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.

Spontaneous Confinement of mRNA Molecules at Biomolecular Condensate Boundaries.

Cellular biomolecular condensates, termed ribonucleoprotein (RNP) granules, are often enriched in messenger RNA (mRNA) molecules relative to the surrounding cytoplasm. Yet, the spatial localization and diffusion of mRNAs in close proximity to phase separated RNP granules are not well understood. In this study, we performed single-molecule fluorescence imaging experiments of mRNAs in live cells in the presence of two types of RNP granules, stress granules (SGs) and processing bodies (PBs), which are distinct in their molecular composition and function. We developed a photobleaching- and noise-corrected colocalization imaging algorithm that was employed to determine the accurate positions of individual mRNAs relative to the granule's boundaries. We found that mRNAs are often localized at granule boundaries, an observation consistent with recently published data. We suggest that mRNA molecules become spontaneously confined at the RNP granule boundary similar to the adsorption of polymer molecules at liquid-liquid interfaces, which is observed in various technological and biological processes. We also suggest that this confinement could be due to a combination of intermolecular interactions associated with, first, the screening of a portion of the RNP granule interface by the polymer and, second, electrostatic interactions due to a strong electric field induced by a Donnan potential generated across the thin interface.

Background-free wide-field fluorescence imaging using edge detection combined with HiLo.

Fluorescence microscopy has been widely used in the field of biological imaging, but the disturbance of background noise has always been an unavoidable phenomenon. To obtain a background free image, a virtual HiLo based on edge detection (V-HiLo-ED) method for background removing is proposed, which is different from the existing popular software algorithms that obtain the background-free image by subtracting the estimated background, but the background-free image is directly reconstructed by estimating the foreground. Compared with two other popular software-based methods, the wavelet-based background and noise subtraction algorithm (WBNS) and the rolling ball algorithm (RBA), the V-HiLo-ED owns a better quality on achieving background-free imaging. Compared with hardware-based method such as HiLo method, V-HiLo-ED exhibits almost the same performance but faster speed. In combination with light sheet microscopy, the V-HiLo-ED further improves the signal-to-noise ratio of images with thick light-sheet. These experiment results indicate that the V-HiLo-ED owns the potentiality in many other image applications such as endoscopy.

In-vivo Single-Molecule Imaging in Yeast: Applications and Challenges.

Single-molecule imaging has gained momentum to quantify the dynamics of biomolecules in live cells, as it provides direct real-time measurements of various cellular activities under their physiological environment. Yeast, a simple and widely used eukaryote, serves as a good model system to quantify single-molecule dynamics of various cellular processes because of its low genomic and cellular complexities, as well as its facile ability to be genetically manipulated. In the past decade, significant developments have been made regarding the intracellular labeling of biomolecules (proteins, mRNA, fatty acids), the microscopy setups to visualize single-molecules and capture their fast dynamics, and the data analysis pipelines to interpret such dynamics. In this review, we summarize the current state of knowledge for the single-molecule imaging in live yeast cells to provide a ready reference for beginners. We provide a comprehensive table to demonstrate how various labs tailored the imaging regimes and data analysis pipelines to estimate various biophysical parameters for a variety of biological processes. Lastly, we present current challenges and future directions for developing better tools and resources for single-molecule imaging in live yeast cells.

HiLo Based Line Scanning Temporal Focusing Microscopy for High-Speed, Deep Tissue Imaging.

High-speed, optical-sectioning imaging is highly desired in biomedical studies, as most bio-structures and bio-dynamics are in three-dimensions. Compared to point-scanning techniques, line scanning temporal focusing microscopy (LSTFM) is a promising method that can achieve high temporal resolution while maintaining a deep penetration depth. However, the contrast and axial confinement would still be deteriorated in scattering tissue imaging. Here, we propose a HiLo-based LSTFM, utilizing structured illumination to inhibit the fluorescence background and, thus, enhance the image contrast and axial confinement in deep imaging. We demonstrate the superiority of our method by performing volumetric imaging of neurons and dynamical imaging of microglia in mouse brains in vivo.

Varifocal Metalens for Optical Sectioning Fluorescence Microscopy.

