Smartphone-based optical sectioning (SOS) microscopy with a telecentric design for fluorescence imaging.

We propose a smartphone-based optical sectioning (SOS) microscope based on the HiLo technique, with a single smartphone replacing a high-cost illumination source and a camera sensor. We built our SOS with off-the-shelf optical, mechanical cage systems with 3D-printed adapters to seamlessly integrate the smartphone with the SOS main body. The liquid light guide can be integrated with the adapter, guiding the smartphone's LED light to the digital mirror device (DMD) with neglectable loss. We used an electrically tuneable lens (ETL) instead of a mechanical translation stage to realise low-cost axial scanning. The ETL was conjugated to the objective lens's back pupil plane (BPP) to construct a telecentric design by a 4f configuration to maintain the lateral magnification for different axial positions. SOS has a 571.5 µm telecentric scanning range and an 11.7 µm axial resolution. The broadband smartphone LED torch can effectively excite fluorescent polystyrene (PS) beads. We successfully used SOS for high-contrast fluorescent PS beads imaging with different wavelengths and optical sectioning imaging of multilayer fluorescent PS beads. To our knowledge, the proposed SOS is the first smartphone-based HiLo optical sectioning microscopy (£1965), which can save around £7035 compared with a traditional HiLo system (£9000). It is a powerful tool for biomedical research in resource-limited areas.

In Vivo Intelligent Fluorescence Endo-Microscopy by Varifocal Meta-Device and Deep Learning.

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.

Multiplane HiLo microscopy with speckle illumination and non-local means denoising.

Significance: HiLo microscopy synthesizes an optically sectioned image from two images, one obtained with uniform and another with patterned illumination, such as laser speckle. Speckle-based HiLo has the advantage of being robust to aberrations but is susceptible to residual speckle noise that is difficult to control. We present a computational method to reduce this residual noise without undermining resolution. In addition, we improve the versatility of HiLo microscopy by enabling simultaneous multiplane imaging (here nine planes). Aim: Our goal is to perform fast, high-contrast, multiplane imaging with a conventional camera-based fluorescence microscope. Approach: Multiplane HiLo imaging is achieved with the use of a single camera and z-splitter prism. Speckle noise reduction is based on the application of a non-local means (NLM) denoising method to perform ensemble averaging of speckle grains. Results: We demonstrate the capabilities of multiplane HiLo with NLM denoising both with synthesized data and by imaging cardiac and brain activity in zebrafish larvae at 40 Hz frame rates. Conclusions: Multiplane HiLo microscopy aided by NLM denoising provides a simple tool for fast optically sectioned volumetric imaging that can be of general utility for fluorescence imaging applications.

Bessel Beams in Ophthalmology: A Review.

The achievable resolution of a conventional imaging system is inevitably limited due to diffraction. Dealing with precise imaging in scattering media, such as in the case of biomedical imaging, is even more difficult owing to the weak signal-to-noise ratios. Recent developments in non-diffractive beams such as Bessel beams, Airy beams, vortex beams, and Mathieu beams have paved the way to tackle some of these challenges. This review specifically focuses on non-diffractive Bessel beams for ophthalmological applications. The theoretical foundation of the non-diffractive Bessel beam is discussed first followed by a review of various ophthalmological applications utilizing Bessel beams. The advantages and disadvantages of these techniques in comparison to those of existing state-of-the-art ophthalmological systems are discussed. The review concludes with an overview of the current developments and the future perspectives of non-diffractive beams in ophthalmology.

Whole-brain Optical Imaging: A Powerful Tool for Precise Brain Mapping at the Mesoscopic Level.

