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Clayton, Andrew H. A. "Phase-Sensitive Fluorescence Image Correlation Spectroscopy." International Journal of Molecular Sciences 25, no. 20 (2024): 11165. http://dx.doi.org/10.3390/ijms252011165.
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Fluorescence lifetime imaging microscopy is sensitive to molecular interactions and environments. In homo-dyne frequency-domain fluorescence lifetime imaging microscopy, images of fluorescence objects are acquired at different phase settings of the detector. The detected intensity as a function of detector phase is a sinusoidal function that is sensitive to the lifetime of the fluorescent species. In this paper, the theory of phase-sensitive fluorescence image correlation spectroscopy is described. In this version of lifetime imaging, image correlation spectroscopy analysis (i.e., spatial autocorrelation) is applied to successive fluorescence images acquired at different phase settings of the detector. Simulations of different types of lifetime distributions reveal that the phase-dependent density of fluorescent objects is dependent on the heterogeneity of lifetimes present in the objects. We provide an example of this analysis workflow to a cervical cancer cell stained with a fluorescent membrane probe.
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Wiseman, Paul. "Introduction to Fluorescence and Image Correlation Spectroscopy." Microscopy and Microanalysis 10, S02 (2004): 246–47. http://dx.doi.org/10.1017/s1431927604886483.
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Bates, Ian R., Paul W. Wiseman, and John W. Hanrahan. "Investigating membrane protein dynamics in living cellsThis paper is one of a selection of papers published in this Special Issue, entitled CSBMCB — Membrane Proteins in Health and Disease." Biochemistry and Cell Biology 84, no. 6 (2006): 825–31. http://dx.doi.org/10.1139/o06-189.
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Live cell imaging is a powerful tool for understanding the function and regulation of membrane proteins. In this review, we briefly discuss 4 fluorescence-microscopy-based techniques for studying the transport dynamics of membrane proteins: fluorescence-correlation spectroscopy, image-correlation spectroscopy, fluorescence recovery after photobleaching, and single-particle and (or) molecule tracking. The advantages and limitations of each approach are illustrated using recent studies of an ion channel and cell adhesion molecules.
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Wiseman, P. W., J. C. Bouwer, S. Peltier, and M. H. Ellisman. "High Speed Two Photon Excitation Microscopy in Live Cell Imaging using Image Correlation Spectroscopy (ICS)." Microscopy and Microanalysis 7, S2 (2001): 22–23. http://dx.doi.org/10.1017/s1431927600026180.
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For live-cell imaging, two-photon excitation microscopy (TPEM) is proving to be a significant technological advancement. The unique features offered by TPEM are the ability to image thick sections, excellent optical sectioning capabilities, low damage to living cells, and less out of focus fluorescence and out of focus photobleaching. of these features, the most useful for the biological microscopist, is optical sectioning. Optical sectioning is an intrinsic property of the two-photon process, whereby, two infrared (IR) photons are absorbed quickly to excite a single UV/blue transition. The probability for exciting a two photon transition is proportional to the instantaneous excitation intensity squared. Therefore, for a focused laser beam, only light at the focal point of the excitation beam excites a fluorescent transition. Thus, the need for confocal apertures and time consuming deconvolution algorithms are, for the most part, eliminated.We have continued to develop and enhance our ability to perform high-speed, two-photon excitation fluorescence microscopy. in 1998, we successfully deployed a prototype, video-rate twophoton laser scanning system (30 frames/sec or faster at reduced scan width) developed with support from Nikon Corporation. That system was built upon a Nikon RCM 8000 confocal microscope.
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Laňková, Martina, Jana Humpolíčková, Stanislav Vosolsobě, et al. "Determination of Dynamics of Plant Plasma Membrane Proteins with Fluorescence Recovery and Raster Image Correlation Spectroscopy." Microscopy and Microanalysis 22, no. 2 (2016): 290–99. http://dx.doi.org/10.1017/s1431927616000568.
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AbstractA number of fluorescence microscopy techniques are described to study dynamics of fluorescently labeled proteins, lipids, nucleic acids, and whole organelles. However, for studies of plant plasma membrane (PM) proteins, the number of these techniques is still limited because of the high complexity of processes that determine the dynamics of PM proteins and the existence of cell wall. Here, we report on the usage of raster image correlation spectroscopy (RICS) for studies of integral PM proteins in suspension-cultured tobacco cells and show its potential in comparison with the more widely used fluorescence recovery after photobleaching method. For RICS, a set of microscopy images is obtained by single-photon confocal laser scanning microscopy (CLSM). Fluorescence fluctuations are subsequently correlated between individual pixels and the information on protein mobility are extracted using a model that considers processes generating the fluctuations such as diffusion and chemical binding reactions. As we show here using an example of two integral PM transporters of the plant hormone auxin, RICS uncovered their distinct short-distance lateral mobility within the PM that is dependent on cytoskeleton and sterol composition of the PM. RICS, which is routinely accessible on modern CLSM instruments, thus represents a valuable approach for studies of dynamics of PM proteins in plants.
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Diaspro, Alberto, Giuseppe Chirico, and Maddalena Collini. "Two-photon fluorescence excitation and related techniques in biological microscopy." Quarterly Reviews of Biophysics 38, no. 2 (2005): 97–166. http://dx.doi.org/10.1017/s0033583505004129.
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1. Introduction 982. Historical background of two-photon effects 992.1 2PE 1002.2 Harmonic generation 1002.3 Fluorescence correlation spectroscopy 1003. Basic principles of two-photon excitation of fluorescent molecules and implications for microscopy and spectroscopy 1013.1 General considerations 1013.2 Fluorescence intensity under the 2PE condition 1033.3 Optical consequences of 2PE 1043.4 Saturation effects in 2PE 1083.5 Fluorescence correlation spectroscopy 1093.5.1 Autocorrelation analysis 1103.5.2 Photon-counting histogram analysis 1124. Two-photon-excited probes 1155. Design considerations for a 2PE fluorescence microscope 1195.1 General aspects 1195.2 Descanned and non-descanned 2PE imaging 1215.3 Lens objectives and pulse broadening 1225.4 Laser sources 1255.5 Example of a practical realization 1276. Applications 1346.1 Biological applications of 2PE 1346.1.1 Brain images 1346.1.2 Applications on the kidney 1396.1.3 Mammalian embryos 1396.1.4 Applications to immuno-response 1416.1.5 Myocytes 1416.1.6 Retina 1426.1.7 DNA imaging 1436.1.8 FISH applications 1446.2 2PE imaging of single molecules 1446.3 FCS applications 1486.4 Signals from nonlinear interactions 1517. Conclusions 1538. Acknowledgements 1549. References 155This review is concerned with two-photon excited fluorescence microscopy (2PE) and related techniques, which are probably the most important advance in optical microscopy of biological specimens since the introduction of confocal imaging. The advent of 2PE on the scene allowed the design and performance of many unimaginable biological studies from the single cell to the tissue level, and even to whole animals, at a resolution ranging from the classical hundreds of nanometres to the single molecule size. Moreover, 2PE enabled long-term imaging of in vivo biological specimens, image generation from deeper tissue depth, and higher signal-to-noise images compared to wide-field and confocal schemes. However, due to the fact that up to this time 2PE can only be considered to be in its infancy, the advantages over other techniques are still being evaluated. Here, after a brief historical introduction, we focus on the basic principles of 2PE including fluorescence correlation spectroscopy. The major advantages and drawbacks of 2PE-based experimental approaches are discussed and compared to the conventional single-photon excitation cases. In particular we deal with the fluorescence brightness of most used dyes and proteins under 2PE conditions, on the optical consequences of 2PE, and the saturation effects in 2PE that mostly limit the fluorescence output. A complete section is devoted to the discussion of 2PE of fluorescent probes. We then offer a description of the central experimental issues, namely: choice of microscope objectives, two-photon excitable dyes and fluorescent proteins, choice of laser sources, and effect of the optics on 2PE sensitivity. An inevitably partial, but vast, overview of the applications and a large and up-to-date bibliography terminate the review. As a conclusive comment, we believe that 2PE and related techniques can be considered as a mainstay of the modern biophysical research milieu and a bright perspective in optical microscopy.