Fluorescence microscopy with optical sectioning capabilities is extensively utilized in biological research to obtain three-dimensional structural images of volumetric samples. Tunable lenses have been applied in microscopy for axial scanning to acquire multiplane images. However, images acquired by conventional tunable lenses suffer from spherical aberration and distortions. Here, we design, fabricate, and implement a dielectric Moiré metalens for fluorescence imaging. The Moiré metalens consists of two complementary phase metasurfaces, with variable focal length, ranging from ∼10 to ∼125 mm at 532 nm by tuning mutual angles. In addition, a telecentric configuration using the Moiré metalens is designed for high-contrast multiplane fluorescence imaging. The performance of our system is evaluated by optically sectioned images obtained from HiLo illumination of fluorescently labeled beads, as well as ex vivo mice intestine tissue samples. The compact design of the varifocal metalens may find important applications in fluorescence microscopy and endoscopy for clinical purposes.

Spatial analysis of water quality parameters in Hilo Bay, Hawai'i, using a combination of interpolated surfaces and hot spot analysis.

Hilo Bay estuary, located on the northeastern side of Hawai'i Island, experiences variability in water quality parameters due to its numerous water inputs. This estuary experiences influxes of water from three sources: groundwater to the east, marine water from the north, and surface water from the Wailuku River to the west. High rainfall and river flow impacts Hilo Bay's water quality including salinity, turbidity, and chlorophyll a concentration. Here, maps of Hilo Bay water quality were examined to assess spatial patterns of these important parameters. Exploring the patterns of these water quality parameters by creating inverse distance weighted (IDW) interpolation surfaces of survey points and clusters based on hot spot analyses during low- and high-flow conditions showed statistically significant differences in spatial water quality in Hilo Bay. Water quality maps after a storm show (1) an overall decrease in salinity, (2) a river plume from the Wailuku River associated with a turbidity hot spot, and (3) a chlorophyll a hot spot offset from the river plume in the center of the bay. Using spatial analysis to analyze water quality throughout the entirety of Hilo Bay before and after storm events can lead to a better understanding of how this ecosystem is affected during these types of events, and furthermore, adopting this method of sampling and analysis allows for a greater representation of water quality all over the bay and can improve the monitoring of water quality in this important ecosystem.

Immediate Impact of Hurricane Lane on Microbiological Quality of Coastal Water in Hilo Bay, Hawaii.

Hurricanes and associated stormwater runoff events are expected to greatly impact coastal marine water quality, yet little is known about their immediate effects on microbiological quality of near-shore water. This study sampled Hilo Bay immediately after the impact of Hurricane Lane to understand the spatial and temporal variations of the abundance and diversity of fecal indicator enterococci, common fecal pathogens, and antibiotic resistance genes (ARGs). Water samples from seven sampling sites over 7 days were collected and analyzed, which showed that the overall microbiological water quality parameters [enterococci geometric mean (GM): 6-22 cfu/100 mL] fell within water quality standards and that the temporal dynamics indicated continuing water quality recovery. However, considerable spatial variation was observed, with the most contaminated site exhibiting impaired water quality (GM = 144 cfu/100 mL). The Enterococcus population also showed distinct genotypic composition at the most contaminated site. Although marker genes for typical fecal pathogens (invA for Salmonella, hipO for Campylobacter, mip for Legionella pneumophila, and eaeA for enteropathogenic Escherichia coli) were not detected, various ARGs (ermB, qurS, tetM, blaTEM, and sul1) and integron-associated integrase intI1 were detected at high levels. Understanding the temporal and spatial variation of microbiological water quality at fine granularity is important for balancing economic and recreational uses of coastal water and the protection of public health post the impact of major hurricane events.

Telecentric design for digital-scanning-based HiLo optical sectioning endomicroscopy with an electrically tunable lens.

Confocal endoscopy has been widely used to obtain fine optically sectioned images. However, confocal endomicroscopic images are formed by point-by-point scanning in both lateral and axial directions, which results in long image acquisition time. Here, an endomicroscope with telecentric configuration is presented to achieve nonmechanical and rapid axial scanning for volumetric fluorescence imaging. In our system, optical sectioning in wide-field fashion is obtained through HiLo imaging with a digital micromirror device. Axial scanning, without mechanical moving parts, is conducted by digital focus adjustment using an electrically tunable lens, offering constant magnification and contrast. We demonstrate imaging performance of our system with optically sectioned images using fluorescently labeled beads, as well as ex vivo mice cardiac tissue samples. Our system provides multiple advantages, in terms of improved scanning range, and reduced image acquisition time, which shows great potentials for three-dimensional biopsies of volumetric biological samples.

