Light-field flow cytometry for high-resolution, volumetric and multiparametric 3D single-cell analysis.

Imaging flow cytometry (IFC) combines flow cytometry and fluorescence microscopy to enable high-throughput, multiparametric single-cell analysis with rich spatial details. However, current IFC techniques remain limited in their ability to reveal subcellular information with a high 3D resolution, throughput, sensitivity, and instrumental simplicity. In this study, we introduce a light-field flow cytometer (LFC), an IFC system capable of high-content, single-shot, and multi-color acquisition of up to 5,750 cells per second with a near-diffraction-limited resolution of 400-600 nm in all three dimensions. The LFC system integrates optical, microfluidic, and computational strategies to facilitate the volumetric visualization of various 3D subcellular characteristics through convenient access to commonly used epi-fluorescence platforms. We demonstrate the effectiveness of LFC in assaying, analyzing, and enumerating intricate subcellular morphology, function, and heterogeneity using various phantoms and biological specimens. The advancement offered by the LFC system presents a promising methodological pathway for broad cell biological and translational discoveries, with the potential for widespread adoption in biomedical research.

Implementation of miniaturized modular-array fluorescence microscopy for long-term live-cell imaging.

Fluorescence microscopy imaging of live cells has provided consistent monitoring of dynamic cellular activities and interactions. However, because current live-cell imaging systems are limited in their adaptability, portable cell imaging systems have been adapted by a variety of strategies, including miniaturized fluorescence microscopy. Here, we provide a protocol for the construction and operational process of miniaturized modular-array fluorescence microscopy (MAM). The MAM system is built in a portable size (15c m×15c m×3c m) and provides in situ cell imaging inside an incubator with a subcellular lateral resolution (∼3µm). We demonstrated the improved stability of the MAM system with fluorescent targets and live HeLa cells, enabling long-term imaging for 12 h without the need for external support or post-processing. We believe the protocol could guide scientists to construct a compact portable fluorescence imaging system and perform time-lapse in situ single-cell imaging and analysis.

Portable light-sheet optofluidic microscopy for 3D fluorescence imaging flow cytometry.

Imaging flow cytometry (IFC) combines conventional flow cytometry with optical microscopy, allowing for high-throughput, multi-parameter screening of single-cell specimens with morphological and spatial information. However, current 3D IFC systems are limited by instrumental complexity and incompatibility with available microfluidic devices or operations. Here, we report portable light-sheet optofluidic microscopy (PLSOM) for 3D fluorescence cytometric imaging. PLSOM exploits a compact, open-top light-sheet configuration compatible with commonly adopted microfluidic chips. The system offers a subcellular resolution (2-4 µm) in all three dimensions, high throughput (∼1000 cells per s), and portability (30 cm (l) × 10 cm (w) × 26 cm (h)). We demonstrated PLSOM for 3D IFC using various phantom and cell systems. The low-cost and custom-built architecture of PLSOM permits easy adaptability and dissemination for broad 3D flow cytometric investigations.

Super-resolution optofluidic scanning microscopy.

Optofluidics enables visualizing diverse anatomical and functional traits of single-cell specimens with new degrees of imaging capabilities. However, the current optofluidic microscopy systems suffer from either low resolution to reveal subcellular details or incompatibility with general microfluidic devices or operations. Here, we report optofluidic scanning microscopy (OSM) for super-resolution, live-cell imaging. The system exploits multi-focal excitation using the innate fluidic motion of the specimens, allowing for minimal instrumental complexity and full compatibility with various microfluidic configurations. The results present effective resolution doubling, optical sectioning and contrast enhancement. We anticipate the OSM system to offer a promising super-resolution optofluidic paradigm for miniaturization and different levels of integration at the chip scale.

Fast and accurate sCMOS noise correction for fluorescence microscopy.

