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1.
J Biomed Opt ; 29(Suppl 2): S22702, 2025 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-38434231

RESUMO

Significance: Advancements in label-free microscopy could provide real-time, non-invasive imaging with unique sources of contrast and automated standardized analysis to characterize heterogeneous and dynamic biological processes. These tools would overcome challenges with widely used methods that are destructive (e.g., histology, flow cytometry) or lack cellular resolution (e.g., plate-based assays, whole animal bioluminescence imaging). Aim: This perspective aims to (1) justify the need for label-free microscopy to track heterogeneous cellular functions over time and space within unperturbed systems and (2) recommend improvements regarding instrumentation, image analysis, and image interpretation to address these needs. Approach: Three key research areas (cancer research, autoimmune disease, and tissue and cell engineering) are considered to support the need for label-free microscopy to characterize heterogeneity and dynamics within biological systems. Based on the strengths (e.g., multiple sources of molecular contrast, non-invasive monitoring) and weaknesses (e.g., imaging depth, image interpretation) of several label-free microscopy modalities, improvements for future imaging systems are recommended. Conclusion: Improvements in instrumentation including strategies that increase resolution and imaging speed, standardization and centralization of image analysis tools, and robust data validation and interpretation will expand the applications of label-free microscopy to study heterogeneous and dynamic biological systems.


Assuntos
Técnicas Histológicas , Microscopia , Animais , Citometria de Fluxo , Processamento de Imagem Assistida por Computador
2.
Anal Chem ; 96(15): 5771-5780, 2024 Apr 16.
Artigo em Inglês | MEDLINE | ID: mdl-38563229

RESUMO

Metabolic abnormalities are at the center of many diseases, and the capability to film and quantify the metabolic activities of a single cell is important for understanding the heterogeneities in these abnormalities. In this paper, a functional plasmonic microscope (FPM) is used to image and measure metabolic activities without fluorescent labels at a single-cell level. The FPM can accurately image and quantify the subnanometer membrane fluctuations with a spatial resolution of 0.5 µm in real time. These active cell membrane fluctuations are caused by metabolic activities across the cell membrane. A three-dimensional (3D) morphology of the bottom cell membrane was imaged and reconstructed with FPM to illustrate the capability of the microscope for cell membrane characterization. Then, the subnanometer cell membrane fluctuations of single cells were imaged and quantified with the FPM using HeLa cells. Cell metabolic heterogeneity is analyzed based on membrane fluctuations of each individual cell that is exposed to similar environmental conditions. In addition, we demonstrated that the FPM could be used to evaluate the therapeutic responses of metabolic inhibitors (glycolysis pathway inhibitor STF 31) on a single-cell level. The result showed that the metabolic activities significantly decrease over time, but the nature of this response varies, depicting cell heterogeneity. A low-concentration dose showed a reduced fluctuation frequency with consistent fluctuation amplitudes, while the high-concentration dose showcased a decreasing trend in both cases. These results have demonstrated the capabilities of the functional plasmonic microscope to measure and quantify metabolic activities for drug discovery.


Assuntos
Corantes , Microscopia , Humanos , Células HeLa , Membrana Celular , Membranas
3.
Zebrafish ; 21(2): 101-108, 2024 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-38621211

RESUMO

Microscopes are essential for research and education in science. Unlike computers and online learning tools, however, microscopes are not currently a fixed element in K-12 classrooms, due to steep cost, needless complexity, and often requiring a prohibitive level of staff training to effectively deploy. In a collaboration with Area 10 Labs, Integrated Science Education Outreach (InSciEd Out) developed a state-of-the-art alternative microscope, the InSciEdRS View, to reduce the financial barrier, prohibitive per-student cost, unnecessary complexity, and extensive staff training. Utilizing a 1080p camera and a lunchbox-style case, this Wi-Fi- and USB-connectable microscope comes with all necessary components for visualization of microscopic specimens (10 × -50 × magnification). While built to handle the rigors of classroom use, its imaging capability and battery-operation can make it flexible for a laboratory or fieldwork as well. We further highlight here K-12 curricula that we have developed using larval zebrafish to enable teachers, science outreach leaders, and parents to support active hands-on science observations. The InSciEdRS View microscope and the InSciEd Out curricula are readily scalable, translatable, and accessible for traditional and neurodiverse students and integrating these in various settings can be an efficient way to achieve better outcomes in science education.


