Abstract
Stimulated emission depletion (STED) microscopy is a typical laser-scanning super-resolution imaging technology, the emergence of which has opened a new research window for studying the dynamic processes of live biological samples on a nanometer scale. According to the characteristics of STED, a high depletion power is required to obtain a high resolution. However, a high laser power can induce severe phototoxicity and photobleaching, which limits the applications for live cell imaging, especially in two-color STED super-resolution imaging. Therefore, we developed a low-power two-color STED super-resolution microscope with a single supercontinuum white-light laser. Using this system, we achieved low-power two-color super-resolution imaging based on digital enhancement technology. Lateral resolutions of 109 and 78 nm were obtained for mitochondria and microtubules in live cells, respectively, with 0.8 mW depletion power. These results highlight the great potential of the novel digitally enhanced two-color STED microscopy for long-term dynamic imaging of live cells.
Highlights
Stimulated emission depletion (STED) is the earliest super-resolution imaging approach to break the optical diffraction limit among many super-resolution imaging technologies [1,2,3,4,5,6]
The spatial resolution of the two-color digitally enhanced STED (DE-STED) microscope was assessed by measuring the effective point spread function (PSF) with two kinds of 100 nm fluorescent beads (488/560 and 633/660, Thermo Fisher)
The above results showed that the DE-STED method with k = 10 at 0.8 mW depletion power can obtain a considerable resolution of STED at 32 mW depletion power
Summary
STED is the earliest super-resolution imaging approach to break the optical diffraction limit among many super-resolution imaging technologies [1,2,3,4,5,6]. STED shrinks the point spread function (PSF) of excited light via the stimulated emission effect, resulting in the breakage of the optical diffraction limit and nanometer-scale spatial resolution [7]. To effectively force the fluorophore into a dark state and achieve the compression of the PSF, the excitation light must reach the sample approximately 200 ps before the depleted light (STED light), and the stimulated emission process has to precede the spontaneous emission. A very high STED light power is required to completely deplete the fluorescence in the overlapping areas due to the short time range and the small stimulated emission cross-section of the fluorescent molecule. Photobleaching [9] and phototoxicity [10] often occur when high spatial resolution is made possible This severely limits the application and development of STED for live cell imaging
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