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Friaa, Ouided, and Cécile Fradin. "Coincidence Measurements in Dual-Color Confocal Microscopy: A Combined Single-Particle and Fluorescence Correlation Approach." Biophysical Reviews and Letters 09, no. 03 (2014): 249–71. http://dx.doi.org/10.1142/s1793048014400074.
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In this paper we discuss how the coincident detection of mobile particles in dual-color confocal images can be improved. Optimal coincidence detection requires a careful choice of experimental conditions and image acquisition parameters in order to maximize the overlap between the two detection volumes. By measuring this overlap with fluorescence cross-correlation spectroscopy, we show in particular that a small confocal field of view is necessary in order to maintain good coincidence. Most importantly, coincidence detection also requires a dedicated image analysis strategy. Traditionally, two approaches have been adopted to assess coincidence of mobile particles: fluorescence fluctuation measurements, notably cross-correlation spectroscopy, and single particle detection. Here we propose to combine these two approaches by calculating a cross-correlation coefficient for each of the detected single particles. We show that this allows to remove accidental coincidence events from a data set, and thus to unambiguously identify particles that instead carry two different fluorophores. This strategy can help increase the available concentration range for confocal coincidence measurements and detect rare binding events. [Formula: see text]Special Issue Comments: This article about coincident detection of mobile particle in two-color confocal images is thematically related to several articles in this Special Issue, namely the review of FRET-based single-molecule fluorescence techniques by Ruedas-Rama et al.,1 the single particle detection work presented by de Keersmaecker et al.2 and the general considerations on the mathematical treatment of single molecule trajectories presented by Flomenbom3. Our study of single liposomes is also relevant to experiments involving proteins and liposomes, such as the enzyme experiments described in the review by Jørgensen and Hatzakis.4
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Pelicci, Simone, Laura Furia, Mirco Scanarini, Pier Giuseppe Pelicci, Luca Lanzanò, and Mario Faretta. "Novel Tools to Measure Single Molecules Colocalization in Fluorescence Nanoscopy by Image Cross Correlation Spectroscopy." Nanomaterials 12, no. 4 (2022): 686. http://dx.doi.org/10.3390/nano12040686.
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Super Resolution Microscopy revolutionized the approach to the study of molecular interactions by providing new quantitative tools to describe the scale below 100 nanometers. Single Molecule Localization Microscopy (SMLM) reaches a spatial resolution less than 50 nm with a precision in calculating molecule coordinates between 10 and 20 nanometers. However new procedures are required to analyze data from the list of molecular coordinates created by SMLM. We propose new tools based on Image Cross Correlation Spectroscopy (ICCS) to quantify the colocalization of fluorescent signals at single molecule level. These analysis procedures have been inserted into an experimental pipeline to optimize the produced results. We show that Fluorescent NanoDiamonds targeted to an intracellular compartment can be employed (i) to correct spatial drift to maximize the localization precision and (ii) to register confocal and SMLM images in correlative multiresolution, multimodal imaging. We validated the ICCS based approach on defined biological control samples and showed its ability to quantitatively map area of interactions inside the cell. The produced results show that the ICCS analysis is an efficient tool to measure relative spatial distribution of different molecular species at the nanoscale.
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Pandzic, E., and R. M. Whan. "A Practical Guide to Fluorescence Temporal and Spatial Correlation Spectroscopy." Biophysicist 2, no. 1 (2021): 40–69. http://dx.doi.org/10.35459/tbp.2019.000143.
