STimulation Emission Depletion (STED) Microscopy
The STED system we built was supported by US taxpayer money via an NIH grant from the National Heart, Lung, and Blood Institute BRG R01 HL088640 (Program Director Dr. Denis Buxton). We will appreciate very much if in a related publication you acknowledge this support.
(Almost all images in this sites are actually gray scale. But in order to better visualize these images we often use the "RED HOT" color map. This color map use the colors Black → Red → Yellow → White to signify intensity from low to high. )
Since its invention in about 1590, resolution of conventional optical microscopy has been always restricted by the diffraction limit. Super-resolution techniques that break such limit started emerging in the 1990’s, including photoactivation localization microscopy (PALM) (Betzig et al., 2006), structured illumination microscopy (SIM) (Gustafsson, 2000), and super-resolution optical fluctuation imaging (SOFI) (Dertinger et al., 2009), etc.
The super-resolution technology we like the best is called stimulated emission depletion (STED) (Hell, 2003), which was invented by a German physicist Stefan Hell. Compared with other super-resolution techniques, its unique feature is that it is a purely optical method that does not require any mathematical manipulation or image processing. The basic idea of STED microscopy is to engineer the point spread function (PSF): in addition to the conventional laser beam that excites the fluorophores, a donut-shaped depletion laser beam is used to de-excite the peripheral fluorescence through stimulated emission, and thus only fluorescence emission from the sub-diffraction-limited center is left out to be recorded.
STED resolution is theoretically unlimited by indefinitely increasing the depletion laser power (Harke et al., 2008a). In practice, about 30-80nm lateral resolution has been reported in various biological samples (Harke et al., 2008a;Lauterbach et al., 2010;Meyer et al., 2008;Nagerl and Bonhoeffer, 2010). Originally based on pulsed lasers, continuous wave (CW) STED has also been made for its technical simplicity and more affordable cost (Willig et al., 2007). STED microscopy is based on confocal microscopy and thus inherits its ability of optical sectioning. STED can also achieve super-resolution in the axial direction simultaneously, realizing so called 3D super-resolution (Harke et al., 2008b). Fast scanning resonant mirrors have been applied to STED microscopy to perform live imaging (Lauterbach et al., 2010;Westphal et al., 2008).
Commercial STED microscopes are available (Leica Microsystems, Germany) but they are by no means cheap. The price tag is easily over $1,000,000. Therefore, we decided to construct a fast scanning STED microscope by ourselves. Custom-built STED systems in laboratories can be much more economical and flexible, but building them are technically challenging. After years of hard work, we have independently built a STED microscope at much lower cost compared with the commercial Leica system without compromising on the image quality.
Photobleaching is always a problem for fluorescence microscope, and it is more so for STED, because the depletion laser beam is quite powerful (~100mW at the pupil of objective). A significant portion of photobleaching is due to triplet state buildup, and Stephen Hell's team once proposed to lower the repetition rate of the pulsed depletion laser to ease it off (Donnert et al., 2007). But this approach significantly slows down the imaging speed. Fast scanning is considered a viable approach to reduce photobleaching (Borlinghaus, 2006;Tsien and Bacskai, p995). We thus utilize an 8 KHz resonant scanning mirror that enables our STED microscope to take images at 16,000 lines per second. Also, compared with piezoelectric stages, a pair of scanning mirror is a much economical solution for scanning.
Another feature of our system is its fast data acquisition. The data acquisition system can work at up to 96 MHz, providing a maximum corrected image size of 3,840 pixels per line. If the pixel size is ~10nm per pixel, we can still have a large 38µm × 38µm field of view. At such fast scanning speed and large field of view, the exposure time of fluorophores is about ~500ns (see Fig. 1.1), considerably shorter than the typical lifetime of triplet states, which is in microseconds. Piezoelectric stages, on the other hand, usually have an exposure time from about 10µs to 10ms. Much shortened exposure time with resonant scanning mirror enables fluorophores to relax from triplets and thus reduces photobleaching (Borlinghaus, 2006;Donnert et al., 2007).
We were able to acquire excellent STED images for both thin biological samples such as mitochondria and neuron cells and for thicker samples such as cardiomyocytes. The resolution achieved by our microscope in these biological samples is proven to be ~46 nm. The system has excellent optical stability. It only needs a fast daily optimization of alignment and it has been constantly up and running for months. The main objective of this site is to provide a clear and thorough summary on how we made a stable fast scanning STED microscope with fast acquisition speed, which can be easily reproduced by other researchers.