The mammalian brain is a highly complex network that consists of millions to billions of densely-interconnected neurons. Precise dissection of neural circuits at the mesoscopic level can provide important structural information for understanding the brain. Optical approaches can achieve submicron lateral resolution and achieve "optical sectioning" by a variety of means, which has the natural advantage of allowing the observation of neural circuits at the mesoscopic level. Automated whole-brain optical imaging methods based on tissue clearing or histological sectioning surpass the limitation of optical imaging depth in biological tissues and can provide delicate structural information in a large volume of tissues. Combined with various fluorescent labeling techniques, whole-brain optical imaging methods have shown great potential in the brain-wide quantitative profiling of cells, circuits, and blood vessels. In this review, we summarize the principles and implementations of various whole-brain optical imaging methods and provide some concepts regarding their future development.

Minimal resin embedding of SBF-SEM samples reduces charging and facilitates finding a surface-linked region of interest.

BACKGROUND: For decoding the mechanism of how cells and organs function information on their ultrastructure is essential. High-resolution 3D imaging has revolutionized morphology. Serial block face scanning electron microscopy (SBF-SEM) offers non-laborious, automated imaging in 3D of up to ~ 1 mm3 large biological objects at nanometer-scale resolution. For many samples there are obstacles. Quality imaging is often hampered by charging effects, which originate in the nonconductive resin used for embedding. Especially, if the imaged region of interest (ROI) includes the surface of the sample and neighbours the empty resin, which insulates the object. This extra resin also obscures the sample's morphology, thus making navigation to the ROI difficult. RESULTS: Using the example of small arthropods and a fish roe we describe a workflow to prepare samples for SBF-SEM using the minimal resin (MR) embedding method. We show that for imaging of surface structures this simple approach conveniently tackles and solves both of the two major problems-charging and ROI localization-that complicate imaging of SBF-SEM samples embedded in an excess of overlying resin. As the surface ROI is not masked by the resin, samples can be precisely trimmed before they are placed into the imaging chamber. The initial approaching step is fast and easy. No extra trimming inside the microscope is necessary. Importantly, charging is absent or greatly reduced meaning that imaging can be accomplished under good vacuum conditions, typically at the optimal high vacuum. This leads to better resolution, better signal to noise ratio, and faster image acquisition. CONCLUSIONS: In MR embedded samples charging is minimized and ROI easily targeted. MR embedding does not require any special equipment or skills. It saves effort, microscope time and eventually leads to high quality data. Studies on surface-linked ROIs, or any samples normally surrounded by the excess of resin, would benefit from adopting the technique.

Application of fluorescence micro-optical sectioning tomography in the cerebrovasculature and applicable vascular labeling methods.

Fluorescence micro-optical sectioning tomography (fMOST) is a three-dimensional (3d) imaging method at the mesoscopic level. The whole-brain of mice can be imaged at a high resolution of 0.32 × 0.32 × 1.00 µm3. It is useful for revealing the fine morphology of intact organ tissue, even for positioning the single vessel connected with a complicated vascular network across different brain regions in the whole mouse brain. Featuring its 3d visualization of whole-brain cross-scale connections, fMOST has a vast potential to decipher brain function and diseases. This article begins with the background of fMOST technology including a widespread 3D imaging methods comparison and the basic technical principal illustration, followed by the application of fMOST in cerebrovascular research and relevant vascular labeling techniques applicable to different scenarios.

Nondestructive 3D Pathology with Light-Sheet Fluorescence Microscopy for Translational Research and Clinical Assays.

In recent years, there has been a revived appreciation for the importance of spatial context and morphological phenotypes for both understanding disease progression and guiding treatment decisions. Compared with conventional 2D histopathology, which is the current gold standard of medical diagnostics, nondestructive 3D pathology offers researchers and clinicians the ability to visualize orders of magnitude more tissue within their natural volumetric context. This has been enabled by rapid advances in tissue-preparation methods, high-throughput 3D microscopy instrumentation, and computational tools for processing these massive feature-rich data sets. Here, we provide a brief overview of many of these technical advances along with remaining challenges to be overcome. We also speculate on the future of 3D pathology as applied in translational investigations, preclinical drug development, and clinical decision-support assays.

Axial resolution and imaging contrast enhancement in inverted light-sheet microscopy by natural illumination modulation.

