STED Microscopy Products

products for STED super-resolution fluorescence microscopy

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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).

Reduction in size of the emitting spot in STED vs. confocal microscopy
Figure 1: Principle of STED microscopy.

Illustration of a 200 nm excitation spot of a classical confocal microscope (A) or the downsized emitting spot (~75 nm) created by a STED microscope (B, inner ring). The spheres represent individual dye molecules in fluorescent (green) or "off" mode (black).

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.

Simplified Jablonski diagram of the STED method
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.

Confocal and STED microscopy images of cells co-stained with vimentin and clathrin
Figure 3: Comparison of conventional, confocal microscopy and STED microscopy.

Vimentin and clathrin were visualized by immunohistological co-staining. The image on the left was prepared using a confocal microscope, while that on the right was produced using a STED microscope. The STED image shows the cell structure proteins much more clearly, and enables discrimination between single filaments. Images courtesy of Leica Microsystems, Germany.


Publications

The use of Chromeo dyes and Fluorescent Antibodies in super-resolution STED microscopy has been described in the following publications: