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Surface plasmona??coupled emission imaging for biological applications
摘要： Fluorescence imaging technology has been extensively applied in chemical and biological research profiting from its high sensitivity and specificity. Much attention has been devoted to breaking the light diffraction–limited spatial resolution. However, it remains a great challenge to improve the axial resolution in a way that is accessible in general laboratories. Surface plasmon–coupled emission (SPCE), generated by the interactions between surface plasmons and excited fluorophores in close vicinity of the thin metal film, offers an opportunity for optical imaging with potential application in analysis of molecular and biological systems. Benefiting from the highly directional and distance-dependent properties, SPCE imaging (SPCEi) has displayed excellent performance in bioimaging with improved sensitivity and axial confinement. Herein, we give a brief overview of the development of SPCEi. We describe the unique optical characteristics and constructions of SPCEi systems and highlight recent advances in the use of SPCEi for biological applications. We hope this review provides readers with both the insights and future prospects of SPCEi as a new promising imaging platform for potentially widespread applications in biological research and medical diagnostics.
关键词： Fluorescence microscopy,Directional emission,Background suppression,Surface plasmon–coupled emission imaging,Biological application,Cell imaging
更新于2025-09-23 15:19:57
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Long Range Energy Transfer in Self-Assembled Stacks of Semiconducting Nanoplatelets
摘要： Fluorescent emitters like ions, dye molecules or semiconductor nanoparticles are widely used in opto-electronic devices, usually within densely-packed layers. Their luminescence properties can then be very different from when they are isolated, because of short-range interparticle interactions such as F?rster resonant energy transfer (FRET). Understanding these interactions is crucial to mitigate FRET-related losses and could also lead to new energy transfer strategies. Exciton migration by FRET hopping between consecutive neighbor fluorophores has been evidenced in various systems but was generally limited to distances of tens of nanometers and involved only a few emitters. Here we image self-assembled linear chains of CdSe nanoplatelets (colloidal quantum wells) and demonstrate exciton migration over 500-nm distances, corresponding to FRET hopping over 90 platelets. By comparing a diffusion-equation model to our experimental data, we measure a (1.5 ps)-1 FRET rate, much faster than all decay mechanisms, so that strong FRET-mediated collective photophysical effects can be expected.
关键词： nanoplatelets,exciton transfer,self-assembly,fluorescence microscopy,FRET
更新于2025-09-23 15:19:57
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Adaptive particle representation of fluorescence microscopy images
摘要： Modern microscopes create a data deluge with gigabytes of data generated each second, and terabytes per day. Storing and processing this data is a severe bottleneck, not fully alleviated by data compression. We argue that this is because images are processed as grids of pixels. To address this, we propose a content-adaptive representation of fluorescence microscopy images, the Adaptive Particle Representation (APR). The APR replaces pixels with particles positioned according to image content. The APR overcomes storage bottlenecks, as data compression does, but additionally overcomes memory and processing bottlenecks. Using noisy 3D images, we show that the APR adaptively represents the content of an image while maintaining image quality and that it enables orders of magnitude benefits across a range of image processing tasks. The APR provides a simple and efficient content-aware representation of fluorescence microscopy images.
关键词： fluorescence microscopy,content-aware representation,image processing,data compression,Adaptive Particle Representation
更新于2025-09-23 15:19:57
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In vivo neurovascular response to focused photoactivation of Channelrhodopsin-2
摘要： The rapid growth in the use of optogenetics for neuroscience applications is largely driven by two important advantages: highly specific cellular targeting through genetic manipulations; and precise temporal control of neuronal activation via temporal modulation of the optical stimulation. The difference between the most commonly used stimulation modalities, namely diffused (i.e. synchronous) and focused (i.e. asynchronous) stimulation has not been described. Furthermore, full realization of optogenetics’ potential is hindered by our incomplete understanding of the cellular and network level response to photoactivation. Here we address these gaps by examining the neuronal and cerebrovascular responses to focused and diffuse photostimulation of channelrhodopsin in the Thy1-ChR2 mouse. We presented the responses of photoactivation via 470-nm fiber optic illumination (diffuse) alongside 458-nm raster-scan (focused) stimulation of the barrel field. Local field potentials (LFP) assessment of intracerebral electrophysiology and two-photon fluorescence microscopy measurements of red blood cell (RBC) speed (vRBC) in cortical penetrating vessels revealed ~40% larger LFP responses (p=0.05) and twice as large cerebrovascular responses (p=0.002) under focused vs. diffuse photostimulation (focused: 1.64±0.84mV LFP amplitude and 75±48% increase in vRBC; diffuse: 1.14±0.75mV LFP amplitude and 35±23% increase in vRBC). Compared to diffuse photostimulation, focused photostimulation resulted in a ~65% increase in the yield of cerebrovascular responses (73±10% for focused and 42±29% for diffuse photostimulation) and a doubling of the signal-to-noise ratio of the cerebrovascular response (20.9±14.7 for focused and 10.4±1.4 for diffuse photostimulation). These data reveal important advantages of focused optogenetic photoactivation, which can be easily integrated into single- or two-photon fluorescence microscopy platforms, as a means of assessing neuronal excitability and cerebrovascular reactivity, thus paving the way for broader application of optogenetics in preclinical models of CNS diseases.