Inverted light-sheet microscopy (ILSM) is widely employed for fast large-volume imaging of biological tissue. However, the scattering especially in an uncleared sample, and the divergent propagation of the illumination beam lead to a trade-off between axial resolution and imaging depth. Herein, we propose naturally modulated ILSM (NM-ILSM) as a technique to improve axial resolution while simultaneously maintaining the wide field-of-view (FOV), and enhancing imaging contrast via background suppression. Theoretical derivations, simulations, and experimental imaging demonstrate 15% axial resolution increases, and fivefold greater image contrast compared with conventional ILSM. Therefore, NM-ILSM allows convenient imaging quality improvement for uncleared tissue and could extend the biological application scope of ILSM.

Mapping the Function of Whole-Brain Projection at the Single Neuron Level.

Axonal projection conveys neural information. The divergent and diverse projections of individual neurons imply the complexity of information flow. It is necessary to investigate the relationship between the projection and functional information at the single neuron level for understanding the rules of neural circuit assembly, but a gap remains due to a lack of methods to map the function to whole-brain projection. Here an approach is developed to bridge two-photon calcium imaging in vivo with high-resolution whole-brain imaging based on sparse labeling with the genetically encoded calcium indicator GCaMP6. Reliable whole-brain projections are captured by the high-definition fluorescent micro-optical sectioning tomography (HD-fMOST). A cross-modality cell matching is performed and the functional annotation of whole-brain projection at the single-neuron level (FAWPS) is obtained. Applying it to the layer 2/3 (L2/3) neurons in mouse visual cortex, the relationship is investigated between functional preferences and axonal projection features. The functional preference of projection motifs and the correlation between axonal length in MOs and neuronal orientation selectivity, suggest that projection motif-defined neurons form a functionally specific information flow, and the projection strength in specific targets relates to the information clarity. This pipeline provides a new way to understand the principle of neuronal information transmission.

Brain-wide mapping of c-Fos expression with fluorescence micro-optical sectioning tomography in a chronic sleep deprivation mouse model.

Chronic sleep deprivation (SD) is a common problem for humans and can lead to many deleterious effects, including depression, anxiety, stroke, permanent cognitive deficits, stress, and other physiological diseases. It is vital to acquire information about the relevant neural activities at the whole-brain level to systematically explore the mechanisms of brain dysfunction related to SD. Expression of the immediate-early gene (IEG) Fos in the mouse brain has been widely used as a functional marker of brain activity in the field of neuroscience. However, most previous studies only analyzed the change of c-Fos in several specific brain regions using traditional research methods or in short-term SD model. Here, we applied c-Fos mapping through the fluorescence micro-optical sectioning tomography (fMOST) technique and AAV-PHP.eB to comprehensive analysis the state of cumulative activation across the whole brain in a mouse model of chronic SD. The chronic rapid eyes movement (REM) SD model was induced by moving mice to a separate holding area filled with water. The experimental period lasted for 6 h per day. The results showed that after 14 days of SD, the mice displayed anxiety-like behaviors in open field test and elevated plus maze test, and displayed depression-like behaviors in tail suspension test and the sucrose preference test. The c-Fos + cells were detected in a maximum of 230 brain regions. SD-induced stress model evoked c-Fos expression in several brain regions compared to the control group. In particular, the isocortex-cerebral cortex plate area, including the retrosplenial, anterior cingulate, agranular insular, gustatory, and parasubiculum, appear to be the most sensitive regions after chronic REM SD.

High-resolution 3D demonstration of regional heterogeneity in the glymphatic system.