The rapid development of scientific CMOS (sCMOS) technology has greatly advanced optical microscopy for biomedical research with superior sensitivity, resolution, field-of-view, and frame rates. However, for sCMOS sensors, the parallel charge-voltage conversion and different responsivity at each pixel induces extra readout and pattern noise compared to charge-coupled devices (CCD) and electron-multiplying CCD (EM-CCD) sensors. This can produce artifacts, deteriorate imaging capability, and hinder quantification of fluorescent signals, thereby compromising strategies to reduce photo-damage to live samples. Here, we propose a content-adaptive algorithm for the automatic correction of sCMOS-related noise (ACsN) for fluorescence microscopy. ACsN combines camera physics and layered sparse filtering to significantly reduce the most relevant noise sources in a sCMOS sensor while preserving the fine details of the signal. The method improves the camera performance, enabling fast, low-light and quantitative optical microscopy with video-rate denoising for a broad range of imaging conditions and modalities.

Miniaturized modular-array fluorescence microscopy.

Fluorescence live-cell imaging allows for continuous interrogation of cellular behaviors, and the recent development of portable live-cell imaging platforms has rapidly transformed conventional schemes with high adaptability, cost-effective functionalities and easy accessibility to cell-based assays. However, broader applications remain restrictive due to compatibility with conventional cell culture workflow and biochemical sensors, accessibility to up-right physiological imaging, or parallelization of data acquisition. Here, we introduce miniaturized modular-array fluorescence microscopy (MAM) for compact live-cell imaging in flexible formats. We advance the current miniscopy technology to devise an up-right modular architecture, each combining a gradient-index (GRIN) objective and individually-addressed illumination and acquisition components. Parallelization of an array of such modular devices allows for multi-site data acquisition in situ using conventional off-the-shelf cell chambers. Compared with existing methods, the device offers a high fluorescence sensitivity and efficiency, exquisite spatiotemporal resolution (∼3 µm and up to 60 Hz), a configuration compatible with conventional cell culture assays and physiological imaging, and an effective parallelization of data acquisition. The system has been demonstrated using various calibration and biological samples and experimental conditions, representing a promising solution to time-lapse in situ single-cell imaging and analysis.

BSSE: An open-source image processing tool for miniaturized microscopy.

Single-photon-excitation-based miniaturized microscope, or miniscope, has recently emerged as a powerful tool for imaging neural ensemble activities in freely moving animals. In the meanwhile, this highly flexible and implantable technology promises great potential for studying a broad range of cells, tissues and organs. To date, however, applications have been largely limited by the properties of the imaging modality. It is therefore highly desirable for a method generally applicable for processing miniscopy images, enabling and extending the applications to diverse anatomical and functional traits, spanning various cell types in the brain and other organs. We report an image processing approach, termed BSSE, for background suppression and signal enhancement for miniscope image processing. The BSSE method provides a simple, automatic solution to the intrinsic challenges of overlapping signals, high background and artifacts in miniscopy images. We validated the method by imaging synthetic structures and various biological samples of brain, tumor, and kidney tissues. The work represents a generally applicable tool for miniscopy technology, suggesting broader applications of the miniaturized, implantable and flexible technology for biomedical research.

Simple anchoring of the penopubic skin to the prepubic deep fascia in surgical correction of buried penis.

PURPOSE: The aim of this study was to categorize concealed penis and buried penis by preoperative physical examination including the manual prepubic compression test and to describe a simple surgical technique to correct buried penis that was based on surgical experience and comprehension of the anatomical components. MATERIALS AND METHODS: From March 2007 to November 2010, 17 patients were diagnosed with buried penis after differentiation of this condition from concealed penis. The described surgical technique consisted of a minimal incision and simple fixation of the penile shaft skin and superficial fascia to the prepubic deep fascia, without degloving the penile skin. RESULTS: The mean age of the patients was 10.2 years, ranging from 8 years to 15 years. The median follow-up was 19 months (range, 5 to 49 months). The mean penile lengths were 1.8 cm (range, 1.1 to 2.5 cm) preoperatively and 4.5 cm (range, 3.3 to 5.8 cm) postoperatively. The median difference between preoperative and postoperative penile lengths was 2.7 cm (range, 2.1 to 3.9 cm). There were no serious intra- or postoperative complications. CONCLUSIONS: With the simple anchoring of the penopubic skin to the prepubic deep fascia, we obtained successful subjective and objective outcomes without complications. We suggest that this is a promising surgical method for selected patients with buried penis.