关键词： Thy1-ChR2,neurovascular coupling,optogenetics,two-photon fluorescence microscopy
更新于2025-09-19 17:15:36
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Fluorescence microscopy image classification of 2D HeLa cells based on the CapsNet neural network
摘要： The development of computer technology now allows the quick and efficient automatic fluorescence microscopy generation of a large number of images of proteins in specific subcellular compartments using fluorescence microscopy. Digital image processing and pattern recognition technology can easily classify these images, identify the subcellular location of proteins, and subsequently carry out related work such as analysis and investigation of protein function. Here, based on a fluorescence microscopy 2D image dataset of HeLa cells, the CapsNet network model was used to classify ten types of images of proteins in different subcellular compartments. Capsules in the CapsNet network model were trained to capture the possibility of certain features and variants rather than to capture the characteristics of a specific variant. The capsule at the same level predicted the instantiation parameters of the higher level capsule through the transformation matrix, and the higher level capsule became active when multiple dynamic routing forecasts were consistent. Experiments show that using the CapsNet network model to classify 2D HeLa datasets can achieve higher accuracy.
关键词： CapsNet,Image classification,2D HeLa,Subcellular localization,Fluorescence microscopy,Neural network
更新于2025-09-19 17:15:36
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Real-time molecular optical micro-imaging of EGFR mutations using a fluorescent erlotinib based tracer
摘要： Background: EGFR mutations are routinely explored in lung adenocarcinoma by sequencing tumoral DNA. The aim of this study was to evaluate a fluorescent-labelled erlotinib based theranostic agent for the molecular imaging of mutated EGFR tumours in vitro and ex vivo using a mice xenograft model and fibred confocal fluorescence microscopy (FCFM). Methods: The fluorescent tracer was synthesized in our laboratory by addition of fluorescein to an erlotinib molecule. Three human adenocarcinoma cell lines with mutated EGFR (HCC827, H1975 and H1650) and one with wild-type EGFR (A549) were xenografted on 35 Nude mice. MTT viability assay was performed after exposure to our tracer. In vitro imaging was performed at 1 μM tracer solution, and ex vivo imaging was performed on fresh tumours excised from mice and exposed to a 1 μM tracer solution in PBS for 1 h. Real-time molecular imaging was performed using FCFM and median fluorescence intensity (MFI) was recorded for each experiment. Results: MTT viability assay confirmed that addition of fluorescein to erlotinib did not suppress the cytotoxic of erlotinib on tumoral cells. In vitro FCFM imaging showed that our tracer was able to distinguish cell lines with mutated EGFR from those lines with wild-type EGFR (p < 0.001). Ex vivo FCFM imaging of xenografts with mutated EGFR had a significantly higher MFI than wild-type (p < 0.001). At a cut-off value of 354 Arbitrary Units, MFI of our tracer had a sensitivity of 100% and a specificity of 96.3% for identifying mutated EGFR tumours. Conclusion: Real time molecular imaging using fluorescent erlotinib is able to identify ex vivo tumours with EGFR mutations.
关键词： Lung cancer,Erlotinib,Theranostic,EGFR,Epidermal growth factor,Fibred confocal fluorescence microscopy,Molecular imaging
更新于2025-09-19 17:15:36
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Monitoring Neuronal Survival via Longitudinal Fluorescence Microscopy
摘要： Standard cytotoxicity assays, which require the collection of lysates or fixed cells at multiple time points, have limited sensitivity and capacity to assess factors that influence neuronal fate. These assays require the observation of separate populations of cells at discrete time points. As a result, individual cells cannot be followed prospectively over time, severely limiting the ability to discriminate whether subcellular events, such as puncta formation or protein mislocalization, are pathogenic drivers of disease, homeostatic responses, or merely coincidental phenomena. Single-cell longitudinal microscopy overcomes these limitations, allowing the researcher to determine differences in survival between populations and draw causal relationships with enhanced sensitivity. This video guide will outline a representative workflow for experiments measuring single-cell survival of rat primary cortical neurons expressing a fluorescent protein marker. The viewer will learn how to achieve high-efficiency transfections, collect and process images enabling the prospective tracking of individual cells, and compare the relative survival of neuronal populations using Cox proportional hazards analysis.
