STED Microscopy Products
products for STED super-resolution fluorescence microscopy
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- Chromeo™ STED Immunofluorescence System
- Chromeo™ 488 Tools for STED Microscopy
- Chromeo™ 505 Tools for STED Microscopy
- Chromeo™ 494 Tools for STED Microscopy
- Fluorescent ATTO (STED) secondary antibody conjugates
The resolution attainable in IF experiments has until recently been limited by a very specific physical property known as the Abbe Law of Diffraction Limiting Resolution. The Abbe limit restricts the ability of the observer to visually resolve objects separated by less than ~200 nm. With STimulated Emission Depletion (STED) microscopy, however, it is possible to exceed the Abbe limit and achieve resolution improvements of up to 12-fold over classical confocal microscopy.
As shown in Figure 1A, all of the dye molecules within the excitation spot emit fluorescent light that is recognized as a single signal by the detector of the microscope; the individual dye molecules can't be resolved separately. In STED microscopy the dye molecules in the outer area of the spot are turned off through use of a second red-shifted "depletion" laser that emits a doughnut-shaped beam. As a result, only dye molecules in the very center of the excitation spot are able to emit a fluorescent signal, which reduces the size of the emitting spot below the diffraction limit (Figure 1B).
Figure 1: Principle of STED microscopy.
To turn the excited dye molecules off, the STED microscope contains two lasers that function in a well-coordinated, paired fashion. As shown in the Jablonski diagram in Figure 2, the excitation laser stimulates the dye molecules to their excited, fluorescent state S1. A second red-shifted depletion laser stimulates the dyes to return down to the ground state S0 without emitting fluorescence. Because the depletion laser has a doughnut-shaped beam, the dye molecules in the center of the excitation spot are not targeted by the depletion laser, so only they can emit detectable fluorescence.
Figure 2: Simplified Jablonski diagram of the STED method.
The key to resolution enhancement in STED is downsizing of the fluorescent spot used to scan the sample, which is achieved through use of an upconverted confocal laser scanning microscope that utilizes two lasers (Figure 3). The first laser (2, green) excites the fluorophores of the sample the same way as a conventional confocal system. These pulses are directly followed by a pair of perpendicularly polarized beams from a red-shifted stimulating depletion laser – the STED pulse (3, red). This induces a depletion of the excited dye molecules, which de-excites them before they can emit any fluorescent light. Due to the depletion beam’s doughnut-like shape (7), fluorescence is inhibited only in the outer regions of the illuminated spot. The result is a small, tightly focused, super-resolution spot that is scanned across the sample (8).
Figure 3: Diagram of the Leica TCS STED microscopy.
The resolution afforded by STED microscopy facilitates the separation of sub-cellular structures that previously could not be resolved, and increases the confidence in the biological roles of proteins and structures that co-localize (Figure 4). For more complete information on STED microscopy, please visit the Leica Microsystems website.
To yield clear, conclusive high-resolution images, it is extremely important to optimize the techniques and reagents used for sample preparation. Proper sample preparation is an extremely important factor for obtaining high-quality images. To help ensure that you consistently achieve the best results possible, Active Motif Chromeon collaborated with Leica Microsystems to develop the Chromeo™ STED Immunofluorescence System. This kit was designed to take the guesswork and challenge out of STED experiments by providing a complete set of proven, QC-tested reagents and an optimized protocol.
In addition to certifying this kit for use with its STED microscopes, Leica recommends Active Motif Chromeon’s Chromeo™ 488, Chromeo™ 505 and Chromeo™ 494 fluorescent dyes and secondary antibody conjugates and its fluorescent ATTO (STED) secondary antibody conjugates for use with its instruments because they meet the specifications required for STED microscopy.
Figure 4: Comparison of conventional, confocal microscopy and STED microscopy.
Leica Microsystems now offers two different types of STED microscopes, continuous wave (CW) and pulsed, that can help in the study of nanostructures within the cell. While both systems utilize fluorescence, they contain different laser systems for excitation and depletion. Therefore, they require fluorescent dyes with different, well-defined properties to label the cellular structures that are to be observed.
With the Leica TCS STED CW system, subcellular details below 80 nm can be visualized. This microscope consists of a continuous argon gas laser (488 nm and 515 nm) for excitation and a continuous 592 nm fiber laser for depletion. The combination of continuous laser excitation and depletion and the capability for fast data acquisition enables live cell imaging in a high-resolution mode. Leica Microsystems has certified Active Motif’s Chromeo™ 488 and Chromeo™ 505 fluorescent dyes and secondary antibody conjugates for use in CW STED.
Figure 5: Immunofluorescent staining of neuromuscular junctions in Drosophila larvae.
The Leica TCS STED system consists of a pulsed 640 nm excitation laser combined with a 750 nm depletion laser. This microscope reaches a spatial resolution of 50-70 nm. For use in this wavelength range, Leica Microsystems recommends Active Motif’s fluorescent ATTO (STED) secondary antibody conjugates. The integration of a second pulsed excitation laser at 531 nm enables the use of a second dye in high-resolution STED microscopy. With dual color STED microscopy, co-localization of proteins can be studied in a novel and reliable way. Chromeo™ 494 fluorescent dye and secondary antibody conjugates have been certified by Leica Microsystems for use in dual color TCS STED.
Figure 6: Nuclear structures visualized by dual color STED experiments.
The use of Chromeo dyes and Fluorescent Antibodies in super-resolution STED microscopy has been described in the following publications:
- “Dual-Color STED Microscopy at 30-nm Focal-Plane Resolution” by Meyer et al (2008) Small 4(8):1095-1100. (Chromeo 488)
- “STED microscopy with a MHz pulsed stimulated-Raman-scattering source” by Rankin et al (2009) Optics Express 17(18):15679-15684. (Chromeo 488)
- “Fast STED microscopy with continuous wave fiber lasers” by Moneron et al (2010) Optics Express 18(12):1302-1309. (Chromeo 488)
- “Direct Synthesis of Lamin A, bypassing Prelamin A processing, Causes Misshapen Nuclei in Fibroblasts but No Detectable Pathology in Mice” by Coffinier et al (2010) J Biol. Chem. 285(27):20818-20826. (ATTO 647N)
- “Confocal Imaging at the Nanoscale with Two-Color STED Microscopy” by Pellett et al (2011) Proc. of SPIE 7905x. (Chromeo 494 and ATTO 647N)
- “Two-color STED microscopy in living cells” by Pellett et al (2011) Biomed. Optics Express 2(8):2364-2371. (Chromeo 494)
- “Sharper low-power STED nanoscopy by time gating” by Vicidomini et al (2011) Nature Methods 8:571-573. (Chromeo 488)
- “Novel Roles of Caenorhabditis elegans Heterochromatin Protein HP1 and Linker Histone in the Regulation of Innate Immune Gene Expression” by Studencka et al (2012) Mol. Cell. Biol. 32:251-265. (Chromeo 488)
- “Local palmitoylation cycles define activity-regulated postsynaptic subdomains” by Fukata et al (2013) J. Cell. Biol. 202(1):145-161. (Chromeo 505)
- “Fast neurotransmitter release regulated by the endocytic scaffold intersectin” by Sakaba et al (2013) PNAS 110(20):8266-8271. (Chromeo 494)
- “Super-resolution microscopy of the neuronal calcium-binding proteins Calneuron-1 and Caldendrin” by Hradsky et al (2013) Methods in Mol. Biol. 963:147-167. (Chromeo 494)