Assuntos
Currículo , Peixe-Zebra , Animais , Humanos , Estudantes , Microscopia
4.
Sensors (Basel) ; 24(7)2024 Mar 25.
Artigo em Inglês | MEDLINE | ID: mdl-38610307

RESUMO

An analysis of the membrane organization and intracellular trafficking of lipids often relies on multiphoton (MP) and super-resolution microscopy of fluorescent lipid probes. A disadvantage of particularly intrinsically fluorescent lipid probes, such as the cholesterol and ergosterol analogue, dehydroergosterol (DHE), is their low MP absorption cross-section, resulting in a low signal-to-noise ratio (SNR) in live-cell imaging. Stimulated emission depletion (STED) microscopy of membrane probes like Nile Red enables one to resolve membrane features beyond the diffraction limit but exposes the sample to a lot of excitation light and suffers from a low SNR and photobleaching. Here, dynamic mode decomposition (DMD) and its variant, higher-order DMD (HoDMD), are applied to efficiently reconstruct and denoise the MP and STED microscopy data of lipid probes, allowing for an improved visualization of the membranes in cells. HoDMD also allows us to decompose and reconstruct two-photon polarimetry images of TopFluor-cholesterol in model and cellular membranes. Finally, DMD is shown to not only reconstruct and denoise 3D-STED image stacks of Nile Red-labeled cells but also to predict unseen image frames, thereby allowing for interpolation images along the optical axis. This important feature of DMD can be used to reduce the number of image acquisitions, thereby minimizing the light exposure of biological samples without compromising image quality. Thus, DMD as a computational tool enables gentler live-cell imaging of fluorescent probes in cellular membranes by MP and STED microscopy.


Assuntos
Corantes Fluorescentes , Microscopia , Membrana Celular , Colesterol , Lipídeos
5.
Mycopathologia ; 189(3): 33, 2024 Apr 16.
Artigo em Inglês | MEDLINE | ID: mdl-38627263

RESUMO

The cutaneous fungal infections in male genitalia are relatively rare, and often present with various atypical clinical symptoms. It was mainly reported in a small number of case reports, while data with large number of patients were rarely reported. In this study, we reported 79 male patients with cutaneous fungal infections on scrotum or penis. The fungal infections were confirmed by microscopic examination directly and fungus culture. Clinical characteristics and predisposing factors were also collected. Of these 79 patients, 72 has lesions on scrotum, 5 on penis and 2 on both scrotum and penis. Trichophyton (T.) rubrum is the most common pathogen, found in 50 (67.6%) patients, which presented diverse clinical manifestation such as majorly erythematous, dry diffused scaly lesions without a clear border, slightly powdery and scutular scalings. Candida (C.) albicans is the secondly common pathogen, found in 21 (28.4%) patients, which also presented diverse lesions such as erythematous with dry whitish scaly lesions and erythematous erosion. The predisposing factors mainly included concomitant fungal infections on sites other than genitalia, especially inguinal region (tinea cruris), application of corticosteroid and high moisture. In conclusion, cutaneous fungal infections in male genitalia could be caused by different fungi, showed atypical or mild clinical appearances in most cases and might be a fungus reservoir, emphasizing the necessity to timely perform the fungi examinations and corresponding therapy.


Assuntos
Dermatomicoses , Humanos , Masculino , Dermatomicoses/patologia , Pele/patologia , Trichophyton , Microscopia , Escroto/microbiologia
6.
Commun Biol ; 7(1): 451, 2024 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-38622287

RESUMO

This report presents an optical fibre-based endo-microscopic imaging tool that simultaneously measures the topographic profile and 3D viscoelastic properties of biological specimens through the phenomenon of time-resolved Brillouin scattering. This uses the intrinsic viscoelasticity of the specimen as a contrast mechanism without fluorescent tags or photoacoustic contrast mechanisms. We demonstrate 2 µm lateral resolution and 320 nm axial resolution for the 3D imaging of biological cells and Caenorhabditis elegans larvae. This has enabled the first ever 3D stiffness imaging and characterisation of the C. elegans larva cuticle in-situ. A label-free, subcellular resolution, and endoscopic compatible technique that reveals structural biologically-relevant material properties of tissue could pave the way toward in-vivo elasticity-based diagnostics down to the single cell level.


