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Wide field-of-view (FOV) and high-resolution imaging requires microscopy modalities to have large space-bandwidth products. Lensfree on-chip microscopy decouples resolution from FOV and can achieve a space-bandwidth product greater than one billion under unit magnification using state-of-the-art opto-electronic sensor chips and pixel super-resolution techniques. However, using vertical illumination, the effective numerical aperture (NA) that can be achieved with an on-chip microscope is limited by a poor signal-to-noise ratio (SNR) at high spatial frequencies and imaging artifacts that arise as a result of the relatively narrow acceptance angles of the sensor's pixels. Here, we report, for the first time, a synthetic aperture-based on-chip microscope in which the illumination angle is scanned across the surface of a dome to increase the effective NA of the reconstructed lensfree image to 1.4, achieving e.g., ∼250-nm resolution at 700-nm wavelength under unit magnification. This synthetic aperture approach not only represents the largest NA achieved to date using an on-chip microscope but also enables color imaging of connected tissue samples, such as pathology slides, by achieving robust phase recovery without the need for multi-height scanning or any prior information about the sample. To validate the effectiveness of this synthetic aperture-based, partially coherent, holographic on-chip microscope, we have successfully imaged color-stained cancer tissue slides as well as unstained Papanicolaou smears across a very large FOV of 20.5 mm 2 . This compact on-chip microscope based on a synthetic aperture approach could be useful for various applications in medicine, physical sciences and engineering that demand high-resolution wide-field imaging. Imaging: on-chip microscopy An on-chip microscope that offers both a high-resolution and a wide field of view looks set to benefit the biological and physical sciences. The lensfree imaging device, developed by researchers at the University of California at Los Angeles, CA, USA, makes use a synthetic aperture approach to provide a very large effective numerical aperture of 1.4 over a field of view of >20 mm 2 ; this is a much larger numerical aperture than previous lensfree approaches had realized (<0.9). Consequently, very high spatial resolution (for example, 250 nm at a wavelength of 700 nm) was achieved. By illuminating samples with light of three different wavelengths (470 nm, 532 nm and 632 nm), the researchers also obtained lens-free color images of samples such as breast cancer tissue.

Heart rate is under the precise control of the autonomic nervous system. However, the wiring of peripheral neural circuits that regulate heart rate is poorly understood. Here, we develop a clearing-imaging-analysis pipeline to visualize innervation of intact hearts in 3D and employed a multi-technique approach to map parasympathetic and sympathetic neural circuits that control heart rate in mice. We identify cholinergic neurons and noradrenergic neurons in an intrinsic cardiac ganglion and the stellate ganglia, respectively, that project to the sinoatrial node. We also report that the heart rate response to optogenetic versus electrical stimulation of the vagus nerve displays different temporal characteristics and that vagal afferents enhance parasympathetic and reduce sympathetic tone to the heart via central mechanisms. Our findings provide new insights into neural regulation of heart rate, and our methodology to study cardiac circuits can be readily used to interrogate neural control of other visceral organs. The wiring of peripheral neural circuits that regulate heart rate is poorly understood. In this study, authors used tissue clearing for high-resolution characterization of nerves in the heart in 3D and transgenic and novel viral vector approaches to identify peripheral parasympathetic and sympathetic neuronal populations involved in heart rate control in mice.

This protocol describes how to fix, embed, clear and stain excised organs or whole organisms to create optically transparent samples. This versatile protocol is able to process a wide range of sample types for high-resolution imaging. To facilitate fine-scale phenotyping of whole specimens, we describe here a set of tissue fixation-embedding, detergent-clearing and staining protocols that can be used to transform excised organs and whole organisms into optically transparent samples within 1–2 weeks without compromising their cellular architecture or endogenous fluorescence. PACT (passive CLARITY technique) and PARS (perfusion-assisted agent release in situ ) use tissue-hydrogel hybrids to stabilize tissue biomolecules during selective lipid extraction, resulting in enhanced clearing efficiency and sample integrity. Furthermore, the macromolecule permeability of PACT- and PARS-processed tissue hybrids supports the diffusion of immunolabels throughout intact tissue, whereas RIMS (refractive index matching solution) grants high-resolution imaging at depth by further reducing light scattering in cleared and uncleared samples alike. These methods are adaptable to difficult-to-image tissues, such as bone (PACT-deCAL), and to magnified single-cell visualization (ePACT). Together, these protocols and solutions enable phenotyping of subcellular components and tracing cellular connectivity in intact biological networks.

