Frequency-domain STED microscopy for selective background noise suppression

Frequency-domain STED microscopy for selective background noise suppression

Imaging results of biological cells. a–c The results of confocal, STED and dmdSTED images, respectively. Scale bar: 2 µm. d–f Partially magnified view of parts indicated by the blue dashed box in a–c. Scale bar: 1 µm. g Image intensity change curve along the blue dashed line. Blue, red, and yellow lines correspond to confocal, STED, and dmdSTED, respectively. The sample used here is vimentin labeled with Star Green. Credit: Wang, Li et al., doi 10.1117/1.AP.4.4.046001

Nanoscopy describes the ability to see beyond the commonly accepted optical limit of 200–300 nm. Stimulated emission reduction (STED) microscopy, developed by Stefan W. Hell and Jan Wichmann in 1994 and experimentally demonstrated by Hell and Thomas Klar in 1999, is a super-resolution technique for nanoscopy. STED microscopy has made significant progress and is widely used in practical research. But its practical use involves some unwanted background noise that negatively affects spatial resolution and image quality. In general, this noise comes from two sources of signal: (i) fluorescence generated by re-excitation caused by ultrahigh doses of light from the depletion beam; and (ii) residual fluorescence, due to insufficient depletion of the inhibitory beam.

Significant background removal approaches have been developed in recent decades. They can be divided into three categories: time domain, spatial domain, and phase domain. Some of these methods have been around for a long time, while others have been developed recently. Although powerful ways to remove unwanted STED noise microscopy images, all of which lead to drawbacks including image distortion, extended acquisition time, or the introduction of shot noise. STED microscopy has not yet reached its full potential.

Nor is it reported in Advanced Photonics, researchers from Zhejiang University recently developed a new method called “dual modulation difference” STED (dmdSTED) to suppress the background selectively and efficiently. The method works by sorting the signals from the spatial domain into the frequency domain, so that non-depleted fluorescence and STED-induced background are conveniently separated from the desired fluorescence signals. The excitation and depletion beams are charged with different time-domain modulations, respectively. Because it avoids the re-excitation caused by the depletion beam, a depletion laser with a wavelength closer to the peak of fluorescence the emission spectrum of the sample can be selected, thereby reducing the required depletion intensity.

The current version of dmdSTED runs with spatial resolution of λ/8, a higher resolution than that of phase-domain methods (e.g., SPLIT, λ/5), which are prone to shot noise. In theory, potential signal loss through time domain approaches (such as time gating) can be avoided by this approach. In addition, dmdSTED is compatible with either pulsed or continuous wave scenarios, and no time-correlated single photon counting (TCSPC) hardware is required. Compared to spatial domain methods, the temporal resolution of dmdSTED is not limited. Thus, dmdSTED is advantageous in obtaining comprehensively fine microscopic images, in spatial resolution, SNR, and temporal resolution.

According to senior author Xu Liu, director of the State Key Laboratory of Advanced Optical Instrumentation, “This frequency-domain method holds great potential for integration into other dual-beam point scanning techniques, such as excited-state saturation microscopy (ESSat) , charge state depletion microscopy (CSD), ground state depletion microscopy (GSD) and so on. In addition, it can accept more sample types with spectral characteristics other than those commonly used fluorescent dyes in STED, like some quantum dots with a wider range of arousal.”


Background suppression for super-resolution light microscopy


More info:
Wensheng Wang et al, Double modulation stimulated emission depletion microscopy for background suppression, Advanced Photonics (2022). DOI: 10.1117/1.AP.4.4.046001

Courtesy of SPIE–International Society for Optics and Photonics

Quote: Frequency Domain STED Microscopy for Selective Background Noise Suppression (2022, July 6), Retrieved July 6, 2022, from https://phys.org/news/2022-07-frequency-domain-sted-microscopy -background-noise.html

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