Real-time image resolution measurement for single molecule localization microscopy.

Recent advancements in single molecule localization microscopy (SMLM) have demonstrated outstanding potential applications in high-throughput and high-content screening imaging. One major limitation to such applications is to find a way to optimize imaging throughput without scarifying image quality, especially the homogeneity in image resolution, during the imaging of hundreds of field-of-views (FOVs) in heterogeneous samples. Here we introduce a real-time image resolution measurement method for SMLM to solve this problem. This method is under the heuristic framework of overall image resolution that counts on localization precision and localization density. Rather than estimating the mean localization density after completing the entire SMLM process, this method uses the spatial Poisson process to model the random activation of molecules and thus determines the localization density in real-time. We demonstrate that the method is valid in real-time resolution measurement and is effective in guaranteeing homogeneous image resolution across multiple representative FOVs with optimized imaging throughput.

Divide and conquer: real-time maximum likelihood fitting of multiple emitters for super-resolution localization microscopy.

Multi-emitter localization has great potential for maximizing the imaging speed of super-resolution localization microscopy. However, the slow image analysis speed of reported multi-emitter localization algorithms limits their usage in mostly off-line image processing with small image size. Here we adopt the well-known divide and conquer strategy in computer science and present a fitting-based method called QC-STORM for fast multi-emitter localization. Using simulated and experimental data, we verify that QC-STORM is capable of providing real-time full image processing on raw images with 100 µm × 100 µm field of view and 10 ms exposure time, with comparable spatial resolution as the popular fitting-based ThunderSTORM and the up-to-date non-iterative WindSTORM. This study pushes the development and practical use of super-resolution localization microscopy in high-throughput or high-content imaging of cell-to-cell differences or discovering rare events in a large cell population.

Quantitative performance evaluation of a back-illuminated sCMOS camera with 95% QE for super-resolution localization microscopy.

Scientific Complementary Metal Oxide Semiconductor (sCMOS) cameras were introduced into the market in 2009 and are now becoming a major type of commercial cameras for low-light imaging. sCMOS cameras provide simultaneously low read noise, high readout speed, and large pixel array; however, the relatively low quantum efficiency (QE) of sCMOS cameras has been a major limitation for its application in single molecule imaging, especially super-resolution localization microscopy which requires high detection sensitivity. Here we report the imaging performance of a newly released back-illuminated sCMOS camera (called Dhyana 95 from Tucsen) which is claimed to be the world's first 95% QE sCMOS camera. The imaging performance evaluation is based on a new methodology which is designed to provide paired images from two tested cameras under almost identical experimental conditions. We verified that this new 95% QE sCMOS camera is able to provide superior imaging performance over a representative front-illuminated sCMOS camera (Hamamatsu Flash 4.0 V2) and a popular back-illuminated EMCCD camera (Andor iXon 897 Ultra) in a wide signal range. We hope this study will inspire more studies on using sCMOS cameras in super-resolution localization microscopy, or even single molecule imaging. © 2017 International Society for Advancement of Cytometry.

High-power homogeneous illumination for super-resolution localization microscopy with large field-of-view.

As a wide-field imaging technique, super-resolution localization microscopy (SRLM) is theoretically capable of increasing field-of-view (FOV) without sacrificing either imaging speed or spatial resolution. There are two key factors for realizing large FOV SRLM: one is high-power illumination over the whole FOV with sufficient illumination homogeneity and the other is large FOV signal detection by a camera that has large number of pixels and sufficient detection sensitivity. However nowadays, even though the state-of-art scientific complementary metal-oxide semiconductor (sCMOS) cameras provide single molecule fluorescence signal detection ability over an FOV of more than 200 µm × 200 µm, large FOV SRLM still has not been achieved due to the lack of high-power homogeneous illumination. In this paper, we report large FOV SRLM with a high-power homogeneous illumination system. We demonstrate experimentally that our illumination system, which is based on a newly designed multimode fiber combiner, is capable of providing sufficient illumination intensity (~4.7 kW/cm2 @ 640 nm) and excellent illumination homogeneity. Compared with the reported approaches, our illumination system is advantageous in laser power scaling and square-shape illumination without beam clipping. As a result, our system makes full use of the sensor of a representative Hamamatsu Flash 4.0 V2 sCMOS camera (2048 × 2048 active pixels) and achieves a FOV as large as 221 µm × 221 µm with homogeneous spatial resolution. The flexible solution for realizing large FOV SRLM reported in this paper pushes a significant step toward the development of SRLM.