Accumulating evidence indicates that the glymphatic system has a critical role in maintaining brain homeostasis. However, the detailed anatomy of the glymphatic pathway is not well understood, mostly due to a lack of high spatial resolution 3D visualization. In this study, a fluorescence micro-optical sectioning tomography (fMOST) was used to characterize the glymphatic architecture in the mouse brain. At 30 and 120 min after intracisternal infusion with fluorescent dextran (Dex-3), lectin was injected to stain the cerebral vasculature. Using fMOST, a high-resolution 3D dataset of the brain-wide distribution of Dex-3 was acquired. Combined with fluorescence microscopy and microplate array, the heterogeneous glymphatic flow and the preferential irrigated regions were identified. These cerebral regions containing large-caliber penetrating arteries and/or adjacent to the subarachnoid space had more robust CSF flow compared to other regions. Moreover, the major glymphatic vessels for CSF influx and fluid efflux in the entire brain were shown in 3D. This study demonstrates the regional heterogeneity in the glymphatic system and provides an anatomical resource for further investigation of the glymphatic function.

Design and Development of a Bimodal Optical Instrument for Simultaneous Vibrational Spectroscopy Measurements.

Vibrational spectroscopy techniques are widely used in analytical chemistry, physics and biology. The most prominent techniques are Raman and Fourier-transform infrared spectroscopy (FTIR). Combining both techniques delivers complementary information of the test sample. We present the design, construction, and calibration of a novel bimodal spectroscopy system featuring both Raman and infrared measurements simultaneously on the same sample without mutual interference. The optomechanical design provides a modular flexible system for solid and liquid samples and different configurations for Raman. As a novel feature, the Raman module can be operated off-axis for optical sectioning. The calibrated system demonstrates high sensitivity, precision, and resolution for simultaneous operation of both techniques and shows excellent calibration curves with coefficients of determination greater than 0.96. We demonstrate the ability to simultaneously measure Raman and infrared spectra of complex biological material using bovine serum albumin. The performance competes with commercial systems; moreover, it presents the additional advantage of simultaneously operating Raman and infrared techniques. To the best of our knowledge, it is the first demonstration of a combined Raman-infrared system that can analyze the same sample volume and obtain optically sectioned Raman signals. Additionally, quantitative comparison of confocality of backscattering micro-Raman and off-axis Raman was performed for the first time.

A phasor-based approach to improve optical sectioning in any confocal microscope with a tunable pinhole.

Confocal fluorescence microscopy is a well-established imaging technique capable of generating thin optical sections of biological specimens. Optical sectioning in confocal microscopy is mainly determined by the size of the pinhole, a small aperture placed in front of a point detector. In principle, imaging with a closed pinhole provides the highest degree of optical sectioning. In practice, the dramatic reduction of signal-to-noise ratio (SNR) at smaller pinhole sizes makes challenging the use of pinhole sizes significantly smaller than 1 Airy Unit (AU). Here, we introduce a simple method to "virtually" perform confocal imaging at smaller pinhole sizes without the dramatic reduction of SNR. The method is based on the sequential acquisition of multiple confocal images acquired at different pinhole aperture sizes and image processing based on a phasor analysis. The implementation is conceptually similar to separation of photons by lifetime tuning (SPLIT), a technique that exploits the phasor analysis to achieve super-resolution, and for this reason we call this method SPLIT-pinhole (SPLIT-PIN). We show with simulated data that the SPLIT-PIN image can provide improved optical sectioning (i.e., virtually smaller pinhole size) but better SNR with respect to an image obtained with closed pinhole. For instance, two images acquired at 2 and 1 AU can be combined to obtain a SPLIT-PIN image with a virtual pinhole size of 0.2 AU but with better SNR. As an example of application to biological imaging, we show that SPLIT-PIN improves confocal imaging of the apical membrane in an in vitro model of the intestinal epithelium. RESEARCH HIGHLIGHTS: We describe a method to boost the optical sectioning power of any confocal microscope. The method is based on the sequential acquisition of multiple confocal images acquired at different pinhole aperture sizes. The resulting image series is analyzed using the phasor-based separation of photons by lifetime tuning (SPLIT) algorithm. The SPLIT-pinhole (SPLIT-PIN) method produces images with improved optical sectioning but preserved SNR. This is the first time that the phasor analysis and SPLIT algorithms are used to exploit the spatial information encoded in a tunable pinhole size and to improve optical sectioning of the confocal microscope.