Recombinant adeno-associated viruses (rAAVs) are efficient gene delivery vectors via intravenous delivery; however, natural serotypes display a finite set of tropisms. To expand their utility, we evolved AAV capsids to efficiently transduce specific cell types in adult mouse brains. Building upon our Cre-recombination-based AAV targeted evolution (CREATE) platform, we developed Multiplexed-CREATE (M-CREATE) to identify variants of interest in a given selection landscape through multiple positive and negative selection criteria. M-CREATE incorporates next-generation sequencing, synthetic library generation and a dedicated analysis pipeline. We have identified capsid variants that can transduce the central nervous system broadly, exhibit bias toward vascular cells and astrocytes, target neurons with greater specificity or cross the blood–brain barrier across diverse murine strains. Collectively, the M-CREATE methodology accelerates the discovery of capsids for use in neuroscience and gene-therapy applications. M-CREATE is an in vivo screening strategy for identifying recombinant AAVs with desired tropism. The approach involves both positive and negative selection and yields vectors with diversified cell-type tropism that can cross the blood–brain barrier in adult mice across strains when delivered intravenously.

Based on partially-coherent digital in-line holography, we report a field-portable microscope that can render lensfree colour images over a wide field-of-view of e.g., >20 mm(2). This computational holographic microscope weighs less than 145 grams with dimensions smaller than 17×6×5 cm, making it especially suitable for field settings and point-of-care use. In this lensfree imaging design, we merged a colorization algorithm with a source shifting based multi-height pixel super-resolution technique to mitigate 'rainbow' like colour artefacts that are typical in holographic imaging. This image processing scheme is based on transforming the colour components of an RGB image into YUV colour space, which separates colour information from brightness component of an image. The resolution of our super-resolution colour microscope was characterized using a USAF test chart to confirm sub-micron spatial resolution, even for reconstructions that employ multi-height phase recovery to handle dense and connected objects. To further demonstrate the performance of this colour microscope Papanicolaou (Pap) smears were also successfully imaged. This field-portable and wide-field computational colour microscope could be useful for tele-medicine applications in resource poor settings.

Pixel-size limitation of lensfree on-chip microscopy can be circumvented by utilizing pixel-super-resolution techniques to synthesize a smaller effective pixel, improving the resolution. Here we report that by using the two-dimensional pixel-function of an image sensor-array as an input to lensfree image reconstruction, pixel-super-resolution can improve the numerical aperture of the reconstructed image by ~3 fold compared to a raw lensfree image. This improvement was confirmed using two different sensor-arrays that significantly vary in their pixel-sizes, circuit architectures and digital/optical readout mechanisms, empirically pointing to roughly the same space-bandwidth improvement factor regardless of the sensor-array employed in our set-up. Furthermore, such a pixel-count increase also renders our on-chip microscope into a Giga-pixel imager, where an effective pixel count of ~1.6–2.5 billion can be obtained with different sensors. Finally, using an ultra-violet light-emitting-diode, this platform resolves 225 nm grating lines and can be useful for wide-field on-chip imaging of nano-scale objects, e.g., multi-walled-carbon-nanotubes.

The ability to automatically detect and classify populations of cells in tissue sections is paramount in a wide variety of applications ranging from developmental biology to pathology. Although deep learning algorithms are widely applied to microscopy data, they typically focus on segmentation which requires extensive training and labor-intensive annotation. Here, we utilized object detection networks (neural networks) to detect and classify targets in complex microscopy images, while simplifying data annotation. To this end, we used a RetinaNet model to classify genetically labeled neurons and glia in the brains of Mosaic Analysis with Double Markers (MADM) mice. Our initial RetinaNet-based model achieved an average precision of 0.90 across six classes of cells differentiated by MADM reporter expression and their phenotype (neuron or glia). However, we found that a single RetinaNet model often failed when encountering dense and saturated glial clusters, which show high variability in their shape and fluorophore densities compared to neurons. To overcome this, we introduced a second RetinaNet model dedicated to the detection of glia clusters. Merging the predictions of the two computational models significantly improved the automated cell counting of glial clusters. The proposed cell detection workflow will be instrumental in quantitative analysis of the spatial organization of cellular populations, which is applicable not only to preparations in neuroscience studies, but also to any tissue preparation containing labeled populations of cells.