关键词： fluorescence microscopy,automation,Cell death,transfection,neurodegeneration,survival analysis
更新于2025-09-19 17:15:36
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[Methods in Molecular Biology] Immune Checkpoint Blockade Volume 1913 (Methods and Protocols) || Multispectral Fluorescence Imaging Allows for Distinctive Topographic Assessment and Subclassification of Tumor-Infiltrating and Surrounding Immune Cells
摘要： Histomorphology has significantly changed over the last decades due to technological achievements in immunohistochemistry (IHC) for the visualization of specific proteins and in molecular pathology, particularly in the field of in situ hybridization of small oligonucleotides and amplification of DNA and RNA amplicons. With an increased availability of suitable methods, the demands regarding the observer of histomorphological slides were the supply of complex quantitative data as well as more information about protein expression and cell-cell interactions in tissue sections. Advances in fluorescence-based multiplexed IHC techniques, such as multispectral imaging (MSI), allow the quantification of multiple proteins at the same tissue section. In histopathology, it is a well-known technique for over a decade yet harboring serious problems concerning quantitative preciseness and tissue autofluorescence of multicolor staining when using formalin-fixed, paraffin-embedded (FFPE) tissue specimen. In recent years, milestones in tissue preparation, fluorescent dyes, hardware imaging, and software analysis were achieved including automated tissue segmentation (e.g., tumor vs. stroma) as well as in cellular and subcellular multiparameter analysis. This chapter covers the role that MSI plays in anatomic pathology for the analysis of FFPE tissue sections, discusses the technical aspects of MSI, and provides a review of its application in the characterization of immune cell infiltrates and beyond regarding its prognostic and predictive value and its use for guidance of clinical decisions for immunotherapeutic strategies.
关键词： Phenotyping,Immune cells,Fluorescence microscopy,Multispectral imaging
更新于2025-09-19 17:15:36
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Attenuation correction for confocal laser scanning microscopy and its application in chromatography
摘要： The applicability of confocal laser scanning microscopy is limited e.g. by attenuation of the excitation and the fluorescence emission beam. As a prerequisite for further processing and analysis of the obtained microscopic images, a new method is presented for correcting this attenuation. The correction is based on beam modeling and on a differential form of the modified Beer-Lambert law. It turns out that the intensity decay can be modeled as a double convolution of the microscopic image with the intensities of the excitation semi-beam and the emission beam. Under weak assumptions made for the intensities of the fluorescent radiation and the detected signal, formulas for the attenuation correction and the attenuation simulation are derived. The method traces back to that one published by Roerdink which is modified concerning a more realistic beam modeling, avoiding the so-called weak attenuation expansion, and considering fluorescence excitation throughout the light cone of the excitation beam. The applicability of the method is demonstrated for synthetic examples as well as microscopic images of chromatographic beads. It is shown that the new method can be successfully applied for reconstructing the true fluorophore distribution in specimens even if the microscopic images are affected by strong attenuation.
关键词： shading correction,protein distribution,chromatographic purification,Fluorescence microscopy
更新于2025-09-19 17:13:59
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The Analysis of Lead Phytotoxicity in Seeds Using CO2 Laser Photoacoustic Spectroscopy
摘要： Lead (Pb) is the most prevalent heavy metal pollutant in the natural environment. Pb is not a fundamental element for plants, but they absorb it when it is present in their environment, having no known physiological activity. The aim of our research was to evaluate the efficacy of laser photoacoustic spectroscopy as a tool to monitor changes induced by Pb in plant respiration by highlighting two molecular markers (C2H4 and CO2). To better understand Pb phytotoxicity, we monitored the plantlets evolution as well as the morphology of the root cells. Firstly, we showed that the treatment hinders the plantlet’s development. Furthermore, using laser photoacoustic spectroscopy, we found a decrease in the concentration of C2H4 and CO2 vapors measured in the respiration of treated plants. Finally, fluorescence microscopy results showed that in Pb treated plantlets, the cell roots morphology is clearly altered as compared with the untreated ones. All the results are well correlated and can help further in understanding Pb phytotoxicity.
关键词： fluorescence microscopy,gas molecules,plant respiration measurement,IR spectroscopy,analysis of gases
更新于2025-09-19 17:13:59