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ABSTRACT The aim of this article is to introduce the basic principles behind the widely used microscopy tool: fluorescence fluctuation correlation spectroscopy (FFCS). We present the fundamentals behind single spot acquisition (FCS) and its extension to spatiotemporal sampling, which is implemented through image correlation spectroscopy (ICS). The article is an educational guide that introduces theoretic concepts of FCS and some of the ICS techniques, followed by interactive exercises in MATLAB. There, the learner can simulate data time series and the application of various FFCS techniques, as well as learn how to measure diffusion coefficients, molecular flow, and concentration of particles. Additionally, each section is followed by a short exercise to reinforce learning concepts by simulating different scenarios, seek verification of outcomes, and make comparisons. Furthermore, we invite the learner throughout the article to consult the literature for different extensions of FFCS techniques that allow measurements of different physicochemical properties of materials. Upon completion of the modules, we anticipate the learner will gain a good understanding in the field of FFCS that will encourage further exploration and adoption of the FFCS tools in future research and educational practices.
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Dal Fovo, Alice, Margherita Morello, Anna Mazzinghi, Caterina Toso, Monica Galeotti, and Raffaella Fontana. "Spectral Mapping Techniques for the Stratigraphic and Compositional Characterisation of a 16th-Century Painting." Heritage 7, no. 3 (2024): 1320–33. http://dx.doi.org/10.3390/heritage7030063.
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Identifying a painting’s pigment palette is crucial for comprehending the author’s technique, as well as for evaluating the degradation of the materials. This paper investigates the stratigraphy and pigments distribution of a 16th-century painting from the Uffizi Galleries collection. Firstly, we obtained compositional information through the cross-sectional analysis of samples using scanning electron microscopy. Secondly, we performed elemental mapping using macro-X-ray fluorescence followed by reflectance imaging spectroscopy. The painting image cube was analysed using the spectral correlation mapping (SCM) classification algorithm to accurately identify the distribution and composition of the pigment mixtures.
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Moens, Pierre D. J., Enrico Gratton, and Iyrri L. Salvemini. "Fluorescence correlation spectroscopy, raster image correlation spectroscopy, and number and brightness on a commercial confocal laser scanning microscope with analog detectors (Nikon C1)." Microscopy Research and Technique 74, no. 4 (2010): 377–88. http://dx.doi.org/10.1002/jemt.20919.
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Cao, Yifan, Behzad Fuladpanjeh-Hojaghan, Milana Trifkovic, and Edward P. L. Roberts. "Operando Visualization of Redox Flow Batteries Using Fluorescence Spectroscopy with Alizarin Red S Electrolyte." ECS Meeting Abstracts MA2024-01, no. 3 (2024): 595. http://dx.doi.org/10.1149/ma2024-013595mtgabs.