Sineprapia, curiosa aportación técnica, del catedrático Sagarra Lascurain 1848-1924) para seccionar los tejidos / Sineprapia, curious technical contribution, of the surgical professor Sagarra Lascurain (1848-1924) to section the injuries

Muchas técnicas se han propuesto con el fin de optimizar la ejecución y desarrollo de los procedimientos en cirugía. Algunas sehan mantenido a lo largo del tiempo y otras han dejado de utilizarse por muy variados motivos. La sinemaprapía fue un procedimiento que, aunque si bien tuvo antecedentes de empleo por algunos autores de forma ocasional, fue el Catedrático de AnatomíaQuirúrgica, Operaciones, Apósitos y Vendajes Vicente Sagarra Lascurain el que la promociono a finales del siglo XIX y principiosdel XX. Quiso destacar en ella su sencillez de uso, eficacia en la diéresis y sobre todo la menor hemorragia que se producía en laherida quirúrgica en su aplicación, que la hacía muy adecuada su empleo en tejidos y órganos muy vascularizados, como eran lasamputaciones de pene y lengua. La técnica se empleo de una forma no muy extendida y con el paso del tiempo quedó olvidada. Eltrabajo trata de recordar desde el punto de vista histórico esta técnica, su empleo, las indicaciones de uso y referencia a sus resultados, profusamente publicados por su promotor. (AU)

Many techniques have been proposed in order to optimize the performance and development of surgical procedures. Some have beenmaintained over time and others have been discontinued for a variety of reasons. Sinemaprasía was a procedure, which althoughit had a history of employment by some authors occasionally, it was the Professor of Surgical Anatomy, Operations, Dressings andBandages, Vicente Sagarra Lascurain who promoted it at the end of the 19th and early 20th centuries. He wanted to highlight in itits simplicity of use, effectiveness in the umlaut and above all the less bleeding that occurred in the surgical wound in its application,which made it very suitable for use in highly vascular tissues and organs, such as penile amputations. and tongue. The techniquewas used in a not very widespread way and with the passage of time it was forgotten. The work tries to remember from the historicalpoint of view this technique, its use, the indications for use and reference to its results, widely published by its promoter. (AU)

Team Reasoning and the Rational Choice of Payoff-Dominant Outcomes in Games.

Standard game theory cannot explain the selection of payoff-dominant outcomes that are best for all players in common-interest games. Theories of team reasoning can explain why such mutualistic cooperation is rational. They propose that teams can be agents and that individuals in teams can adopt a distinctive mode of reasoning that enables them to do their part in achieving Pareto-dominant outcomes. We show that it can be rational to play payoff-dominant outcomes, given that an agent group identifies. We compare team reasoning to other theories that have been proposed to explain how people can achieve payoff-dominant outcomes, especially with respect to rationality. Some authors have hoped that it would be possible to develop an argument that it is rational to group identify. We identify some large-probably insuperable-problems with this project and sketch some more promising approaches, whereby the normativity of group identification rests on morality.

Variação morfológica no padrão lobar e nas fissuras dos pulmões: relato de caso / Morphological variation in the lobar pattern and in the fissures of the lungs: a case report

Modelo do estudo: Relato de caso. Importância do problema: Lobos e fissuras acessórias nos pulmões podem induzir a erros de interpretação e diagnóstico, além de favorecer a disseminação de patologias como a pneumonia para lobos adjacentes. Dessa forma, a constatação dessas alterações anatômicas pode colaborar em diagnósticos clínicos e procedimentos cirúrgicos. Comentários: Relatamos um caso raro de modificação anatômica nos lobos e fis-suras dos pulmões. O pulmão esquerdo exibia um lobo médio acessório e uma fissura horizontal completa. Por outro lado, o pulmão direito apresentava duas fissuras acessórias incompletas situadas inferiormente na face costal do lobo médio. Além disso, observamos que o hilo do pulmão esquerdo continha dois brônquios lobares (superior e inferior), um brônquio lobar acessório e três artérias pulmonares. O conhecimento das variações anatômicas aqui relatadas é fundamental, pois auxiliam os profissionais da saúde nos diagnósticos e nas decisões terapêuticas e cirúrgicas.(AU)

Study: Case Report. Importance: Lobos and accessory fissures in the lungs can induce errors of interpretation and diagnosis, as well as favor the spread of pathologies such as pneumonia to adjacent lobes. Thus, the confirmation of these anatomical alterations can collaborate in clinical diagnoses and surgical procedures. Comments: We report a rare case of anatomical variation in the lobes and fissures of the lungs. The left lung showed an accessory middle lobe and a complete horizontal fissure. On the other hand, the right lung had two incomplete accessory fissures located inferiorly in the costal face of the middle lobe. In addition, we observed that the left lung hilum contained two lobar bronchi (upper and lower), one accessory lobar bronchus and three pulmonary arteries. Knowledge of the anatomical variations reported here is fundamental, as they help health professionals in diagnoses and therapeutic and surgical decisions (AU)

Clinical Breathing Mechanics Differ Based on Test and Position.

