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1.
J Biophotonics ; : e202400258, 2024 Oct 10.
Artigo em Inglês | MEDLINE | ID: mdl-39389582

RESUMO

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. We also custom-designed FISH probes for quail embryos, demonstrating the ease of fabricating probes for use with less common animal models. Furthermore, we show high-quality 3D images using a conventional fluorescence microscope, without using more advanced depth sectioning instruments such as confocal or light-sheet microscopy. For broader adoption, we simplified and optimized 3D-LIMPID-FISH to minimize the barrier to entry, and we provide a detailed protocol to aid users with navigating the thick and thin of 3D microscopy.

2.
Biomed Opt Express ; 14(6): 2416-2431, 2023 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-37342724

RESUMO

Recent advances in optical tissue clearing and three-dimensional (3D) fluorescence microscopy have enabled high resolution in situ 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. We achieved high vessel detection accuracy and obtained accurate vascular morphometrics such as vessel length density and orientation. In the future, such digital labeling approach could easily be transferred to other biological structures.

3.
Neuromodulation ; 26(8): 1757-1771, 2023 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-36707292

RESUMO

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. Therefore, we hypothesized that other heating methods, such as resistive heating (RH) in a cuff, could reproduce the selective inhibition observed in INI. MATERIALS AND METHODS: We conducted ex vivo nerve-heating experiments on pleural-abdominal connective nerves of Aplysia californica using both IR and RH. We fabricated a transparent silicone nerve cuff for simultaneous IR heating, RH, and temperature measurements. Temperature elevations (ΔT) on the nerve surface were recorded for both heating modalities, which were tested over a range of power levels that cover a similar ΔT range. We recorded electrically evoked compound action potentials (CAPs) and segmented them into fast and slow subcomponents on the basis of conduction velocity differences between the large and small-diameter axonal subpopulations. We calculated the normalized inhibition strength and inhibition selectivity index on the basis of the rectified area under the curve of each subpopulation. RESULTS: INI and RH showed a similar selective inhibition effect on CAP subcomponents for slow-conducting axons, confirmed by the inhibition probability vs ΔT dose-response curve based on approximately 2000 CAP measurements. The inhibition selectivity indexes of the two heating modalities were similar across six nerves. RH only required half the total electrical power required by INI to achieve a similar ΔT. SIGNIFICANCE: We show that selective INI can be reproduced by other heating modalities such as RH. RH, because of its high energy efficiency and simple design, can be a good candidate for future implantable neural interface designs.


Assuntos
Calefação , Condução Nervosa , Humanos , Condução Nervosa/fisiologia , Inibição Neural , Potenciais de Ação/fisiologia , Axônios/fisiologia
4.
J Comp Neurol ; 530(17): 3072-3103, 2022 12.
Artigo em Inglês | MEDLINE | ID: mdl-35988033

RESUMO

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. We observed no obvious somatotopy in the somata distribution within the nodose ganglion. The central termination patterns of afferents from different organs within the NTS overlap substantially. Convergence of vagal afferent inputs from different organs onto single NTS neurons is observed. Abdominal and thoracic afferents terminate throughout the NTS, including in the rostral NTS, where the 7th cranial nerve inputs are known to synapse. To address whether the axonal labeling produced by viral transduction is so widespread because it fills axons traveling to their targets, and not just terminal fields, we labeled pre and postsynaptic elements of vagal afferents in the NTS . Vagal afferents form multiple putative synapses as they course through the NTS, with each vagal afferent neuron distributing sensory signals to multiple second-order NTS neurons. We observe little selectivity between vagal afferents from different visceral targets and NTS neurons with common neurochemical phenotypes, with afferents from different organs making close appositions with the same NTS neuron. We conclude that specific viscerosensory information is distributed widely within the NTS and that the coding of this input is probably determined by the intrinsic properties and projections of the second-order neuron.


Assuntos
Núcleo Solitário , Nervo Vago , Animais , Neurônios Motores , Neurônios Aferentes/fisiologia , Gânglio Nodoso , Ratos , Núcleo Solitário/fisiologia , Nervo Vago/fisiologia
5.
Sci Rep ; 12(1): 10205, 2022 06 17.
Artigo em Inglês | MEDLINE | ID: mdl-35715554

RESUMO

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. For 2D imaging, fixed samples are imaged on a conventional MUSE system either label free (via auto-fluorescence) or after staining with fluorescent dyes. This method provides a simple and rapid technique to visualize myelinated nerve fibers at specific locations along the length of the nerve and perform measurements of fiber morphology (e.g., axon diameter and g-ratio). For 3D imaging, a whole-mount staining and MUSE block-face imaging method is developed that can be used to characterize peripheral nerve micro-anatomy and improve the accuracy of computational models in neuromodulation. Images of rat sciatic and human cadaver tibial nerves are presented, illustrating the applicability of the method in different preclinical models.


