Dual-modulation difference stimulated emission depletion microscopy to suppress the background signal

Wensheng Wang; Chuankang Li; Zhengyi Zhan; Zhimin Zhang; Yubing Han; Cuifang Kuang; Xu Liu

doi:10.1117/1.AP.4.4.046001

Abstract

Stimulated emission depletion (STED) nanoscopy is one of the most well-developed nanoscopy techniques that can provide subdiffraction spatial resolution imaging. Here, we introduce dual-modulation difference STED microscopy (dmdSTED) to suppress the background noise in traditional STED imaging. By applying respective time-domain modulations to the two continuous-wave lasers, signals are distributed discretely in the frequency spectrum and thus are obtained through lock-in demodulation of the corresponding frequencies. The background signals can be selectively eliminated from the effective signal without compromise of temporal resolution. We used nanoparticle, fixed cell, and perovskite coating experiments, as well as theoretical demonstration, to confirm the effectiveness of this method. We highlight dmdSTED as an idea and approach with simple implementation for improving the imaging quality, which substantially enlarges the versatility of STED nanoscopy.

Far-field fluorescence optical microscopy is an important tool for understanding the microscopic world, benefitting from its low damage to biological tissues and imaging specificity in biomedical research. However, the resolution of traditional far-field optical microscopy is limited to approximately half the wavelength owing to the diffraction limit. In the past three decades, super-resolution microscopy (or nanoscopy) was developed to break through this bottleneck.1^–4 As one of the mainstream nanoscopy techniques, stimulated emission depletion (STED) microscopy has made considerable progress and has been widely used in practical researches.5^–7 STED is typically implemented using confocal laser scanning microscopy. Apart from the Gaussian excitation beam, STED introduces another doughnut-shaped depletion beam whose intensity profile is ideally zero in the central area and increases toward the periphery. The excitation and depletion beams should be of precise alignment in the focus volume. Through stimulated emission effect, the periphery region of the original fluorescence excited by the Gaussian excitation beam is de-excited. Hence, the fluorescence of the outer ring of the point spread function (PSF) disappears, and only fluorescence in the central area of the excitation beam is retained. Thus, the FWHM of the system PSF is compressed by the doughnut-shaped beam, and the spatial resolution is enhanced accordingly.