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Redox flow batteries (RFBs) charge and discharge by transferring electrons between two pairs of redox species separated by a proton exchange membrane. Unlike traditional batteries that store energy in redox-active materials within an enclosed cell, RFBs store energy in liquid electrolytes housed in external reservoirs1. These electrolytes are pumped through the cell, enabling redox reactions on the surface of electrodes. The energy storage capacity of RFBs is determined by the concentration of the active species and the size of the reservoirs, making them a promising technology for large-scale energy storage applications2. While RFBs possess advantages such as long lifetime, flexible design for battery capacity and power, and rapid response, they suffer from drawbacks like low energy density, power density, and energy efficiency3. Efforts to address these issues have primarily focused on modifications to the electrode materials or the flow field4 , 5. However, evidence supporting performance enhancements often relies on global parameters such as energy efficiency, energy density, and voltage drop, lacking detailed mechanistic and microscopic studies. Real-time observation of the electrode-electrolyte interface during redox reactions and the transport of redox species in the flow field holds the potential to significantly contribute to understanding the electrode structure and mass transport phenomena in RFBs6. This understanding, in turn, facilitates prediction and purposeful enhancement of battery performance by developing desirable structures for both electrode and flow fields. Microscopy, particularly confocal microscopy, serves as a powerful tool for observing microscopic structures. Confocal microscopes yield sharper images with higher resolution than widefield microscopy, enabling rapid acquisition of high-resolution images. In this study, we employed a confocal laser scanning microscope for operando visualization of RFBs. We designed a transparent framework for the battery, allowing light to pass through, and covered its surface with a glass cover to visualize the porous carbon electrode. We selected a fluorescent redox-active species, Alizarin Red S to enable visualization using fluorescence spectroscopy. Leveraging its unique fluorescence properties, we established a correlation between fluorescence intensity and the state of charge. This innovative approach enables us to observe the distribution of electrolytes, the dynamics of redox reactions on the electrode surface (with fast speed of imaging ＜0.2s), the mass transfer behavior of the electrolyte on the electrode surface, and the uniformity of electrolyte flow in the flow field, including areas of stagnation or recirculation. Zhang, J. et al. An all-aqueous redox flow battery with unprecedented energy density. Energy Environ. Sci. 11, 2010–2015 (2018). Rubio-Garcia, J., Kucernak, A. & Charleson, A. Direct visualization of reactant transport in forced convection electrochemical cells and its application to redox flow batteries. Electrochem. commun. 93, 128–132 (2018). Tolmachev, Y. V. Flow Batteries From 1879 To 2022 And Beyond. Qeios 1–79 (2023). Wang, R. et al. Gradient-distributed NiCo2O4 nanorod electrode for redox flow batteries: Establishing the ordered reaction interface to meet the anisotropic mass transport. Appl. Catal. B Environ. 332, 122773 (2023). Kumar, S. & Jayanti, S. Effect of flow field on the performance of an all-vanadium redox flow battery. J. Power Sources 307, 782–787 (2016). Wong, A. A., Rubinstein, S. M. & Aziz, M. J. Direct visualization of electrochemical reactions and heterogeneous transport within porous electrodes in operando by fluorescence microscopy. Cell Reports Phys. Sci. 2, 100388 (2021).
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Waharte, François, Karine Steenkeste, Romain Briandet, and Marie-Pierre Fontaine-Aupart. "Diffusion Measurements inside Biofilms by Image-Based Fluorescence Recovery after Photobleaching (FRAP) Analysis with a Commercial Confocal Laser Scanning Microscope." Applied and Environmental Microbiology 76, no. 17 (2010): 5860–69. http://dx.doi.org/10.1128/aem.00754-10.
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ABSTRACT Research about the reactional and structural dynamics of biofilms at the molecular level has made great strides, owing to efficient fluorescence imaging methods in terms of spatial resolution and fast acquisition time but also to noninvasive conditions of observation consistent with in situ biofilm studies. In addition to conventional fluorescence intensity imaging, the fluorescence recovery after photobleaching (FRAP) module can now be routinely implemented on commercial confocal laser scanning microscopes (CLSMs). This method allows measuring of local diffusion coefficients in biofilms and could become an alternative to fluorescence correlation spectroscopy (FCS). We present here an image-based FRAP protocol to improve the accuracy of FRAP measurements inside “live” biofilms and the corresponding analysis. An original kymogram representation allows control of the absence of perturbing bacterial movement during image acquisition. FRAP data analysis takes into account molecular diffusion during the bleach phase and uses the image information to extract molecular diffusion coefficients. The fluorescence spatial intensity profile analysis used here for the first time with biofilms is supported both by our own mathematical model and by a previously published one. This approach was validated to FRAP experiments on fluorescent-dextran diffusion inside Lactoccocus lactis and Stenotrophomonas maltophilia biofilms, and the results were compared to previously published FCS measurements.
