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.
Structured Illumination Microscopy (SIM)
Live-cell labeling with fluorescent proteins
Because SIM can be used for thicker sections and live-cell imaging, many publications demonstrate the use of fluorescent proteins for high-resolution studies using GFP or tagRFP. 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 SIM applications as conjugates of antibodies for fixed-cell applications, and proteins, dextrans, or other biomolecules for fixed or live cells. Use the table below to select the fluorophore with the best wavelength and SIM rating for your application—citations are listed for each. Several Alexa Fluor® probes are outstanding in this application. By following the links, 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.
DAPI is one of the most widely used reagents for DNA staining, and it is highly rated for SIM applications. Because of its high affinity for DNA, it is also frequently used for counting cells, measuring apoptosis, and as a nuclear segmentation tool in high-content imaging analysis. Because DNA is highly abundant, DAPI's limited brightness produces adequate signal and the blue fluorescence is well separated from signals in the FITC channel. Since DAPI is cell-impermeant, it is generally used to stain fixed cells, although the stain will enter live cells when used at higher concentrations.
Organic dyes are easily multiplexed with fluorescent proteins for either live- or fixed-cell imaging, and some organelle-specific probes are suitable for both. MitoTracker® Red dye is a far-red–fluorescent dye that stains mitochondria in live cells, and its accumulation is dependent upon membrane potential. It serves as an effective SIM probe, and is retained in cells after fixation.
CellMask™ Orange Plasma Membrane Stain allows fast and uniform labeling of the plasma membrane without the cell-type differences exhibited by lectins. This stain is often used as a segmentation tool for HCS (high-content screening) as well as to stain cellular plasma membranes for standard fluorescence microscopy—it also rates very highly for SIM. The stain survives fixation, but not permeabilization, so it is not suitable for experiments that also involve probing internal targets with antibodies.
|Alexa Fluor® 405||Alexa Fluor® 488||Alexa Fluor® 546||Alexa Fluor® 555||Alexa Fluor® 568||Alexa Fluor® 594|
|Laser line (nm)||350/405||488||488||488||561||594|
|Standard filter set||DAPI||FITC||TRITC||TRITC||RFP||Texas Red® dye|
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Molecular Probes® Handbook