Introduction: One major obstacle in validating drugs for the treatment or prevention of hearing loss is the limited data available on the distribution and concentration of drugs in the human inner ear. Although small animal models offer some insights into inner ear pharmacokinetics, their smaller organ size and different barrier (round window membrane) permeabilities compared to humans can complicate study interpretation. Therefore, developing a reliable large animal model for inner ear drug delivery is crucial. The inner and middle ear anatomy of domestic pigs closely resembles that of humans, making them promising candidates for studying inner ear pharmacokinetics. However, unlike humans, the anatomical orientation and tortuosity of the porcine external ear canal frustrates local drug delivery to the inner ear. Methods: In this study, we developed a surgical technique to access the tympanic membrane of pigs. To assess hearing pre- and post-surgery, auditory brainstem responses to click and pure tones were measured. Additionally, we performed 3D segmentation of the porcine inner ear images and used this data to simulate the diffusion of dexamethasone within the inner ear through fluid simulation software (FluidSim). Results: We have successfully delivered dexamethasone and dexamethasone sodium phosphate to the porcine inner ear via the intratympanic injection. The recorded auditory brainstem measurements revealed no adverse effects on hearing thresholds attributable to the surgery. We have also simulated the diffusion rates for dexamethasone and dexamethasone sodium phosphate into the porcine inner ear and confirmed the accuracy of the simulations using in-vivo data. Discussion: We have developed and characterized a method for conducting pharmacokinetic studies of the inner ear using pigs. This animal model closely mirrors the size of the human cochlea and the thickness of its barriers. The diffusion time and drug concentrations we reported align closely with the limited data available from human studies. Therefore, we have demonstrated the potential of using pigs as a large animal model for studying inner ear pharmacokinetics.

We report Giga-pixel lensfree holographic microscopy and tomography using color sensor-arrays such as CMOS imagers that exhibit Bayer color filter patterns. Without physically removing these color filters coated on the sensor chip, we synthesize pixel super-resolved lensfree holograms, which are then reconstructed to achieve ~350 nm lateral resolution, corresponding to a numerical aperture of ~0.8, across a field-of-view of ~20.5 mm(2). This constitutes a digital image with ~0.7 Billion effective pixels in both amplitude and phase channels (i.e., ~1.4 Giga-pixels total). Furthermore, by changing the illumination angle (e.g., ± 50°) and scanning a partially-coherent light source across two orthogonal axes, super-resolved images of the same specimen from different viewing angles are created, which are then digitally combined to synthesize tomographic images of the object. Using this dual-axis lensfree tomographic imager running on a color sensor-chip, we achieve a 3D spatial resolution of ~0.35 µm × 0.35 µm × ~2 µm, in x, y and z, respectively, creating an effective voxel size of ~0.03 µm(3) across a sample volume of ~5 mm(3), which is equivalent to >150 Billion voxels. We demonstrate the proof-of-concept of this lensfree optical tomographic microscopy platform on a color CMOS image sensor by creating tomograms of micro-particles as well as a wild-type C. elegans nematode.

Mammalian organs comprise a variety of cells that interact with each other and have distinct biological roles. Access to evaluate and perturb intact biological systems at the cellular and molecular levels is essential to fully understand their functioning in normal and diseased conditions, yet technical limitations have constrained most research to small pieces of tissue. Tissue clearing and optogenetics can help overcome this hurdle: tissue clearing affords optical interrogation of whole organs at the molecular level, and optogenetics enables the scalable control and measurement of cellular activity with light. In this Q&A, we delineate recent advances and practical challenges associated with these two techniques when applied body-wide.