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- Chromeo™ 488 Tools for STED Microscopy
- Chromeo™ 505 Tools for STED Microscopy
- Chromeo™ 494 Tools for STED Microscopy
How STED microscopy overcomes the Abbe limit
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.
Optimized sample preparation for STED microscopy
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 3: Comparison of conventional, confocal microscopy and STED microscopy.
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)