Assuntos
Imageamento Tridimensional , Microscopia , Animais , Microscopia/métodos , Imageamento Tridimensional/métodos , Caenorhabditis elegans , Elasticidade , Biologia
7.
J Biomed Opt ; 29(Suppl 2): S22704, 2024 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-38584966

RESUMO

Significance: Full-field optical coherence microscopy (FF-OCM) is a prevalent technique for backscattering and phase imaging with epi-detection. Traditional methods have two limitations: suboptimal utilization of functional information about the sample and complicated optical design with several moving parts for phase contrast. Aim: We report an OCM setup capable of generating dynamic intensity, phase, and pseudo-spectroscopic contrast with single-shot full-field video-rate imaging called bichromatic tetraphasic (BiTe) full-field OCM with no moving parts. Approach: BiTe OCM resourcefully uses the phase-shifting properties of anti-reflection (AR) coatings outside the rated bandwidths to create four unique phase shifts, which are detected with two emission filters for spectroscopic contrast. Results: BiTe OCM overcomes the disadvantages of previous FF-OCM setup techniques by capturing both the intensity and phase profiles without any artifacts or speckle noise for imaging scattering samples in three-dimensional (3D). BiTe OCM also utilizes the raw data effectively to generate three complementary contrasts: intensity, phase, and color. We demonstrate BiTe OCM to observe cellular dynamics, image live, and moving micro-animals in 3D, capture the spectroscopic hemodynamics of scattering tissues along with dynamic intensity and phase profiles, and image the microstructure of fall foliage with two different colors. Conclusions: BiTe OCM can maximize the information efficiency of FF-OCM while maintaining overall simplicity in design for quantitative, dynamic, and spectroscopic characterization of biological samples.


Assuntos
Microscopia , Tomografia de Coerência Óptica , Animais , Microscopia/métodos , Tomografia de Coerência Óptica/métodos , Microscopia de Contraste de Fase
8.
Nat Commun ; 15(1): 2932, 2024 Apr 04.
Artigo em Inglês | MEDLINE | ID: mdl-38575577

RESUMO

Ultrasound localization microscopy (ULM) enables deep tissue microvascular imaging by localizing and tracking intravenously injected microbubbles circulating in the bloodstream. However, conventional localization techniques require spatially isolated microbubbles, resulting in prolonged imaging time to obtain detailed microvascular maps. Here, we introduce LOcalization with Context Awareness (LOCA)-ULM, a deep learning-based microbubble simulation and localization pipeline designed to enhance localization performance in high microbubble concentrations. In silico, LOCA-ULM enhanced microbubble detection accuracy to 97.8% and reduced the missing rate to 23.8%, outperforming conventional and deep learning-based localization methods up to 17.4% in accuracy and 37.6% in missing rate reduction. In in vivo rat brain imaging, LOCA-ULM revealed dense cerebrovascular networks and spatially adjacent microvessels undetected by conventional ULM. We further demonstrate the superior localization performance of LOCA-ULM in functional ULM (fULM) where LOCA-ULM significantly increased the functional imaging sensitivity of fULM to hemodynamic responses invoked by whisker stimulations in the rat brain.


Assuntos
Aprendizado Profundo , Microscopia , Ratos , Animais , Microscopia/métodos , Microbolhas , Ultrassonografia/métodos , Microscopia Intravital , Microvasos/diagnóstico por imagem
9.
Nat Commun ; 15(1): 2935, 2024 Apr 05.
Artigo em Inglês | MEDLINE | ID: mdl-38580633

