Nanoscopy describes the ability to see beyond the generally accepted optical limit of 200 to 300 nanometers. Stimulated emission depletion (STED) microscopy, developed by Stefan W. Hell and Jan Wichmann in 1994, and experimentally demonstrated 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 some undesirable background noise, which negatively affects spatial resolution and image quality. In general, this noise comes from two signal sources: fluorescence generated by re-excitation caused by ultrahigh light doses from the depletion beam; and residual fluores­cence, due to insufficient depletion of the inhi­bition beam.

Significant background removal approaches have been developed over the past decades. These can be divided into three categories: time-domain, space-domain, and phasor-domain. Some of these methods are long-standing and some are more recently developed. While powerful ways to remove undesirable noise from STED micro­scopy images, they all entail drawbacks, including image distortion, prolonged acquisi­tion times, or introduction of shot noise. STED microscopy has yet to achieve its full potential. Now, researchers from Zhejiang University recently developed a novel method – “dual-modu­lation difference” STED (dmdSTED) – to suppress backgrounds selectively and effec­tively.

The method works by sorting space-domain signals into the frequency domain so that the nondepleted fluorescence and STED-induced background are con­veniently separated from the desired fluorescent signals. The excitation and the depletion beams are loaded respectively with different time-domain modu­lations. Since it avoids the re-excitation caused by the depletion beam, a depletion laser with a wavelength closer to the peak of the fluorescence emission spectrum of the sample can be selected, thus reducing the required depletion intensity.

The current version of dmdSTED performs with spatial resolution of λ/8, higher resolution than that of the phasor-domain methods which are prone to shot noise. Theoretically, potential signal loss by time-domain approaches like time-gating can be avoided by this approach. In addition, dmdSTED is compatible with either pulsed or continuous-wave scenarios, and hardware for time-correlated single-photon counting (TCSPC) is not required. Compared with space-domain methods, the time resolution of dmdSTED is not confined. Thus, dmdSTED is advan­tageous in acquisition of compre­hensively fine microscopy images, in spatial resolution, SNR, and time resolution.

According to Xu Liu, director of the State Key Laboratory of Modern Optical Instru­mentation, “this frequency-domain method possesses great potential to integrate into other dual-beam point-scanning techniques, like excited state satura­tion micro­scopy (ESSat), charge state depletion microscopy (CSD), ground state depletion micro­scopy (GSD) and so on.” Liu remarks, “In addition, it can accept more types of samples with spectral characteristics different from commonly used fluorescent dyes in STED, such as some quantum dots with a wider excitation spectrum.” (Source: SPIE)

Reference: W. Wang et al.: Dual-modulation difference stimulated emission depletion microscopy to suppress the background signal, Adv. Phot. 4, 046001 (2022); DOI: 10.1117/1.AP.4.4.046001

Link: State Key Laboratory of Modern Optical Instrumentation, Zhejiang University, Hangzhou, China