CONTEXT: Altered diaphragm function is linked to decreased core stabilization, postural changes, and decreased function. Two clinical tests used to assess breathing are the Hi-lo and lateral rib expansion (LRE) tests. It is currently unknown how breathing classification based on these tests differ and how their results are affected by varying test positions. OBJECTIVE: To compare the results of breathing tests when conducted in varying test positions. DESIGN: Prospective cross-sectional study. SETTING: University laboratory. PARTICIPANTS: A total of 50 healthy adults (females 31 and males 29; age 29.3 [4.1] y; height 170.0 [10.4] cm; weight70.7 [15.1] kg). INTERVENTION(S): Hi-lo and LRE tests in supine, seated, standing, and half-kneeling body positions. All tests were recorded and later scored by a single examiner. A generalized estimating equations approach with breathing test and body position as factors was used for analysis. Pairwise comparison with Bonferroni correction was used to adjust for multiple tests. Statistical significance was set at P = .05, 2 tailed. MAIN OUTCOME MEASURES: Hi-lo and LRE tests were scored based on the presence or absence of abdominal excursion, LRE, and superior rib cage migration. Following scoring, results were classified as functional or dysfunctional based on observation of these criteria. RESULTS: A significant breathing test × test position interaction (P < .01) was noted, as well as main effects for test (P < .01) and test position (P < .01). All Hi-lo test positions identified significantly more dysfunctional breathers in positions of increased stability demand (P < .01), except between standing and half-kneeling positions (P = .52). In the LRE test, all positions were similar (P > .99) except for half-kneeling, which was significantly different from all other positions (P < .01). CONCLUSIONS: The Hi-lo test and LRE tests assess different breathing mechanics. Clinicians should use these tests in combination to gain a comprehensive understanding of a person's breathing pattern. The Hi-lo test should be administered in multiple testing positions.

Rinomodelación con hilo - Fundamento anatómico / Nasal modelling with thread - Anatomical basis

Este trabajo está enfocado en pacientes que presentan una rinodeformidad mínima, en quienes creo, como cirujanos plásticos, que podemos realizar una intervención quirúrgica mínimamente invasiva mediante una rinomodelación simple con hilo (vicril - mononylon). Para casos de mayor rinodeformidad (giba ósea y cartilaginosa de cualquier tipo) podemos realizar una rinomodelación combinada (resección de giba) con hilo. Existen varias técnicas descriptas de rinomodelación. Esta técnica se basa en la esqueletización de los cartílagos alares con transección lateral, disección limitada del dorso, miotomía del musculo depresor, preservando el ligamento suspensor de la punta a través de incisiones mínimas colocando un hilo, que actúa como tutor y no tensor, desde la columela al rádix nasal, por lo que se obtiene un refi namiento de la punta nasal proyectando la misma y acortando la longitud nasal. También indicada en casos de resección de giba ósea y cartilaginosa realizando una rinomodelación combinada.

Single-Molecule Tracking and Its Application in Biomolecular Binding Detection.

In the past two decades significant advances have been made in single-molecule detection, which enables the direct observation of single biomolecules at work in real time and under physiological conditions. In particular, the development of single-molecule tracking (SMT) microscopy allows us to monitor the motion paths of individual biomolecules in living systems, unveiling the localization dynamics and transport modalities of the biomolecules that support the development of life. Beyond the capabilities of traditional camera-based tracking techniques, state-of-the-art SMT microscopies developed in recent years can record fluorescence lifetime while tracking a single molecule in the 3D space. This multiparameter detection capability can open the door to a wide range of investigations at the cellular or tissue level, including identification of molecular interaction hotspots and characterization of association/dissociation kinetics between molecules. In this review, we discuss various SMT techniques developed to date, with an emphasis on our recent development of the next generation 3D tracking system that not only achieves ultrahigh spatiotemporal resolution but also provides sufficient working depth suitable for live animal imaging. We also discuss the challenges that current SMT techniques are facing and the potential strategies to tackle those challenges.

Single Molecule Approaches in RNA-Protein Interactions.

RNA-protein interactions govern every aspect of RNA metabolism, and aberrant RNA-binding proteins are the cause of hundreds of genetic diseases. Quantitative measurements of these interactions are necessary in order to understand mechanisms leading to diseases and to develop efficient therapies. Existing methods of RNA-protein interactome capture can afford a comprehensive snapshot of RNA-protein interaction networks but lack the ability to characterize the dynamics of these interactions. As all ensemble methods, their resolution is also limited by statistical averaging. Here we discuss recent advances in single molecule techniques that have the potential to tackle these challenges. We also provide a thorough overview of single molecule colocalization microscopy and the essential protein and RNA tagging and detection techniques.

