Stimulated Emission Depletion Microscopy (STED)
|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.
Live-cell labeling with fluorescent proteins
STED is performed predominantly with organic fluorophores, but live cells can be imaged using fluorescent proteins, particularly GFP. Molecular Probes® CellLight® reagents are ready-to-use fluorescent protein constructs (GFP or tagRFP) targeted to specific subcellular structures. These reagents provide a simple and effective method for introducing targeted intracellular labels within living cells. Specific organelles such as mitochondria, lysosomes, membranes, cytoskeleton, and nucleus can all be visualized using CellLight® reagents in live cells or after fixation. CellLight® reagents can be multiplexed with a range of fluorophores in live cells or fixed and imaged using immunofluorescence techniques.
Antibody conjugates and labels
Molecular Probes® fluorophores have been tried and tested in STED applications as conjugates of antibodies for fixed-cell applications, and proteins, dextrans, or other biomolecules for fixed or live cells. Use the STED product selection guide below to select the fluorophore with the best wavelength and STED rating for your application—citations are listed for each. Several Alexa Fluor® probes are outstanding in this application, giving you options in each channel for multiplexing. By following the product links in the selection guide, you can also find all of the bioconjugates of each fluorophore currently available to target your molecule of interest; the list ranges from antibodies and phalloidins to growth factors and lectins. If you don't find a conjugate you need on the list, the links will also show you reactive dye forms or optimized labeling kits to enable you to create your own bioconjugate probes.
CellTrace™ Calcein Green AM dye
CellTrace™ Calcein Green AM dye is a cell-permeant dye that is often used to determine cell viability in eukaryotic cells. In live cells the nonfluorescent CellTrace™ Calcein Green AM dye is converted to a green-fluorescent calcein after intracellular esterases remove the acetoxymethyl (AM) esters.
|Alexa Fluor® 488||Oregon Green®||Pacific Orange®||Alexa Fluor® 594|
|Laser line (nm)||488||488||405||594|
|Standard filter set||FITC||FITC||TRITC||Texas Red® dye|
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Molecular Probes® Handbook