A Simple and Fast Optical Clearing Method for Whole-Mount Fluorescence In Situ Hybridization (FISH) Imaging.

We report a single-step optical clearing method that is compatible with RNA fluorescence in situ hybridization (FISH) imaging. We previously demonstrated microscopy imaging with immunohistochemistry and genetic reporters using a technique called lipid-preserving refractive index matching for prolonged imaging depth (LIMPID). Our protocol reliably produces high-resolution three-dimensional (3D) images with minimal aberrations using high magnification objectives, captures large field-of-view images of whole-mount tissues, and supports co-labeling with antibody and FISH probes.

Digital labeling for 3D histology: segmenting blood vessels without a vascular contrast agent using deep learning.

Recent advances in optical tissue clearing and three-dimensional (3D) fluorescence microscopy have enabled high resolution imaging of intact tissues. Using simply prepared samples, we demonstrate here "digital labeling," a method to segment blood vessels in 3D volumes solely based on the autofluorescence signal and a nuclei stain (DAPI). We trained a deep-learning neural network based on the U-net architecture using a regression loss instead of a commonly used segmentation loss to achieve better detection of small vessels.

Two single-axis scanners make a virtual gimbaled scanner.

A 2D scan generated from two single-axis scanning mirrors often has the beam steered about two distant axes that lead to scan artifacts, such as displacement jitters, telecentric errors, and spot variations. Previously, this problem has been addressed with complicated optical and mechanical designs, such as 4f relays and gimbaled mechanics, which ultimately limit the performance of the system. Here, we show that two single-axis scanners alone can produce a 2D scanning pattern nearly identical to a single-pivot gimbaled scanner through an apparently previously undiscovered simple geometry.

Selective Infrared Neural Inhibition Can Be Reproduced by Resistive Heating.

Objectives: Small-diameter afferent axons carry various sensory signals that are critical for vital physiological conditions but sometimes contribute to pathologies. Infrared (IR) neural inhibition (INI) can induce selective heat block of small-diameter axons, which holds potential for translational applications such as pain management. Previous research suggested that IR-heating-induced acceleration of voltage-gated potassium channel kinetics is the mechanism for INI.

Analysis of the distribution of vagal afferent projections from different peripheral organs to the nucleus of the solitary tract in rats.

Anatomical tracing studies examining the vagal system can conflate details of sensory afferent and motor efferent neurons. Here, we used a serotype of adeno-associated virus that transports retrogradely and exhibits selective tropism for vagal afferents, to map their soma location and central termination sites within the nucleus of the solitary tract (NTS). We examined the vagal sensory afferents innervating the trachea, duodenum, stomach, or heart, and in some animals, from two organs concurrently.

Imaging peripheral nerve micro-anatomy with MUSE, 2D and 3D approaches.

Understanding peripheral nerve micro-anatomy can assist in the development of safe and effective neuromodulation devices. However, current approaches for imaging nerve morphology at the fiber level are either cumbersome, require substantial instrumentation, have a limited volume of view, or are limited in resolution/contrast. We present alternative methods based on MUSE (Microscopy with Ultraviolet Surface Excitation) imaging to investigate peripheral nerve morphology, both in 2D and 3D.

3D imaging of the vagus nerve fascicular anatomy with cryo-imaging and UV excitation.

Vagus nerve stimulation (VNS) is a method to treat drug-resistant epilepsy and depression, but therapeutic outcomes are often not ideal. Newer electrode designs such as intra-fascicular electrodes offer potential improvements in reducing off-target effects but require a detailed understanding of the fascicular anatomy of the vagus nerve. We have adapted a section-and-image technique, cryo-imaging, with UV excitation to visualize fascicles along the length of the vagus nerve.

Slide Over: Advances in Slide-Free Optical Microscopy as Drivers of Diagnostic Pathology.

Conventional analysis using clinical histopathology is based on bright-field microscopy of thinly sliced tissue specimens. Although bright-field microscopy is a simple and robust method of examining microscope slides, the preparation of the slides needed is a lengthy and labor-intensive process. Slide-free histopathology, however, uses direct imaging of intact, minimally processed tissue samples using advanced optical-imaging systems, bypassing the extended workflow now required for the preparation of tissue sections.

