High-speed optical imaging with sCMOS pixel reassignment.

Fluorescence microscopy has undergone rapid advancements, offering unprecedented visualization of biological events and shedding light on the intricate mechanisms governing living organisms. However, the exploration of rapid biological dynamics still poses a significant challenge due to the limitations of current digital camera architectures and the inherent compromise between imaging speed and other capabilities. Here, we introduce sHAPR, a high-speed acquisition technique that leverages the operating principles of sCMOS cameras to capture fast cellular and subcellular processes. sHAPR harnesses custom fiber optics to convert microscopy images into one-dimensional recordings, enabling acquisition at the maximum camera readout rate, typically between 25 and 250 kHz. We have demonstrated the utility of sHAPR with a variety of phantom and dynamic systems, including high-throughput flow cytometry, cardiomyocyte contraction, and neuronal calcium waves, using a standard epi-fluorescence microscope. sHAPR is highly adaptable and can be integrated into existing microscopy systems without requiring extensive platform modifications. This method pushes the boundaries of current fluorescence imaging capabilities, opening up new avenues for investigating high-speed biological phenomena.

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.

Small separation frequency-domain near-infrared spectroscopy for the recovery of tissue optical properties at millimeter depths.

Millimeter-depth sensitivity with frequency domain near-infrared spectroscopy has been challenging due to the breakdown of the diffusion equation for source-detection separations < 1cm. To overcome this challenge, we employ a Monte-Carlo lookup table-based inverse algorithm to fit small separation (3-6 mm) frequency-domain near-infrared spectroscopy (FDNIRS) data for absorption and reduced scattering coefficients. We verify this small separation FDNIRS method through a series of in vitro and in vivo studies. In vitro, we observed a root mean squared percent error (RMSE) in estimation of the reduced scattering coefficient and absorption coefficient of 2.8% and 7.6%, respectively, in liquid phantoms consisting of Intralipid and Indian ink, and a RMSE in estimation of oxygen saturation and total hemoglobin concentrations of 7.8 and 11.2%, respectively, in blood-mixed liquid phantoms. Next, we demonstrate one particularly valuable in vivo application of this technique wherein we non-invasively measure the optical properties of the mouse brain (n = 4). We find that the measured resting state cerebral oxygen saturation and hemoglobin concentration are consistent with literature reported values, and we observe expected trends during a hyper-/hypoxia challenge that qualitatively mimic changes in partial pressure of oxygen (pO2) measured simultaneously with an invasive pO2 sensor. Further, through simulations of the mouse head geometry, we demonstrate that the skull and scalp exert minimal influence on the estimate oxygen saturation, while leading to small but systematic underestimation of total hemoglobin concentration. In total, these results demonstrate the robustness of small separation FDNIRS to assess tissue optical properties at millimeter depth resolution.

Dual-display laparoscopic laser speckle contrast imaging for real-time surgical assistance.

Laser speckle contrast imaging (LSCI) utilizes the speckle pattern of a laser to determine the blood flow in tissues. The current approaches for its use in a clinical setting require a camera system with a laser source on a separate optical axis making it unsuitable for minimally invasive surgery (MIS). With blood flow visualization, bowel viability, for example, can be determined. Thus, LSCI can be a valuable tool in gastrointestinal surgery. In this work, we develop the first-of-its-kind dual-display laparoscopic vision system integrating LSCI with a commercially available 10mm rigid laparoscope where the laser has the same optical axis as the laparoscope. Designed for MIS, our system permits standard color RGB, label-free vasculature imaging, and fused display modes. A graphics processing unit accelerated algorithm enables the real-time display of three different modes at the surgical site. We demonstrate the capability of our system for imaging relative flow rates in a microfluidic phantom with channels as small as 200 µm at a working distance of 1-5 cm from the laparoscope tip to the phantom surface. Using our system, we reveal early changes in bowel perfusion, which are invisible to standard color vision using a rat bowel occlusion model. Furthermore, we apply our system for the first time for imaging intestinal vasculature during MIS in a swine.