Effects of optical aberrations on localization of MINFLUX super-resolution microscopy.

A novel super-resolution imaging technique based on the minimum photon flux (MINFLUX), can achieve nanometer-scale localization precision and sub-5-nm imaging. However, aberrations can affect the localization performance and degrade the quality of reconstructed images. In this study, we analyze the effects of different low-order aberrations on the MINFLUX system through both theoretical limits and Monte Carlo methods. We report that 1) defocus and spherical aberration have little effect on 2D localization performance, whereas astigmatism and coma have significant negative effects; 2) system aberrations that can be measured in advance cause changes primarily in the magnitude and angular uniformity of localization precision, whereas sample-induced aberrations that cannot be a priori introduce large biases and reduce localization accuracy.

Simultaneous super-resolution estimation of single-molecule position and orientation with minimal photon fluxes.

The orientation of a single molecule provides valuable information on fundamental biological processes. We report a technique for the simultaneous estimation of single-molecule 2D position and 2D orientation with ultra-high localization precision (∼2-nm precision with ∼500 photons under a typical 100-nm diameter of excitation beam pattern), which is also compatible with tracking in living cells. In the proposed method, the theoretical precision limits are calculated, and the localization and orientation performance along with potential applications are explored using numerical simulations. Compared to other camera-based orientation measurement methods, it is confirmed that the proposed method can obtain reasonable estimates even under very weak signals (∼15 photons). Moreover, the maximum likelihood estimator (MLE) is found to converge to the theoretical limit when the total number of photons is less than 100.

3D resolution enhancement in saturated competition microscopy.

To overcome the diffraction barrier, super-resolution microscopy is contrived and has witnessed scientific developments in varying fields, especially in last few decades, such as stochastic optical reconstruction microscopy, stimulated emission depletion microscopy (STED), mirror-enhanced super-resolution microscopy (MEANS), and fluorescence emission difference microscopy (FED). Recently, saturated competition microscopy (SAC) was developed to realize high sub-diffraction resolution in either fluorescent or non-fluorescent imaging. Compared with STED, SAC features non-constraint in fluorescent dye selection. Nevertheless, the lateral resolution is limited in consideration of photobleaching side effects. Also, the axial resolution enhancement of SAC has not been demonstrated. In this study, a method, combining FED, MEANS, and SAC, is presented to improve the three-dimensional (3D) resolution. The numerical study reveals that the lateral resolution is close to 0.085λ and axial resolution can be enhanced to 0.184λ. In addition, the SNR is improved simultaneously. The availability to improve 3D resolution of SAC is believed to be significant for biological imaging in the future.

Prospects for Fluorescence Nanoscopy.

Overcoming Abbe's diffraction limit has been a challenging task and one of great interest for biological investigations. The emergence of fluorescence nanoscopy circumvents the diffraction barrier with nearly limitless power for optical microscopy, which enables investigations of the microscopic world in the 1-100 nm range. Proposed variants, such as expansion microscopy (ExM), stimulated emission depletion microscopy (STED), and Airyscan, are innovative in three aspects: sampling, illumination, and detection. These techniques show increasing strength in bioimaging subcellular structures. In this Perspective, we highlight advances in and prospects of fluorescence nanoscopy.