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We present a mm-sized, ultrasonically powered lensless CMOS image sensor as a progress towards wireless fluorescence microscopy. Access to biological information within the tissue has the potential to provide insights guiding diagnosis and treatment across numerous medical conditions including cancer therapy. This information, in conjunction with current clinical imaging techniques that have limitations in obtaining images continuously and lack wireless compatibility, can improve continual detection of multicell clusters deep within tissue. The proposed platform incorporates a 2.4 × 4.7 mm 2 integrated circuit (IC) fabricated in TSMC 0.18 µm, a micro laser diode (µLD), a single piezoceramic and off-chip storage capacitors. The IC consists of a 36 × 40 array of capacitive trans-impedance amplifier-based pixels, wireless power management and communication via ultrasound and a laser driver all controlled by a Finite State Machine. The piezoceramic harvests energy from the acoustic waves at a depth of 2 cm to power up the IC and transfer 11.5 kbits/frame via backscattering. During Charge-Up, the off-chip capacitor stores charge to later supply a high-power 78 mW µLD during Imaging. Proof of concept of the imaging front end is shown by imaging distributions of CD8 T-cells, an indicator of the immune response to cancer, ex vivo, in the lymph nodes of a functional immune system (BL6 mice) against colorectal cancer consistent with the results of a fluorescence microscope. The overall system performance is verified by detecting 140 µm features on a USAF resolution target with 32 ms exposure time and 389 ms ultrasound backscattering.

Single-molecule localization microscopy (SMLM) enables imaging scientists to visualize biological structures with unprecedented resolution. Particularly powerful implementations of SMLM are capable of three-dimensional, multicolor and high-throughput imaging and can yield key biological insights. However, widespread access to these technologies is limited, primarily by the cost of commercial options and complexity of de novo development of custom systems. Here we provide a comprehensive guide for interested researchers who wish to establish a high-end, custom-built SMLM setup in their laboratories. We detail the initial configuration and subsequent assembly of the SMLM, including the instructions for the alignment of all the optical pathways, the software and hardware integration, and the operation of the instrument. We describe the validation steps, including the preparation and imaging of test and biological samples with structures of well-defined geometries, and assist the user in troubleshooting and benchmarking the system's performance. Additionally, we provide a walkthrough of the reconstruction of a super-resolved dataset from acquired raw images using the Super-resolution Microscopy Analysis Platform. Depending on the instrument configuration, the cost of the components is in the range US$95,000-180,000, similar to other open-source advanced SMLMs, and substantially lower than the cost of a commercial instrument. A builder with some experience of optical systems is expected to require 4-8 months from the start of the system construction to attain high-quality three-dimensional and multicolor biological images., (© 2024. Springer Nature Limited.)

We present a versatile extended depth-of-field (EDOF) wide-field fluorescence microscopy using a new, to the best of our knowledge, active device, micro-mirror array lens system (MALS) for calibration-free and orientation-insensitive EDOF imaging. The MALS changed the focal plane during image acquisition, and the system could be operated in any orientation. Two EDOF imaging modes of high-speed accumulation and low-speed surface sectioning were implemented. The performance was demonstrated in non-contact imaging of conjunctival goblet cells in live mice and depth-resolved cellular examination of ex-vivo human cancer specimens. MALS-based EDOF microscopy has potential for versatile cellular examination.

Despite widespread adoption of tissue clearing techniques in recent years, poor access to suitable light-sheet fluorescence microscopes remains a major obstacle for biomedical end-users. Here, we present descSPIM (desktop-equipped SPIM for cleared specimens), a low-cost ($20,000-50,000), low-expertise (one-day installation by a non-expert), yet practical do-it-yourself light-sheet microscope as a solution for this bottleneck. Even the most fundamental configuration of descSPIM enables multi-color imaging of whole mouse brains and a cancer cell line-derived xenograft tumor mass for the visualization of neurocircuitry, assessment of drug distribution, and pathological examination by false-colored hematoxylin and eosin staining in a three-dimensional manner. Academically open-sourced ( https://github.com/dbsb-juntendo/descSPIM ), descSPIM allows routine three-dimensional imaging of cleared samples in minutes. Thus, the dissemination of descSPIM will accelerate biomedical discoveries driven by tissue clearing technologies., (© 2024. The Author(s).)

By superlocalizing the positions of millions of single molecules over many camera frames, a class of super-resolution fluorescence microscopy methods known as single-molecule localization microscopy (SMLM) has revolutionized how we understand subcellular structures over the past decade. In this review, we highlight emerging studies that transcend the outstanding structural (shape) information offered by SMLM to extract and map physicochemical parameters in living mammalian cells at single-molecule and super-resolution levels. By encoding/decoding high-dimensional information-such as emission and excitation spectra, motion, polarization, fluorescence lifetime, and beyond-for every molecule, and mass accumulating these measurements for millions of molecules, such multidimensional and multifunctional super-resolution approaches open new windows into intracellular architectures and dynamics, as well as their underlying biophysical rules, far beyond the diffraction limit.