Avaliação física e ultrassonográfica da cicatriz em 12 equinos submetidos à celiotomia mediana com fio de poliéster para tratamento de cólica / Physical and ultrasonographic assessment of scar from celiorrhaphy with polyester thread in horses undergoing treatment of acute abdominal colic / Evaluación física y ecografia de cicatriz en 12 equinos sometidos a celiotomia mediana con hilo de poliéster para tratamiento del cólico

Foi realizado um levantamento das complicações incisionais em 12 equinos submetidos à celiotomia mediana seguida de celiorrafia com fio de poliéster. Os animais foram submetidos a exame clínico e complementar. O exame clínico consistiu na inspeção e palpação da linha média. O método de exame complementar foi a ultrassonografia da linha media na área referente à cicatriz abdominal. Foram encontrados complicações incisionais em sete animais (58,3%). As complicações incisionais encontradas foram fibrose (41,6%), reação ao fio de sutura (41,6%), fístulas (16,6%), edema peri-incisional (25%), infecção incisional (16,6%), drenagem incisional (16,6%). No exame indireto foi observado edema pós-operatório, pequenas áreas focais de líquido ao redor do fio de sutura. Mesmo com as complicações incisionais encontradas e o número de animais examinados, o fio de poliéster mostrou-se resistente e seguro podendo ser utilizado na laparorrafia mediana em equinos.

A survey on the incisional complications in twelve horses submitted to median celiotomy followed with celiorrhaphy with polyester suture was performed. The animals were submitted to clinical and complementary examination. Inspection and palpation of the midline for clinical evaluation was performed. Ultrasonography of the abdominal scar of the midline area was used as an indirect test. Seven animals presented incisional complications (58.3%), such as fibrosis (41.6%), suture material reaction (41.6%), fistulas (16.6%), peri-incisional edema (25%), wound infection (16.6%), and incisional drainage (16.6%). In indirect evaluation, post-operative edema was observed, as well as small focal areas with fluid around the suture. Despite the incisional complications found and the small number of animals examined, it was concluded that polyester suture proved to be resilient and safe, and can be used in median celiorrhaphy in horses.

Una encuesta sobre las complicaciones incisionales en 12 equinos sujetados a una celiotomía mediana seguida de una celiorrafia con hilo de poliéster. Los animales fueron sometidos a exámenes clínicos y complementarios. El examen clínico incluyó la inspección y palpación de la línea mediana. Otro método evaluativo fue la ecografía de la línea media en la zona referente a la cicatriz abdominal. Complicaciones incisionales fueron encontradas en siete animales (58,3%). Las complicaciones incisionales encontradas fueron fibrosis (41,6%), reacción al hilo de sutura (41,6%), fístulas (16,6%), edema perincisional (25%), infecciones incisionales (16,6%) y drenaje incisional (16,6%). En el examen indirecto se ha observado edema postoperatorio, pequeñas áreas focales de líquido alrededor del hilo de sutura. A pesar de las complicaciones incisionales encontradas y el número de animales examinados, el hilo de poliéster demostró resistencia, seguridad y puede ser utilizado en celiorrafia mediana en equinos.

Retinal detachment caused by Arruga suture scleral intrusion. Treatment.

CASE REPORT: We present the case of an 81-year-old man with retinal detachment caused by intrusion of an Arruga suture. The encircling buckle was located in the sub-retinal space and caused retinal breaks with retinal detachment DISCUSSION: A pars plana vitrectomy was performed along with intraocular cutting of the Arruga suture with retinal re-attachment.

Single-molecule imaging of cytoplasmic dynein in vivo.

While early fluorescence microscopy experiments employing fluorescent probes afforded snapshots of the cell, the power of live-cell microscopy is required to understand complex dynamics in biological processes. The first successful cloning of green fluorescent protein in the 1990s paved the way for development of approaches that we now utilize for visualization in a living cell. In this chapter, we discuss a technique to observe fluorescently tagged single molecules in fission yeast. With a few simple modifications to the established total internal reflection fluorescence microscopy, cytoplasmic dynein molecules in the cytoplasm and on the microtubules can be visualized and their intracellular dynamics can be studied. We illustrate a technique to study motor behavior, which is not apparent in conventional ensemble studies of motors. In general, this technique can be employed to study single-molecule dynamics of fluorescently tagged proteins in the cell interior.