CompassLSM: axially swept light-sheet microscopy made simple.

Axially swept light-sheet microscopy (ASLM) is an effective method of generating a uniform light sheet across a large field of view (FOV). However, current ASLM designs are more complicated than conventional light-sheet systems, limiting their adaptation in less experienced labs. By eliminating difficult-to-align components and reducing the total number of components, we show that high-performance ASLM can be accomplished much simpler than existing designs, requiring less expertise and effort to construct, align, and operate.

Development of an arteriolar niche and self-renewal of breast cancer stem cells by lysophosphatidic acid/protein kinase D signaling.

Breast cancer stem cells (BCSCs) are essential for cancer growth, metastasis and recurrence. The regulatory mechanisms of BCSC interactions with the vascular niche within the tumor microenvironment (TME) and their self-renewal are currently under extensive investigation. We have demonstrated the existence of an arteriolar niche in the TME of human BC tissues.

Pocket MUSE: an affordable, versatile and high-performance fluorescence microscope using a smartphone.

Smartphone microscopes can be useful tools for a broad range of imaging applications. This manuscript demonstrates the first practical implementation of Microscopy with Ultraviolet Surface Excitation (MUSE) in a compact smartphone microscope called Pocket MUSE, resulting in a remarkably effective design. Fabricated with parts from consumer electronics that are readily available at low cost, the small optical module attaches directly over the rear lens in a smartphone.

Three-dimensional alignment of microvasculature and cardiomyocytes in the developing ventricle.

While major coronary artery development and pathologies affecting them have been extensively studied, understanding the development and organization of the coronary microvasculature beyond the earliest developmental stages requires new tools. Without techniques to image the coronary microvasculature over the whole heart, it is likely we are underestimating the microvasculature's impact on normal development and diseases. We present a new imaging and analysis toolset to visualize the coronary microvasculature in intact embryonic hearts and quantify vessel organization.

Heterogeneity of Vascular Endothelial Cells, De Novo Arteriogenesis and Therapeutic Implications in Pancreatic Neuroendocrine Tumors.

Arteriogenesis supplies oxygen and nutrients in the tumor microenvironment (TME), which may play an important role in tumor growth and metastasis. Pancreatic neuroendocrine tumors (pNETs) are the second most common pancreatic malignancy and are frequently metastatic on presentation. Nearly a third of pNETs secrete bioactive substances causing debilitating symptoms.

SLIME: robust, high-speed 3D microvascular mapping.

Three dimensional (3D) microvascular imaging of cubic millimeter to centimeter size volumes often requires much time and expensive instruments. By combining optical clearing with a novel scatter-based optical coherence tomography (OCT) contrast agent, we have greatly extended OCT imaging depth in excised tissues while maintaining a simple and low cost approach that does not require in-depth OCT knowledge. The new method enables fast 3D microvascular mapping in large tissue volumes, providing a promising tool for investigating organ level microvascular abnormalities in large cohorts.

Method to extract minimally damaged collagen fibrils from tendon.

A new method is presented to extract collagen fibrils from mammalian tendon tissue. Mammalian tendons are treated with a trypsin-based extraction medium and gently separated with tweezers in an aqueous solution. Collagen fibrils released in the solution are imaged using both dark-field light microscopy and scanning electron microscopy.

Tension tests on mammalian collagen fibrils.

A brief overview of isolated collagen fibril mechanics testing is followed by presentation of the first results testing fibrils isolated from load-bearing mammalian tendons using a microelectromechanical systems platform. The in vitro modulus (326 ± 112 MPa) and fracture stress (71 ± 23 MPa) are shown to be lower than previously measured on fibrils extracted from sea cucumber dermis and tested with the same technique. Scanning electron microscope images show the fibrils can fail with a mechanism that involves circumferential rupture, whereas the core of the fibril stays at least partially intact.