Frequency-Domain STED Microscopy for Selective Background Suppression

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

Important approaches to background noise removal have been developed over the past few decades. These can be divided into three categories: time domain, spatial domain and phasor domain. Some of these methods have been around for a long time and others have been developed more recently. Although powerful ways to remove unwanted noise from STED microscopy images, they all have drawbacks, including image distortion, long acquisition times, or the introduction of shot noise. STED microscopy has not yet reached its full potential.

As reported in Advanced photonics, researchers from Zhejiang University recently developed a new method called “double modulation difference” STED (dmdSTED) to suppress background noise selectively and effectively. The method works by sorting signals from the spatial domain into the frequency domain such that undepleted fluorescence and STED-induced background are conveniently separated from the desired fluorescent signals. The excitation and depletion beams are loaded respectively with different temporal modulations. Since it avoids re-excitation caused by the depletion beam, a depletion laser with a wavelength closer to the peak of the sample’s fluorescence emission spectrum can be selected, thereby reducing the intensity of the sample. impoverishment required.

The current version of dmdSTED works with a spatial resolution of λ/8, a higher resolution than phasor domain methods (eg, SPLIT, λ/5), which are prone to shot noise. Theoretically, the potential loss of signal by time domain approaches (such as time-gating) can be avoided by this approach. Additionally, dmdSTED is compatible with pulsed or continuous wave scenarios, and hardware for time-correlated single photon counting (TCSPC) is not required. Compared to spatial domain methods, the temporal resolution of dmdSTED is not confined. Thus, dmdSTED is advantageous in acquiring very fine microscopy images, in spatial resolution, SNR and temporal resolution.

According to lead author Xu Liu, director of the State Key Laboratory of Modern Optical Instrumentation, “This frequency-domain method has great potential for integration into other dual-beam point-scanning techniques, such as optical saturation microscopy. state (ESSat), charge state depletion microscopy (CSD), ground state depletion microscopy (GSD), etc. Liu remarks, “In addition, it can accept more types of samples with different spectral characteristics from fluorescent dyes commonly used in STED, such as some quantum dots with a broader excitation spectrum.

– This press release was originally published on the SPIE website