RESUMO

Histopathology plays a critical role in the diagnosis and surgical management of cancer. However, access to histopathology services, especially frozen section pathology during surgery, is limited in resource-constrained settings because preparing slides from resected tissue is time-consuming, labor-intensive, and requires expensive infrastructure. Here, we report a deep-learning-enabled microscope, named DeepDOF-SE, to rapidly scan intact tissue at cellular resolution without the need for physical sectioning. Three key features jointly make DeepDOF-SE practical. First, tissue specimens are stained directly with inexpensive vital fluorescent dyes and optically sectioned with ultra-violet excitation that localizes fluorescent emission to a thin surface layer. Second, a deep-learning algorithm extends the depth-of-field, allowing rapid acquisition of in-focus images from large areas of tissue even when the tissue surface is highly irregular. Finally, a semi-supervised generative adversarial network virtually stains DeepDOF-SE fluorescence images with hematoxylin-and-eosin appearance, facilitating image interpretation by pathologists without significant additional training. We developed the DeepDOF-SE platform using a data-driven approach and validated its performance by imaging surgical resections of suspected oral tumors. Our results show that DeepDOF-SE provides histological information of diagnostic importance, offering a rapid and affordable slide-free histology platform for intraoperative tumor margin assessment and in low-resource settings.


Assuntos
Aprendizado Profundo , Microscopia , Corantes Fluorescentes , Hematoxilina , Amarelo de Eosina-(YS)
10.
J Vis Exp ; (205)2024 Mar 29.
Artigo em Inglês | MEDLINE | ID: mdl-38619235

RESUMO

Two-photon microscopy has emerged as a potent tool for evaluating deep tissue cells and characterizing the alignment of the extracellular matrix (ECM) in various biological systems. This technique relies on nonlinear light-matter interactions to detect two distinct signals: the second harmonic generated (SHG) diffusion signal, which facilitates the visualization of collagen fibers and their orientation, and the near-infrared excitation signal for imaging ultraviolet excited autofluorescence. SHG imaging proves especially effective in visualizing collagen fibers due to the non-centrosymmetric crystalline structure of fibrillar collagen I. Given that tendons are matrix-rich tissues with a limited number of cells, their high collagen content makes them ideal candidates for analysis using two-photon microscopy. Consequently, two-photon microscopy offers a valuable means to analyze and characterize collagen abnormalities in tendons. Its application extends to studying tendon development, injuries, healing, and aging, enabling the comprehensive characterization of tendon cells and their interactions with the ECM under various conditions using two-photon microscopy tools. This protocol outlines the use of two-photon microscopy in tendon biology and presents an adapted methodology to achieve effective imaging and characterization of tendon cells during development and after injury. The method allows the utilization of thin microscopic sections to create a comprehensive image of the ECM within tendons and the cells that interact with this matrix. Most notably, the article showcases a technique to generate 3D images using two-photon microscopy in animal models.


Assuntos
Envelhecimento , Microscopia , Animais , Difusão , Tendões/diagnóstico por imagem , Colágeno
11.
J Vis Exp ; (205)2024 Mar 29.
Artigo em Inglês | MEDLINE | ID: mdl-38619234

RESUMO

Light-sheet microscopy (LSM) plays a pivotal role in comprehending the intricate three-dimensional (3D) structure of the heart, providing crucial insights into fundamental cardiac physiology and pathologic responses. We hereby delve into the development and implementation of the LSM technique to elucidate the micro-architecture of the heart in mouse models. The methodology integrates a customized LSM system with tissue clearing techniques, mitigating light scattering within cardiac tissues for volumetric imaging. The combination of conventional LSM with image stitching and multiview deconvolution approaches allows for the capture of the entire heart. To address the inherent trade-off between axial resolution and field of view (FOV), we further introduce an axially swept light-sheet microscopy (ASLM) method to minimize out-of-focus light and uniformly illuminate the heart across the propagation direction. In the meanwhile, tissue clearing methods such as iDISCO enhance light penetration, facilitating the visualization of deep structures and ensuring a comprehensive examination of the myocardium throughout the entire heart. The combination of the proposed LSM and tissue clearing methods presents a promising platform for researchers in resolving cardiac structures in rodent hearts, holding great potential for the understanding of cardiac morphogenesis and remodeling.