Assuntos
Alprostadil , Nervos Periféricos , Animais , Axônios , Imageamento Tridimensional/métodos , Fibras Nervosas Mielinizadas , Nervos Periféricos/diagnóstico por imagem , Ratos , Nervo Isquiático/diagnóstico por imagem
6.
Opt Lett ; 47(21): 5712-5714, 2022 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-37219310

RESUMO

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. This finding broadens the design parameter space of beam steering applications.

7.
Biomed Opt Express ; 13(11): 5599-5615, 2022 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-36733755

RESUMO

Of all congenital heart defects (CHDs), anomalies in heart valves and septa are among the most common and contribute about fifty percent to the total burden of CHDs. Progenitors to heart valves and septa are endocardial cushions formed in looping hearts through a multi-step process that includes localized expansion of cardiac jelly, endothelial-to-mesenchymal transition, cell migration and proliferation. To characterize the development of endocardial cushions, previous studies manually measured cushion size or cushion cell density from images obtained using histology, immunohistochemistry, or optical coherence tomography (OCT). Manual methods are time-consuming and labor-intensive, impeding their applications in cohort studies that require large sample sizes. This study presents an automated strategy to rapidly characterize the anatomy of endocardial cushions from OCT images. A two-step deep learning technique was used to detect the location of the heart and segment endocardial cushions. The acellular and cellular cushion regions were then segregated by K-means clustering. The proposed method can quantify cushion development by measuring the cushion volume and cellularized fraction, and also map 3D spatial organization of the acellular and cellular cushion regions. The application of this method to study the developing looping hearts allowed us to discover a spatial asymmetry of the acellular cardiac jelly in endocardial cushions during these critical stages, which has not been reported before.

8.
Am J Pathol ; 192(2): 180-194, 2022 02.
Artigo em Inglês | MEDLINE | ID: mdl-34774514

RESUMO

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. This article explains the technical basis of slide-free microscopy, reviews common slide-free optical microscopy techniques, and discusses the opportunities and challenges involved in clinical implementation.


Assuntos
Processamento de Imagem Assistida por Computador , Microscopia , Patologia Clínica , Humanos
9.
Biomed Opt Express ; 12(10): 6571-6589, 2021 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-34745757

RESUMO

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. Despite the high simplicity, our design achieved 3.5-µm uniform optical sectioning across a >6-mm FOV, surpassing existing light-sheet designs with similar optical sectioning. With well-corrected chromatic aberration, multi-channel fluorescence imaging can be performed without realignment. This manuscript provides a comprehensive tutorial on building the system and demonstrates the imaging performance with optically cleared whole-mount tissue samples.

10.
Commun Biol ; 4(1): 780, 2021 06 24.
Artigo em Inglês | MEDLINE | ID: mdl-34168243

RESUMO

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. Intriguingly, BCSCs tend to be enriched within the arteriolar niche in human estrogen receptor positive (ER+) BC and bi-directionally interact with arteriolar endothelial cells (ECs). Mechanistically, this interaction is driven by the lysophosphatidic acid (LPA)/protein kinase D (PKD-1) signaling pathway, which promotes both arteriolar differentiation of ECs and self-renewal of CSCs likely via differential regulation of CD36 transcription. This study indicates that CSCs may enjoy blood perfusion to maintain their stemness features. Targeting the LPA/PKD-1 -CD36 signaling pathway may have therapeutic potential to curb tumor progression by disrupting the arteriolar niche and effectively eliminating CSCs.


Assuntos
Neoplasias da Mama/patologia , Lisofosfolipídeos/fisiologia , Células-Tronco Neoplásicas/fisiologia , Proteína Quinase C/fisiologia , Nicho de Células-Tronco/fisiologia , Antígenos CD36/análise , Comunicação Celular , Diferenciação Celular , Células Endoteliais/citologia , Feminino , Humanos , Proteína Quinase C/análise , Transdução de Sinais/fisiologia , Microambiente Tumoral
11.
Commun Biol ; 4(1): 334, 2021 03 12.
Artigo em Inglês | MEDLINE | ID: mdl-33712728

RESUMO

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. It enables high-quality multichannel fluorescence microscopy with submicron resolution over a 10× equivalent field of view. In addition to the novel optical configuration, Pocket MUSE is compatible with a series of simple, portable, and user-friendly sample preparation strategies that can be directly implemented for various microscopy applications for point-of-care diagnostics, at-home health monitoring, plant biology, STEM education, environmental studies, etc.


