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Advanced fluorescence microscopy techniques for the life sciences
摘要： The development of super-resolved fluorescence microscopy, for which the Nobel Prize was awarded in 2014, has been a topic of interest to physicists and biologists alike. It is inevitable that numerous questions in biomedical research cannot be answered by means other than direct observation. In this review, advances to fluorescence microscopy are covered in a widely accessible fashion to facilitate its use in decisions related to its acquisition and utilization in biomedical research.
关键词： Nobel Prize,direct observation,fluorescence microscopy,super-resolution,biomedical research
更新于2025-09-09 09:28:46
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Protein Phosphorylation || FRET-Based Biosensors: Genetically Encoded Tools to Track Kinase Activity in Living Cells
摘要： Fluorescence microscopy is widely used in biology to localize, to track, or to quantify proteins in single cells. However, following particular events in living cells with good spatio-temporal resolution is much more complex. In this context, Forster resonance energy transfer (FRET) biosensors are tools that have been developed to monitor various events such as dimerization, cleavage, elasticity, or the activation state of a protein. In particular, genetically encoded FRET biosensors are strong tools to study mechanisms of activation and activity of a large panel of kinases in living cells. Their principles are based on a conformational change of a genetically encoded probe that modulates the distance between a pair of fluorescent proteins leading to FRET variations. Recent advances in fluorescence microscopy such as fluorescence lifetime imaging microscopy (FLIM) have made the quantification of FRET efficiency easier. This review aims to address the different kinase biosensors that have been developed, how they allow specific tracking of the activity or activation of a kinase, and to give an overview of the future challenging methods to simultaneously track several biosensors in the same system.
关键词： multiplex,biosensor,fluorescence microscopy,FRET,protein conformation,kinase
更新于2025-09-09 09:28:46
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Deep learning in imaging
摘要： Machine learning approaches that include deep learning are moving beyond image classification to change the way images are made. Computers are powerful tools for carrying out tasks such as image classification or identification as well as or better than human experts. Conventional machine learning approaches are widely used for segmentation and phenotyping in fluorescence microscopy. These tools are now being largely outperformed by their deep-learning-based counterparts, some of which are available as user-friendly tools. But a perhaps more astonishing wave of developments has recently come about through the use of deep learning not for image analysis but for image transformation. In these cases, deep convolutional networks are trained to transform one type of image into another. For example, two studies have shown the power of deep learning for the creation of fluorescence micrographs of cells directly from bright-field or phase images, to facilitate multiplexed and longitudinal imaging. Researchers have also used deep learning to go from low signal-to-noise images to high-quality images, which opens the door to extended imaging of even very light-sensitive living organisms. Deep learning can similarly overcome obstacles associated with super-resolution microscopy. Two approaches, ANNA-PALM and DeepSTORM, were developed to improve the speed of localization microscopy, which is one of the major hurdles of the technique. Deep learning can also enable cross-modality imaging, where applications such as a shift from confocal images to stimulated-emission-depletion-microscopy-resolution images could democratize super-resolution imaging. As with any method, the caveats associated with deep learning in such applications, such as the potential for artifacts, must be carefully considered and analyzed. Nevertheless, we think we have seen only the tip of the iceberg, and that deep learning stands to improve all aspects of imaging, from acquisition to analysis.
关键词： image transformation,machine learning,fluorescence microscopy,deep learning,super-resolution microscopy,imaging
更新于2025-09-04 15:30:14
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DNA-Based Super-Resolution Microscopy: DNA-PAINT
摘要： Super-resolution microscopies, such as single molecule localization microscopy (SMLM), allow the visualization of biomolecules at the nanoscale. The requirement to observe molecules multiple times during an acquisition has pushed the field to explore methods that allow the binding of a fluorophore to a target. This binding is then used to build an image via points accumulation for imaging nanoscale topography (PAINT), which relies on the stochastic binding of a fluorescent ligand instead of the stochastic photo-activation of a permanently bound fluorophore. Recently, systems that use DNA to achieve repeated, transient binding for PAINT imaging have become the cutting edge in SMLM. Here, we review the history of PAINT imaging, with a particular focus on the development of DNA-PAINT. We outline the different variations of DNA-PAINT and their applications for imaging of both DNA origamis and cellular proteins via SMLM. Finally, we reflect on the current challenges for DNA-PAINT imaging going forward.
