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Fluorescence

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.

 

Principles of the STED microscope

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).

Diagram illustrating the principle behind Stimulated Emission Depletion microscopy

Figure 3: Diagram of the Leica TCS STED microscopy.

The basis of STED microscopy is the coupling of the excitation laser with the STED depletion laser, resulting in the doughnut-shaped depletion. The two perfectly aligned laser systems minimize the size of the fluorescence spot, overcoming the resolution-limiting effects of diffraction. Diagram courtesy of Leica Microsystems, Germany.

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.

Optimized sample preparation for STED microscopy

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.

Confocal and STED microscopy images of cells co-stained with vimentin and clathrin
Figure 4: 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.

Available STED microscopes

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.

Staining of Bruchpilot in Drosophila larvae with fluorescent Chromeo 488 dye using the Leica TCS STED CW system
Figure 5: Immunofluorescent staining of neuromuscular junctions in Drosophila larvae.

Bruchpilot was stained with Chromeo 488 and imaged by STED microscopy using the TCS STED CW system. Image courtesy of Stephan Sigrist and Werner Fouquet, Freie Universität Berlin, Germany.

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.

Comparison of nuclear structures visualized by dual color microscopy images with Chromeo 494 (green) and ATTO 647N fluorescent dyes using confocal and STED microscopes
Figure 6: Nuclear structures visualized by dual color STED experiments.

The image on the left was prepared using a confocal microscope, while that on the right was produced using a STED microscope. The nuclear structures have been visualized with Chromeo 494 (green) and ATTO 647N (red). Images courtesy of Dr. L. Schermelleh, LMU Biozentrum Munich, Germany.

Product Citations

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