Assuntos
Coração , Microscopia , Animais , Camundongos , Coração/diagnóstico por imagem , Miocárdio , Modelos Animais de Doenças , Reprodução
12.
PLoS Negl Trop Dis ; 18(4): e0012041, 2024 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-38602896

RESUMO

BACKGROUND: Infections caused by soil-transmitted helminths (STHs) are the most prevalent neglected tropical diseases and result in a major disease burden in low- and middle-income countries, especially in school-aged children. Improved diagnostic methods, especially for light intensity infections, are needed for efficient, control and elimination of STHs as a public health problem, as well as STH management. Image-based artificial intelligence (AI) has shown promise for STH detection in digitized stool samples. However, the diagnostic accuracy of AI-based analysis of entire microscope slides, so called whole-slide images (WSI), has previously not been evaluated on a sample-level in primary healthcare settings in STH endemic countries. METHODOLOGY/PRINCIPAL FINDINGS: Stool samples (n = 1,335) were collected during 2020 from children attending primary schools in Kwale County, Kenya, prepared according to the Kato-Katz method at a local primary healthcare laboratory and digitized with a portable whole-slide microscopy scanner and uploaded via mobile networks to a cloud environment. The digital samples of adequate quality (n = 1,180) were split into a training (n = 388) and test set (n = 792) and a deep-learning system (DLS) developed for detection of STHs. The DLS findings were compared with expert manual microscopy and additional visual assessment of the digital samples in slides with discordant results between the methods. Manual microscopy detected 15 (1.9%) Ascaris lumbricoides, 172 (21.7%) Tricuris trichiura and 140 (17.7%) hookworm (Ancylostoma duodenale or Necator americanus) infections in the test set. Importantly, more than 90% of all STH positive cases represented light intensity infections. With manual microscopy as the reference standard, the sensitivity of the DLS as the index test for detection of A. lumbricoides, T. trichiura and hookworm was 80%, 92% and 76%, respectively. The corresponding specificity was 98%, 90% and 95%. Notably, in 79 samples (10%) classified as negative by manual microscopy for a specific species, STH eggs were detected by the DLS and confirmed correct by visual inspection of the digital samples. CONCLUSIONS/SIGNIFICANCE: Analysis of digitally scanned stool samples with the DLS provided high diagnostic accuracy for detection of STHs. Importantly, a substantial number of light intensity infections were missed by manual microscopy but detected by the DLS. Thus, analysis of WSIs with image-based AI may provide a future tool for improved detection of STHs in a primary healthcare setting, which in turn could facilitate monitoring and evaluation of control programs.


Assuntos
Helmintíase , Helmintos , Criança , Animais , Humanos , Inteligência Artificial , Solo/parasitologia , Microscopia , Região de Recursos Limitados , Fezes/parasitologia , Trichuris , Helmintíase/diagnóstico , Helmintíase/parasitologia , Ascaris lumbricoides , Ancylostomatoidea , Prevalência
13.
Sci Rep ; 14(1): 8418, 2024 04 10.
Artigo em Inglês | MEDLINE | ID: mdl-38600062

RESUMO

Accumulation of bioavailable heavy metals in aquatic environment poses a serious threat to marine communities and human health due to possible trophic transfers through the food chain of toxic, non-degradable, exogenous pollutants. Copper (Cu) is one of the most spread heavy metals in water, and can severely affect primary producers at high doses. Here we show a novel imaging test to assay the dose-dependent effects of Cu on live microalgae identifying stress conditions when they are still capable of sustaining a positive growth. The method relies on Fourier Ptychographic Microscopy (FPM), capable to image large field of view in label-free phase-contrast mode attaining submicron lateral resolution. We uniquely combine FPM with a new multi-scale analysis method based on fractal geometry. The system is able to provide ensemble measurements of thousands of diatoms in the liquid sample simultaneously, while ensuring at same time single-cell imaging and analysis for each diatom. Through new image descriptors, we demonstrate that fractal analysis is suitable for handling the complexity and informative power of such multiscale FPM modality. We successfully tested this new approach by measuring how different concentrations of Cu impact on Skeletonema pseudocostatum diatom populations isolated from the Sarno River mouth.