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Lou, Jieqiong, Lorenzo Scipioni, Belinda K. Wright, et al. "Phasor histone FLIM-FRET microscopy quantifies spatiotemporal rearrangement of chromatin architecture during the DNA damage response." Proceedings of the National Academy of Sciences 116, no. 15 (2019): 7323–32. http://dx.doi.org/10.1073/pnas.1814965116.
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To investigate how chromatin architecture is spatiotemporally organized at a double-strand break (DSB) repair locus, we established a biophysical method to quantify chromatin compaction at the nucleosome level during the DNA damage response (DDR). The method is based on phasor image-correlation spectroscopy of histone fluorescence lifetime imaging microscopy (FLIM)-Förster resonance energy transfer (FRET) microscopy data acquired in live cells coexpressing H2B-eGFP and H2B-mCherry. This multiplexed approach generates spatiotemporal maps of nuclear-wide chromatin compaction that, when coupled with laser microirradiation-induced DSBs, quantify the size, stability, and spacing between compact chromatin foci throughout the DDR. Using this technology, we identify that ataxia–telangiectasia mutated (ATM) and RNF8 regulate rapid chromatin decompaction at DSBs and formation of compact chromatin foci surrounding the repair locus. This chromatin architecture serves to demarcate the repair locus from the surrounding nuclear environment and modulate 53BP1 mobility.
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Marshall, Wallace F. "Quantitative High-Throughput Assays for Flagella-Based Motility in Chlamydomonas Using Plate-Well Image Analysis and Transmission Correlation Spectroscopy." Journal of Biomolecular Screening 14, no. 2 (2009): 133–41. http://dx.doi.org/10.1177/1087057108328131.
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Cilia are motile and sensory organelles with important roles in human development, physiology, and disease. Genetic defects in cilia produce a host of disease symptoms, including polycystic kidney disease, hydrocephalus, retinal degeneration, chronic bronchiectasis, infertility, and polydactyly. Currently, there are no known drugs for pharmacological remediation of ciliary defects. Small-molecule modulators of ciliary assembly or function would provide potential lead compounds for drug discovery efforts and would immediately be invaluable tools for a chemical biology approach to studying cilia. Here the author describes 2 assays for ciliary motility that are quantitative, automatable, cost-effective, and simple to implement. Both assays exploit cell-based strategies using the model organism Chlamydomonas reinhardtii. The first assay scores cilia-dependent gravitaxis by analyzing the cell distribution in wells of U-bottom microplates, using a simple and robust image analysis algorithm. The second assay measures motility directly by estimating the time required for cells to swim across a small illuminated aperture using a method equivalent to fluorescence correlation spectroscopy adapted to transmitted-light microscopy. The 2 assays have different advantages in terms of speed and sensitivity to small reductions in motility and may be most efficiently used in combination. ( Journal of Biomolecular Screening 2009:133-141)
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Khare, Devanshi, Pallavi Chandwadkar, and Celin Acharya. "Structural Analysis of Gliding Motility of a Bacteroidetes Bacterium by Correlative Light and Scanning Electron Microscopy (CLSEM)." Microscopy and Microanalysis 28, no. 2 (2022): 515–21. http://dx.doi.org/10.1017/s1431927622000095.
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The members of the Bacteroidetes phylum move on surfaces by gliding motility in the absence of external motility appendages, leading to the formation of spreading colonies. Here, the structural features of the spreading colony were assessed in a uranium-tolerant Bacteroidetes bacterium, Chryseobacterium sp. strain PMSZPI, by using correlative light and scanning electron microscopy (CLSEM). We developed a simple and convenient workflow for CLSEM using a shuttle and find software module and a correlative sample holding slide designed to transport samples between the light/fluorescence microscope (LM/FM) and the scanning electron microscope (SEM) to image spreading colony edges. The datasets from the CLSEM studies allowed convenient examination of the colonial organization by LM/FM followed by ultrastructural analysis by SEM. The regions of interest (ROIs) of the spreading colony edges that were observed in LM/FM in the absence and presence of uranium could be re-identified in the SEM quickly without prolonged searching. Perfect correlation between LM and SEM could be achieved with minimum preparation steps. Subsequently, imaging of the correlated regions was done at higher resolution in SEM to obtain more comprehensive information. We further showed the association of uranium with the gliding PMSZPI cells by energy-dispersive X-ray spectroscopy (EDS) attached to SEM.