High-Resolution Digital Panorama of Multiple Structures in Whole Brain of Alzheimer's Disease Mice.

Simultaneously visualizing Amyloid-ß (Aß) plaque with its surrounding brain structures at the subcellular level in the intact brain is essential for understanding the complex pathology of Alzheimer's disease, but is still rarely achieved due to the technical limitations. Combining the micro-optical sectioning tomography (MOST) system, whole-brain Nissl staining, and customized image processing workflow, we generated a whole-brain panorama of Alzheimer's disease mice without specific labeling. The workflow employed the steps that include virtual channel splitting, feature enhancement, iso-surface rendering, direct volume rendering, and feature fusion to extract and reconstruct the different signals with distinct gray values and morphologies. Taking advantage of this workflow, we found that the denser-distribution areas of Aß plaques appeared with relatively more somata and smaller vessels, but show a dissimilar distributing pattern with nerve tracts. In addition, the entorhinal cortex and adjacent subiculum regions present the highest density and biggest diameter of plaques. The neuronal processes in the vicinity of these Aß plaques showed significant structural alternation such as bending or abrupt branch ending. The capillaries inside or adjacent to the plaques were observed with abundant distorted micro-vessels and abrupt ending. Depicting Aß plaques, somata, nerve processes and tracts, and blood vessels simultaneously, this panorama enables us for the first time, to analyze how the Aß plaques interact with capillaries, somata, and processes at a submicron resolution of 3D whole-brain scale, which reveals potential pathological effects of Aß plaques from a new cross-scale view. Our approach opens a door to routine systematic studies of complex interactions among brain components in mouse models of Alzheimer's disease.

Optical sectioning of unlabeled samples using bright-field microscopy.

The bright-field (BF) optical microscope is a traditional bioimaging tool that has been recently tested for depth discrimination during evaluation of specimen morphology; however, existing approaches require dedicated instrumentation or extensive computer modeling. We report a direct method for three-dimensional (3D) imaging in BF microscopy, applicable to label-free samples, where we use Köhler illumination in the coherent regime and conventional digital image processing filters to achieve optical sectioning. By visualizing fungal, animal tissue, and plant samples and comparing with light-sheet fluorescence microscopy imaging, we demonstrate the accuracy and applicability of the method, showing how the standard microscope is an effective 3D imaging device.

Imaging plant tissues: advances and promising clearing practices.

The study of the organ structure of plants and understanding their physiological complexity requires 3D imaging with subcellular resolution. Most plant organs are highly opaque to light, and their study under optical sectioning microscopes is therefore difficult. In animals, many protocols have been developed to make organs transparent to light using clearing protocols (CPs). By contrast, clearing plant tissues is challenging because of the presence of fibers and pigments. We describe progress in the development of plant CPs over the past 20 years through a modified taxonomy of CPs based on their physical and optical parameters that affect tissue properties. We also discuss successful approaches that combine CPs with new microscopy methods and their future applications in plant science research.

Minimum-dose phase-contrast tomography by successive numerical optical sectioning employing the aberration-corrected STEM and a pixelated detector.

Aberration correction combined with a pixelated detector enable atomic-resolution phase-contrast imaging in the scanning transmission electron microscope (STEM) using all elastically scattered electrons within the illumination cone. The review describes this possibility in detail revisiting the image formation in the STEM on a fundamental quantum-mechanical treatment of electron scattering within the object and the effect of the lenses on the electron wave. Describing electron scattering by means of scattering amplitudes enables a straightforward derivation of a) the reciprocity theorem, b) the optical theorem of electron scattering, and c) the precise formulation of the image intensity distribution in the STEM for different modes of operation. The second part of the review describes in detail a novel method for obtaining pure phase-contrast images in the STEM using the integrated differential phase-contrast (IDPC) procedure. The incorporation of a chromatic (Cc) and spherically (Cs) corrected objective lens and a pixelated detector in the STEM combined with numerical through-focusing enables optical sectioning with atomic 3D resolution of thick objects with about the same dose as that for a 2D object, at least in principle. Numerical simulations of the IDPC transfer function and the point spread function for the focal plane and several reconstructed defocused planes demonstrate the feasibility of the method.

