rapidly developing field with regular updates to both products and publications.
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.
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.
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.
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.
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