Assuntos
Microscopia de Fluorescência/instrumentação , Aplicativos Móveis , Smartphone , Animais , Bactérias , Desenho de Equipamento , Imunofluorescência , Humanos , Processamento de Imagem Assistida por Computador , Camundongos , Mucosa Bucal , Plantas , Reprodutibilidade dos Testes , Manejo de Espécimes , Coloração e Rotulagem
12.
Artigo em Inglês | MEDLINE | ID: mdl-35313654

RESUMO

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. In addition to offering optical sectioning at the surface via reduced penetration depth, UV illumination also produces sufficient contrast between fascicular structures and connective tissue. Here we demonstrate the utility of this approach in pilot experiments. We imaged fixed, cadaver vagus nerve samples, segmented fascicles, and demonstrated 3D tracking of fascicles. Such data can serve as input for computer models of vagus nerve stimulation.

13.
Sci Rep ; 10(1): 14955, 2020 09 11.
Artigo em Inglês | MEDLINE | ID: mdl-32917915

RESUMO

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. The fluorescent dyes DiI and DAPI were used to stain the coronary vasculature and cardiomyocyte nuclei in quail embryo hearts during rapid growth and morphogenesis of the left ventricular wall. Vessel and cardiomyocytes orientation were automatically extracted and quantified, and vessel density was calculated. The coronary microvasculature was found to follow the known helical organization of cardiomyocytes in the ventricular wall. Vessel density in the left ventricle did not change during and after compaction. This quantitative and automated approach will enable future cohort studies to understand the microvasculature's role in diseases such as hypertrophic cardiomyopathy where misalignment of cardiomyocytes has been observed in utero.


Assuntos
Vasos Coronários/embriologia , Coturnix/embriologia , Microvasos/embriologia , Modelos Cardiovasculares , Miócitos Cardíacos/metabolismo , Animais , Ventrículos do Coração/embriologia
14.
J Clin Med ; 8(11)2019 Nov 14.
Artigo em Inglês | MEDLINE | ID: mdl-31739580

RESUMO

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. Current treatment options for metastatic pNETs are limited. Importantly, these tumors are highly vascularized and heterogeneous neoplasms, in which the heterogeneity of vascular endothelial cells (ECs) and de novo arteriogenesis may be critical for their progression. Current anti-angiogenetic targeted treatments have not shown substantial clinical benefits, and they are poorly tolerated. This review article describes EC heterogeneity and heterogeneous tumor-associated ECs (TAECs) in the TME and emphasizes the concept of de novo arteriogenesis in the TME. The authors also emphasize the challenges of current antiangiogenic therapy in pNETs and discuss the potential of tumor arteriogenesis as a novel therapeutic target. Finally, the authors prospect the clinical potential of targeting the FoxO1-CD36-Notch pathway that is associated with both pNET progression and arteriogenesis and provide insights into the clinical implications of targeting plasticity of cancer stem cells (CSCs) and vascular niche, particularly the arteriolar niche within the TME in pNETs, which will also provide insights into other types of cancer, including breast cancer, lung cancer, and malignant melanoma.

15.
Sci Rep ; 9(1): 893, 2019 01 29.
Artigo em Inglês | MEDLINE | ID: mdl-30696870

RESUMO

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.


Assuntos
Imageamento Tridimensional , Condicionamento Físico Animal , Tomografia de Coerência Óptica/métodos , Animais , Meios de Contraste , Modelos Animais de Doenças , Transtornos do Espectro Alcoólico Fetal/diagnóstico por imagem , Transtornos do Espectro Alcoólico Fetal/patologia , Microvasos , Nanopartículas , Tomografia de Coerência Óptica/normas
16.
Interface Focus ; 6(1): 20150080, 2016 Feb 06.
Artigo em Inglês | MEDLINE | ID: mdl-26855757

RESUMO

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.

17.
J Biol Methods ; 3(4): e54, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-31453217

RESUMO

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. The method successfully extracts isolated fibrils from rat tail and patellar tendons. To examine whether the method is likely to damage fibrils during extraction, sea cucumber dermis fibril lengths are compared against those obtained using only distilled water. The two methods produce fibrils of similar lengths. This is contrasted with fibrils being shortened when extracted using a tissue homogenizer. Scanning electron microscopy shows the new method preserves D-banding features on fibril surfaces and that fibril diameter does not vary substantially compared with water extracted fibrils.

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