Assuntos
Diatomáceas , Metais Pesados , Humanos , Cobre/farmacologia , Microscopia , Fractais , Metais Pesados/farmacologia
14.
Opt Lett ; 49(7): 1725-1728, 2024 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-38560847

RESUMO

Ultrasound coupling is one of the critical challenges for traditional photoacoustic (or optoacoustic) microscopy (PAM) techniques transferred to the clinical examination of chronic wounds and open tissues. A promising alternative potential solution for breaking the limitation of ultrasound coupling in PAM is photoacoustic remote sensing (PARS), which implements all-optical non-interferometric photoacoustic measurements. Functional imaging of PARS microscopy was demonstrated from the aspects of histopathology and oxygen metabolism, while its performance in hemodynamic quantification remains unexplored. In this Letter, we present an all-optical thermal-tagging flowmetry approach for PARS microscopy and demonstrate it with comprehensive mathematical modeling and ex vivo and in vivo experimental validations. Experimental results demonstrated that the detectable range of the blood flow rate was from 0 to 12 mm/s with a high accuracy (measurement error:±1.2%) at 10-kHz laser pulse repetition rate. The proposed all-optical thermal-tagging flowmetry offers an effective alternative approach for PARS microscopy realizing non-contact dye-free hemodynamic imaging.


Assuntos
Técnicas Fotoacústicas , Tecnologia de Sensoriamento Remoto , Técnicas Fotoacústicas/métodos , Reologia/métodos , Ultrassonografia/métodos , Microscopia/métodos
15.
Int. microbiol ; 27(2): 559-569, Abr. 2024. ilus
Artigo em Inglês | IBECS | ID: ibc-ADZ-166

RESUMO

Nervous necrosis virus (NNV) is the causative agent of viral nervous necrosis in freshwater and marine fishes. In this study, NNV circulating among wild and farmed Nile tilapia (Oreochromis niloticus) was genetically and morphologically characterized using reverse transcription polymerase chain reaction (RT-PCR), sequencing analysis, and transmission electron microscopy (TEM). Brain, eye, and other organ (spleen, kidney, heart, and liver) specimens were collected from 87 wild (66) and farmed (21) Nile tilapia fish during their adult or juvenile stage at different localities in Qena and Sohag governorates in southern Egypt. Among them, 57/87 fish showed suspected NNV clinical signs, and 30/87 were healthy. The results revealed that NNV was detected in 66 out of 87 fish (58.62% in the wild and 17.24% in farmed Nile tilapia by RT-PCR), and the prevalence was higher among diseased (55.17%) than in healthy (20.69%) fish. NNV was detected in the brain, eye, and other organs. Using TEM, virion size variations based on the infected organs were observed. Nucleotide sequence similarity indicated that NNVs had a divergence of 75% from other fish nodaviruses sequenced in Egypt and worldwide. Phylogenetic analysis distinguished them from other NNV genotypes, revealing the emergence of a new NNV genotype in southern Egypt. In conclusion, NNV is circulating among diseased and healthy Nile tilapia, and a new NNV genotype has emerged in southern Egypt. (AU)


Assuntos
Animais , Necrose , Peixes , Água Doce , Genética , RNA Polimerases Dirigidas por DNA , Microscopia
16.
Rev. ORL (Salamanca) ; 15(1)25-03-2024. tab
Artigo em Espanhol | IBECS | ID: ibc-231855

RESUMO

Introducción y objetivo: La otosclerosis es una causa de hipoacusia en jóvenes, con mayor frecuencia en mujeres. La cirugía del estribo es un procedimiento correctivo ampliamente aceptado, con el advenimiento de la tecnología y cambios en la técnica, surge la interrogante de si existen diferencias entre ellas. Objetivo: Evaluar si existen diferencias en la ganancia auditiva entre técnicas y abordajes de las cirugías del estribo en pacientes con otosclerosis Método: Se recabaron variables demográficas, clínicas y quirúrgicas. Se aplicó estadística descriptiva. Se empleó prueba U de Mann-Whitney para variables numéricas, así como Kruskal Wallis para comparación diferencias en tres o más grupos. Se consideró significativo un valor de p ≤ a 0.05. Resultados: Entre los años 2020 y 2023 se realizaron 55 cirugías de estribo por otosclerosis, de las cuales 20 se tuvieron que excluir. De 35 cirugías en 31 pacientes, la media de edad de 41.16 ± 8.64 años, 77.4% fueron mujeres, el 51.4 % fueron en el oído derecho; se presentaron comorbilidades en el 25.7%, las complicaciones 5 presentaron hipoacusia, el 88.6 % de los procedimientos se encontró un cierre satisfactorio de la brecha aérea y ósea. No se presentaron diferencia entre las técnicas de la cirugía de estribo y resultados audiológicos postquirúrgicos p=0.872, ni con el tipo de abordaje de visualización p=0.636. Discusión: Nuestros resultados son similares a lo que encontraron algunos autores, no obstante, aún sigue existiendo incertidumbre sobre la mejor técnica. Conclusiones: No se encontraron diferencias estadísticamente significativas en cuanto a la ganancia auditiva con el abordaje de visualización y el tipo de procedimiento en el estribo para la colocación de la prótesis. (AU)