关键词： DNA PAINT,SMLM,DNA origami,DNA,fluorescence microscopy,super-resolution microscopy
更新于2025-09-04 15:30:14
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Confocal Fluorescence Microscopy and Kinetics of the Cr3+-Chromate Ion Oxidation Equilibria at the Solid Liquid Interface
摘要： Silica-borax pearl samples impregnated with 0.17 and 0.64% Cr3+ were characterized by specific surface area measurements, UV-Vis spectroscopy, energy-dispersive X-ray fluorescence and laser-scanning confocal microscopy. Pearl stability against oxidizing conditions was tested by adding samples to an aqueous hydrogen peroxide solution. The reaction was examined by UV-Vis spectroscopic measurements of the supernatant and laser-scanning confocal microscopy images of the substrate. Overall, hydrogen peroxide-induced Cr3+ to Cr6+ oxidation across the solid-liquid interface promoted solid matrix cleavage pearl degradation and concomitant formation of multiple scattering centers was observed. A dual-detection scheme was employed in the confocal microscopy measurements allowing us to separate scattering and absorptive contributions to the observed signals. The confocal microscopy images indicate that Cr3+ oxidation induced by hydrogen peroxide solutions occurs throughout the entire pearl sample and indicate that oxidation reactions induce leakage of chromate ion into aqueous solutions.
关键词： energy dispersive X-ray fluorescence,confocal fluorescence microscopy,chromium mobility,H2O2 oxidation equilibria,mass transfer phenomena,solid liquid interfaces
更新于2025-09-04 15:30:14
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Automated Neuron Reconstruction from 3D Fluorescence Microscopy Images Using Sequential Monte Carlo Estimation
摘要： Microscopic images of neuronal cells provide essential structural information about the key constituents of the brain and form the basis of many neuroscientific studies. Computational analyses of the morphological properties of the captured neurons require first converting the structural information into digital tree-like reconstructions. Many dedicated computational methods and corresponding software tools have been and are continuously being developed with the aim to automate this step while achieving human-comparable reconstruction accuracy. This pursuit is hampered by the immense diversity and intricacy of neuronal morphologies as well as the often low quality and ambiguity of the images. Here we present a novel method we developed in an effort to improve the robustness of digital reconstruction against these complicating factors. The method is based on probabilistic filtering by sequential Monte Carlo estimation and uses prediction and update models designed specifically for tracing neuronal branches in microscopic image stacks. Moreover, it uses multiple probabilistic traces to arrive at a more robust, ensemble reconstruction. The proposed method was evaluated on fluorescence microscopy image stacks of single neurons and dense neuronal networks with expert manual annotations serving as the gold standard, as well as on synthetic images with known ground truth. The results indicate that our method performs well under varying experimental conditions and compares favorably to state-of-the-art alternative methods.
关键词： Sequential Monte Carlo estimation,Bayesian filtering,Neuron reconstruction,Fluorescence microscopy,Particle filtering
更新于2025-09-04 15:30:14
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[IEEE 2018 IEEE International Conference on Electrical Engineering and Photonics (EExPolytech) - Saint Petersburg, Russia (2018.10.22-2018.10.23)] 2018 IEEE International Conference on Electrical Engineering and Photonics (EExPolytech) - Total Internal Reflection Fluorescence Microscopy for Investigation of Dynamics of Single Molecules
摘要： In the work, the study of dynamics of single molecules by means of total internal reflection fluorescence microscopy is suggested. The fluorescence microscopy is directed to detect the single molecules in different fluids. We developed a new experimental scheme of fluorescence microscopy. The developed system based on total internal regime allows studying movements of single chlorophyll particles in the aquatic environment and opens a great prospect for single molecules investigation.