Time-resolved fluorescence imaging techniques, like confocal fluorescence lifetime imaging microscopy, are powerful photonic instrumentation tools of modern science with diverse applications, including: biology, medicine, and chemistry. However, complexities of the systems, both at specimen and device levels, cause difficulties in quantifying soft biomarkers. To address the problems, we first aim to understand and model the underlying photophysics of fluorescence decay curves. For this purpose, we provide a set of mathematical functions, called "life models", fittable with the real temporal recordings of histogram of photon counts. For each model, an equivalent electrical circuit, called a "life circuit", is derived for explaining the whole process. In confocal endomicroscopy, the components of excitation laser, specimen, and fluorescence-emission signal as the histogram of photon counts are modelled by a power source, network of resistor-inductor-capacitor circuitry, and multimetre, respectively. We then design a novel pixel-level temporal classification algorithm, called a "fit-flexible approach", where qualities of "intensity", "fall-time", and "life profile" are identified for each point. A model selection mechanism is used at each pixel to flexibly choose the best representative life model based on a proposed Misfit-percent metric. A two-dimensional arrangement of the quantified information detects some kind of structural information. This approach showed a potential of separating microbeads from lung tissue, distinguishing the tri-sensing from conventional methods. We alleviated by 7% the error of the Misfit-percent for recovering the histograms on real samples than the best state-of-the-art competitor. Codes are available online.

High-speed wide-field fluorescence microscopy has the potential to capture biological processes with exceptional spatiotemporal resolution. However, conventional cameras suffer from low signal-to-noise ratio at high frame rates, limiting their ability to detect faint fluorescent events. Here, we introduce an image sensor where each pixel has individually programmable sampling speed and phase, so that pixels can be arranged to simultaneously sample at high speed with a high signal-to-noise ratio. In high-speed voltage imaging experiments, our image sensor significantly increases the output signal-to-noise ratio compared to a low-noise scientific CMOS camera (~2-3 folds). This signal-to-noise ratio gain enables the detection of weak neuronal action potentials and subthreshold activities missed by the standard scientific CMOS cameras. Our camera with flexible pixel exposure configurations offers versatile sampling strategies to improve signal quality in various experimental conditions., (© 2024. The Author(s).)

Multicellular systems grow over the course of weeks from single cells to tissues or even full organisms, making live imaging challenging. To bridge spatiotemporal scales, we present an open-top dual-view and dual-illumination light-sheet microscope dedicated to live imaging of large specimens at single-cell resolution. The configuration of objectives together with a customizable multiwell mounting system combines dual view with high-throughput multiposition imaging. We use this microscope to image a wide variety of samples and highlight its capabilities to gain quantitative single-cell information in large specimens such as mature intestinal organoids and gastruloids., (© 2024. The Author(s).)

Endo-microscopy is crucial for real-time 3D visualization of internal tissues and subcellular structures. Conventional methods rely on axial movement of optical components for precise focus adjustment, limiting miniaturization and complicating procedures. Meta-device, composed of artificial nanostructures, is an emerging optical flat device that can freely manipulate the phase and amplitude of light. Here, an intelligent fluorescence endo-microscope is developed based on varifocal meta-lens and deep learning (DL). The breakthrough enables in vivo 3D imaging of mouse brains, where varifocal meta-lens focal length adjusts through relative rotation angle. The system offers key advantages such as invariant magnification, a large field-of-view, and optical sectioning at a maximum focal length tuning range of ≈2 mm with 3 µm lateral resolution. Using a DL network, image acquisition time and system complexity are significantly reduced, and in vivo high-resolution brain images of detailed vessels and surrounding perivascular space are clearly observed within 0.1 s (≈50 times faster). The approach will benefit various surgical procedures, such as gastrointestinal biopsies, neural imaging, brain surgery, etc., (© 2024 The Authors. Advanced Science published by Wiley‐VCH GmbH.)

The mitochondrial translocases of the outer membrane (TOM) and of the inner membrane (TIM) act together to facilitate the import of nuclear-encoded proteins across the mitochondrial membranes. Stimulated Emission Depletion (STED) super-resolution microscopy enables the in situ imaging of such complexes in single cells at sub-diffraction resolution. STED microscopy requires only conventional sample preparation techniques and provides super-resolved raw data without the need for further image processing. In this chapter, we provide a detailed example protocol for STED microscopy of TOM20 and mitochondrial DNA in fixed mammalian cells. The protocol includes instructions on sample preparation for immunolabeling, including cell line selection, fixation, permeabilization, blocking, labeling and mounting, but also recommendations for sample and microscope performance evaluation. The protocol is supplemented by considerations on key factors that influence the quality of the final image and also includes some considerations for the analysis of the acquired images. While the protocol described here is aimed at imaging TOM20 and DNA, it contains all the information for an immediate adaptation to other cellular targets., (Copyright © 2024. Published by Elsevier Inc.)