Introduction and objective: Otosclerosis is a cause of hearing loss in young people, more frequently in women. Stapes surgery is a widely accepted corrective procedure, with the advent of technology and changes in technique, the question arises as to whether there are differences between them. Objective: To evaluate whether there are differences in hearing gain between techniques and approaches of stapes surgeries in patients with otosclerosis. Method: Demographic, clinical and surgical variables were collected. Descriptive statistics were applied. The Mann-Whitney U test was used for numerical variables, as well as the Kruskal Wallis test to compare differences in three or more groups. A p value ≤ 0.05 was considered significant. Results: Between 2020 and 2023, 55 stapes surgeries were performed for otosclerosis, of which 20 had to be excluded. Of 35 surgeries in 31 patients, mean age 41.16 ± 8.64 years, 77.4% were women, 51.4% were in the right ear; Comorbidities were present in 25.7%, 5 complications presented hearing loss, in 88.6% of the procedures a satisfactory closure of the air-bone gap was found. There was no difference between stapes surgery techniques and postsurgical audiological results p=0.872, nor with the type of visualization approach p=0.636. Discussion: Our results are similar to what some authors found, however, there is still uncertainty about the best technique. Conclusions: No statistically significant differences were found in terms of hearing gain with the visualization approach and the type of procedure in the stapes for placement of the prosthesis. (AU)


Assuntos
Humanos , Masculino , Feminino , Adulto , Pessoa de Meia-Idade , Cirurgia do Estribo , Otosclerose/complicações , Perda Auditiva , Cirurgia do Estribo/métodos , Cirurgia do Estribo/tendências , Microscopia
17.
Theranostics ; 14(5): 1794-1814, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38505609

RESUMO

Rationale: The acoustic stimulation of microbubbles within microvessels can elicit a spectrum of therapeutically relevant bioeffects from permeabilization to perfusion shutdown. These bioeffects ultimately arise from complex interactions between microbubbles and microvascular walls, though such interactions are poorly understood particularly at high pressure, due to a paucity of direct in vivo observations. The continued development of focused ultrasound methods hinges in large part on establishing links between microbubble-microvessel interactions, cavitation signals, and bioeffects. Methods: Here, a system was developed to enable simultaneous high-speed intravital imaging and cavitation monitoring of microbubbles in vivo in a chorioallantoic membrane model. Exposures were conducted using the clinical agent DefinityTM under conditions previously associated with microvascular damage (1 MHz, 0.5-3.5 MPa, 5 ms pulse length). Results: Ultrasound-activated microbubbles could be observed and were found to induce localized wall deformations that were more pronounced in smaller microvessels and increased with pressure. A central finding was that microbubbles could extravasate from microvessels (from 34% of vessels at 1 MPa to 79% at 3 MPa) during insonation (94% within 0.5 ms) and that this occurred more frequently and in progressively larger microvessels (up to 180 µm) as pressure was increased. Following microbubble extravasation, transient or sustained red blood cell leakage ensued at the extravasation site in 96% of cases for pressures ≥1 MPa. Conclusions: The results here represent the first high-speed in vivo investigation of high-pressure focused ultrasound-induced microbubble-microvessel interactions. This data provides direct evidence that the process of activated microbubble extravasation can occur in vivo and that it is linked to producing microvessel wall perforations of sufficient size to permit red blood cell leakage. The association of red blood cell leakage with microbubble extravasation provides mechanistic insight into the process of microvessel rupture, which has been widely observed in histology.