关键词： single molecules,total internal reflection,fluorescence,microscopy
更新于2025-09-04 15:30:14
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High-throughput, non-equilibrium studies of single biomolecules using glass-made nanofluidic devices
摘要： Single-molecule detection schemes offer powerful means to overcome static and dynamic heterogeneity inherent to complex samples. However, probing biomolecular interactions and reactions with high throughput and time resolution remains challenging, often requiring surface-immobilized entities. Here, we introduce glass-made nanofluidic devices for the high-throughput detection of freely-diffusing single biomolecules by camera-based fluorescence microscopy. Nanochannels of 200 nm height and a width of several micrometers confine the movement of biomolecules. Using pressure-driven flow through an array of parallel nanochannels and by tracking the movement of fluorescently labelled DNA oligonucleotides, we observe conformational changes with high throughput. In a device geometry featuring a T-shaped junction of nanochannels, we drive steady-state non-equilibrium conditions by continuously mixing reactants and triggering chemical reactions. We use the device to probe the conformational equilibrium of a DNA hairpin as well as to continuously observe DNA synthesis in real time. Our platform offers a straightforward and robust method for studying reaction kinetics at the single-molecule level.
关键词： fluorescence microscopy,DNA,nanofluidic devices,single-molecule detection,biomolecular interactions
更新于2025-09-04 15:30:14
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SENSING IMMOBILIZED MOLECULES OF STREPTAVIDIN ON A SILICON SURFACE BY MALDI-TOF MASS SPECTROMETRY AND FLUORESCENCE MICROSCOPY
摘要： A hydrogen-terminated Si (111) surface was modified to form an aminoterminated monolayer for immobilization of streptavidin. Cleavage of an N-(ω-undecylenyl)-phthalimide covered surface using hidrazine yields an amino group-modified surface, which serves as a substrate for the attachment of biotin and subsequently streptavidin. We used surface analytical techniques to characterize the surface and to control the course of functionalization before the immobilization of streptavidin. To confirm the presence of the streptavidin Texas red on the surface two powerful techniques available in a standard biochemical laboratory are used, Fluorescence Microscopy and MALDI-TOF that allow us to detect and determine the immobilized streptavidin. This work provides an avenue for the development of devices in which the exquisite binding specificity of biomolecular recognition is directly coupled to a biosensor. In addition, we have demonstrated that MALDI-TOF and fluorescence microscopy are useful techniques for the characterization of silicon functionalized surfaces.
关键词： streptavidin,fluorescence microscopy,MALDI-TOF,biosensor,silicon surface
更新于2025-09-04 15:30:14
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A Comparison of Different Corneal Iontophoresis Protocols for Promoting Transepithelial Riboflavin Penetration
摘要： PURPOSE. To measure corneal riboflavin penetration using different transepithelial iontophoresis protocols. METHODS. Freshly enucleated rabbit eyes were divided into nine treatment groups of 4 eyes. One group, in which 0.1% wt/vol riboflavin was applied for 30 minutes without iontophoresis after corneal epithelial debridement, acted as a control. The remaining groups were treated with an intact epithelium using different riboflavin formulations and varying iontophoresis current, soak, and rinse times. After riboflavin application, eyes were snap frozen in liquid nitrogen. Corneal cross sections 35 lm thick were then imaged immediately by two-photon fluorescence microscopy, using image processing software to quantify stromal riboflavin concentration at different corneal depths. RESULTS. In the epithelium-on iontophoresis treatment groups, greater stromal riboflavin penetration was achieved with higher-concentration riboflavin solutions, greater iontophoresis dosage, and longer solution contact times. A protocol utilizing 0.25% wt/vol riboflavin with benzalkonium chloride (BAC) 0.01% and two cycles of applied current and subsequent soaking (1 mA 5 minutes, soak 5 minutes; 0.5 mA 5 minutes, soak 5 minutes) achieved similar stromal riboflavin penetration to epithelium-off controls. The best-performing non-BAC-containing protocol produced stromal riboflavin penetration approximately 60% that of epithelium-off controls. Riboflavin solutions containing saline resulted in minimal stromal penetration. Riboflavin loading within the epithelium was equivalent to or higher than that in the subjacent stroma, despite rinsing the ocular surface with balanced salt solution. CONCLUSIONS. Modified iontophoresis protocols can significantly improve transepithelial riboflavin penetration in experimental corneal collagen cross-linking.
关键词： iontophoresis,riboflavin,two-photon fluorescence microscopy,corneal cross-linking,transepithelial
更新于2025-09-04 15:30:14