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Dobrinskikh, Evgenia, Luca Lanzano, Joanna Rachelson, et al. "Shank2 contributes to the apical retention and intracellular redistribution of NaPiIIa in OK cells." American Journal of Physiology-Cell Physiology 304, no. 6 (2013): C561—C573. http://dx.doi.org/10.1152/ajpcell.00189.2012.
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In renal proximal tubule (PT) cells, sodium-phosphate cotransporter IIa (NaPiIIa) is normally concentrated within the apical membrane where it reabsorbs ∼70% of luminal phosphate (Pi). NaPiIIa activity is acutely regulated by moderating its abundance within the apical membrane. Under low-Pi conditions, NaPiIIa is retained within the apical membrane. Under high-Pi conditions, NaPiIIa is retrieved from the apical membrane and trafficked to the lysosomes for degradation. The present study investigates the role of Shank2 in regulating the distribution of NaPiIIa. In opossum kidney cells, a PT cell model, knockdown of Shank2 in cells maintained in low-Pi media resulted in a marked decrease in NaPiIIa abundance. After being transferred into high-Pi media, live-cell imaging showed that mRFP-Shank2E and GFP-NaPiIIa underwent endocytosis and trafficked together through the subapical domain. Fluorescence cross-correlation spectroscopy demonstrated that GFP-NaPiIIa and mRFP-Shank2 have indistinguishable diffusion coefficients and migrated through the subapical domain in temporal synchrony. Raster image cross-correlation spectroscopy demonstrated these two proteins course through the subapical domain in temporal-spatial synchrony. In the microvilli of cells under low-Pi conditions and in the subapical domain of cells under high-Pi conditions, fluorescence lifetime imaging microscopy-Forster resonance energy transfer analysis of Cer-NaPiIIa and EYFP-Shank2E found these fluors reside within 10 nm of each other. Demonstrating a complexity of functions, in cells maintained under low-Pi conditions, Shank2 plays an essential role in the apical retention of NaPiIIa while under high-Pi conditions Shank2 remains associated with NaPiIIa and escorts NaPiIIa through the cell interior.
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Zhou, Mian, and Yu-Li Wang. "Distinct Pathways for the Early Recruitment of Myosin II and Actin to the Cytokinetic Furrow." Molecular Biology of the Cell 19, no. 1 (2008): 318–26. http://dx.doi.org/10.1091/mbc.e07-08-0783.
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Equatorial organization of myosin II and actin has been recognized as a universal event in cytokinesis of animal cells. Current models for the formation of equatorial cortex favor either directional cortical transport toward the equator or localized de novo assembly. However, this process has never been analyzed directly in dividing mammalian cells at a high resolution. Here we applied total internal reflection fluorescence microscope (TIRF-M), coupled with spatial temporal image correlation spectroscopy (STICS) and a new analytical approach termed temporal differential microscopy (TDM), to image the dynamics of myosin II and actin during the assembly of equatorial cortex. Our results indicated distinct and at least partially independent mechanisms for the early equatorial recruitment of myosin and actin filaments. Cortical myosin showed no detectable directional flow during early cytokinesis. In addition to equatorial assembly, we showed that localized inhibition of disassembly contributed to the formation of the equatorial myosin band. In contrast to myosin, actin filaments underwent a striking flux toward the equator. Myosin motor activity was required for the actin flux, but not for actin concentration in the furrow, suggesting that there was a flux-independent, de novo mechanism for actin recruitment along the equator. Our results indicate that cytokinesis involves signals that regulate both assembly and disassembly activities and argue against mechanisms that are coupled to global cortical movements.