Assuntos
Membrana Corioalantoide , Microbolhas , Animais , Microscopia , Ultrassonografia/métodos , Microscopia Intravital
18.
Elife ; 122024 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-38488831

RESUMO

Nondestructive pathology based on three-dimensional (3D) optical microscopy holds promise as a complement to traditional destructive hematoxylin and eosin (H&E) stained slide-based pathology by providing cellular information in high throughput manner. However, conventional techniques provided superficial information only due to shallow imaging depths. Herein, we developed open-top two-photon light sheet microscopy (OT-TP-LSM) for intraoperative 3D pathology. An extended depth of field two-photon excitation light sheet was generated by scanning a nondiffractive Bessel beam, and selective planar imaging was conducted with cameras at 400 frames/s max during the lateral translation of tissue specimens. Intrinsic second harmonic generation was collected for additional extracellular matrix (ECM) visualization. OT-TP-LSM was tested in various human cancer specimens including skin, pancreas, and prostate. High imaging depths were achieved owing to long excitation wavelengths and long wavelength fluorophores. 3D visualization of both cells and ECM enhanced the ability of cancer detection. Furthermore, an unsupervised deep learning network was employed for the style transfer of OT-TP-LSM images to virtual H&E images. The virtual H&E images exhibited comparable histological characteristics to real ones. OT-TP-LSM may have the potential for histopathological examination in surgical and biopsy applications by rapidly providing 3D information.


Assuntos
Microscopia , Neoplasias , Masculino , Humanos , Microscopia/métodos , Corantes Fluorescentes , Pele , Amarelo de Eosina-(YS) , Imageamento Tridimensional/métodos
19.
Soft Matter ; 20(12): 2804-2811, 2024 Mar 20.
Artigo em Inglês | MEDLINE | ID: mdl-38446076

RESUMO

The peels of lime, lemon, pomelo and citron are investigated at macroscopic and microscopic level. The structural composition of the peels is compared and properties such as peel thickness, proportion of flavedo, density and proportion of intercellular spaces are determined. µCT images are used to visualize vascular bundles and oil glands. SEM images provide information about the appearance of the cellular tissue in the outer flavedo and inner albedo. The proportion of intercellular spaces is quantitatively determined by manual and software-assisted analysis (ilastik). While there are macroscopic differences in the fruits, they differ only slightly in the orientation of the vascular bundles and the arrangement of the oil glands. However, in peel thickness and flavedo thickness, the fruit peels differ significantly from each other. There are no significant differences between the two analysis methods used, although the use of ilastik is preferred due to time reduction of up to 70%. The large amount of intercellular spaces in the albedo but also the denser flavedo both have a mechanical protective function to prevent damage to the fruit. In addition, the entire peel structure is mechanically reinforced by vascular bundles. This combination of penetration protection (flavedo) and energy dissipation (albedo) makes Citrus spp. peels a promising inspiration for technical material systems.


Assuntos
Citrus , Citrus/química , Citrus/ultraestrutura , Microscopia , Frutas/química , Frutas/ultraestrutura
20.
PLoS One ; 19(3): e0299875, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38498588

RESUMO

The widespread availability and diversity of open-source microcontrollers paired with off-the-shelf electronics and 3D printed technology has led to the creation of a wide range of low-cost scientific instruments, including microscopes, spectrometers, sensors, data loggers, and other tools that can be used for research, education, and experimentation. These devices can be used to explore a wide range of scientific topics, from biology and chemistry to physics and engineering. In this study, we designed and built a multifunction fluorescent open source in vivo/in vitro imaging system (openIVIS) system that integrates a Raspberry Pi with commercial cameras and LEDs with 3D printed structures combined with an acrylic housing. Our openIVIS provides three excitation wavelengths of 460 nm, 520 nm, and 630 nm integrated with Python control software to enable fluorescent measurements across the full visible light spectrum. To demonstrate the potential applications of our system, we tested its performance against a diverse set of experiments including laboratory assays (measuring fluorescent dyes, using optical nanosensors, and DNA gel electrophoresis) to potentially fieldable applications (plant and mineral imaging). We also tested the potential use for a high school biology environment by imaging small animals and tracking their development over the course of ten days. Our system demonstrated its ability to measure a wide dynamic range fluorescent response from millimolar to picomolar concentrations in the same sample while measuring responses across visible wavelengths. These results demonstrate the power and flexibility of open-source hardware and software and how it can be integrated with customizable manufacturing to create low-cost scientific instruments with a wide range of applications. Our study provides a promising model for the development of low-cost instruments that can be used in both research and education.


Assuntos
Eletrônica , Microscopia , Animais , Luz , Software , Tecnologia
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