Deconvolution microscopy: A platform for rapid on-site evaluation of fine needle aspiration specimens that enables recovery of the sample.

CONTEXT: Rapid on-site evaluation (ROSE) optimises the performance of cytology, but requires skilled handling, and smearing can make the material unavailable for some ancillary tests. There is a need to facilitate ROSE without sacrificing part of the sample. OBJECTIVE: We evaluated the image quality of inexpensive deconvolution fluorescence microscopy for optically sectioning non-smeared fine needle aspiration (FNA) tissue fragments. DESIGN: A portion of residual material from 14 FNA samples was stained for 3 min in Hoechst 33342 and Sypro™ Red to label DNA and protein respectively, transferred to an imaging chamber, and imaged at 200× or 400× magnification at 1 micron intervals using a GE DeltaVision inverted fluorescence microscope. A deconvolution algorithm was applied to remove out-of-plane signal, and the resulting images were inverted and pseudocoloured to resemble H&E sections. Five cytopathologists blindly diagnosed 2 to 4 representative image stacks per case (total 70 evaluations), and later compared them to conventional epifluorescent images. RESULTS: Accurate definitive diagnoses were rendered in 45 (64%) of 70 total evaluations; equivocal diagnoses (atypical or suspicious) were made in 21 (30%) of the 70. There were two false positive and two false negative "definite" diagnoses in three cases (4/70; 6%). Cytopathologists preferred deconvolved images compared to raw images (P < 0.01). The imaged fragments were recovered and prepared into a ThinPrep or cell block without discernible alteration. CONCLUSIONS: Deconvolution improves image quality of FNA fragments compared to epifluorescence, often allowing definitive diagnosis while enabling the ROSE material to be subsequently triaged.

Using saturated absorption for superresolution laser scanning transmission microscopy.

We improved the three-dimensional spatial resolution of laser scanning transmission microscopy by exploiting the saturated absorption of dye molecules. The saturated absorption is induced by the high-intensity light irradiation and localises the signal within the centre of the focal spot. Our numerical calculation indicates that the spatial resolution in transmission imaging is significantly improved for both lateral and axial directions using nonlinear transmitted signals induced by saturated absorption. We experimentally demonstrated the improvement of the three-dimensional resolution by observing fine structures of stained rat kidney tissues, which were not able to be visualised by conventional laser scanning transmission microscopy.

Confocal laser scanning microscopy is a powerful technique for three-dimensional imaging to study structures in a specimen. The use of confocal pinhole provides three-dimensional spatial resolution in various types of optical microscopes, such as fluorescence, reflection and scattering. However, in transmission microscopy, the confocal pinhole cannot provide the same effect because the spatial information on the optical axial is not transferred in the imaging system. Therefore, the three-dimensional distribution of light absorbers cannot be observed by laser scanning transmission microscopy. In this paper, we propose the use of saturated absorption to image the three-dimensional distribution of light absorbers in a sample by laser scanning transmission microscopy. The saturated absorption is induced by the high-intensity light irradiation and occurs prominently at the centre of a focal spot. The information of the saturated absorption signal within the focal spot is transferred to the transmitted light, providing the capability of optical sectioning in transmission imaging. In our research, we theoretically and experimentally confirmed that light absorption by dye molecules is saturable at the high illumination intensity, and the saturated absorption signal can be extracted by harmonic demodulation. We obtained the images of a stained rat kidney tissue by selectively detecting the nonlinear transmission signals induced by saturable absorption of the dye molecules. We confirmed the high depth discrimination capability of our technique clearly visualised the fine structures in the specimen that cannot be observed by a conventional laser scanning absorption microscope.