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Saha, Ipsita, and Saveez Saffarian. "Dynamics in HIV Gag Lattice Detected by Time-Lapse iPALM." Proceedings 50, no. 1 (2020): 65. http://dx.doi.org/10.3390/proceedings2020050065.
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Immature HIV virions have a lattice of Gag and Gag-Pol proteins anchored to the lumen of their envelope. This lattice has significant void spaces that were previously characterized by fluorescence correlation spectroscopy, cryoelectron tomography, and iPALM imaging. In the current study, we demonstrate that HIV virus-like particles (VLPs,) assembled by the viral protein Gag tagged at its C terminus with the photoactivable fluorescent protein Dendra, and are of the same size as virus-like particles assembled using only HIV Gag (140 ± 15 nm). We show that the Gag-Dendra lattice observed within these VLPs has similar gaps as those observed in Gag-only VLPs. We further used time-lapse iPALM microscopy to image the Gag–Dendra lattice within the lumen of VLPs at two timepoints. The reconstruction of these time-lapse images shows significant lattice dynamics within the lumen of purified VLPs. The addition of disuccinimidyl suberate (DSS) to the purified VLPs completely abrogated these dynamics. A method to quantify the dynamics of the Gag–Dendra lattice using correlation function analysis is further presented. The HIV Gag lattice, along with the structural lattices of many other viruses, have been mostly considered static. Our study provides an important tool to investigate the dynamics within these lattices and also highlights the effects of fluorescent tags on virion structures.
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Majors, P. D., J. S. McLean, J. K. Fredrickson, and R. A. Wind. "NMR methods for in-situ biofilm metabolism studies: spatial and temporal resolved measurements." Water Science and Technology 52, no. 7 (2005): 7–12. http://dx.doi.org/10.2166/wst.2005.0174.
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We are developing novel nuclear magnetic resonance (NMR) microscopy, spectroscopy and combined NMR/optical techniques for the study of biofilms under known, controlled growth conditions. Objectives include: time and depth-resolved metabolite concentrations with isotropic spatial resolution on the order of 10 microns, metabolic pathways and flux rates, mass transport and ultimately their correlation with gene expression by optical microscopy in biofilms. We describe the implementation of ex-situ grown biofilms to improve growth environment control and NMR analysis. In-situ NMR depth resolved metabolite profiling techniques are introduced and demonstrated for a Shewanella oneidensis strain MR-1 biofilm. Finally, initial combined confocal fluorescence and magnetic resonance images are shown for a GFP-labeled Shewanella biofilm. These methods are equally applicable to other biofilm systems of interest; thus they may provide a significant contribution toward the understanding of adherent cell metabolism.
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Clayton, Andrew H. A. "Spectral Relaxation Imaging Microscopy II: Complex Dynamics." International Journal of Molecular Sciences 24, no. 15 (2023): 12271. http://dx.doi.org/10.3390/ijms241512271.
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The dynamics of condensed matter can be measured by the time-dependent Stokes shift of a suitable fluorescent probe. The time-dependent spectral correlation function is typically described by one or more spectral relaxation correlation times, which, in liquid solvents, characterize the timescales of the dipolar relaxation processes around the excited-state probe. The phasor plot provides a powerful approach to represent and analyze time and frequency-domain data acquired as images, thus providing a spatial map of spectral dynamics in a complex structure such as a living cell. Measurements of the phase and modulation at two emission wavelength channels were shown to be sufficient to extract a single excited-state lifetime and a single spectral relaxation correlation time, supplying estimates of the mean rate of excited-state depopulation and the mean rate of spectral shift. In the present contribution, two more issues were addressed. First, the provision of analytic formulae allowing extraction of the initial generalized polarization and the relaxed generalized polarization, which characterize the fluorescence spectrum of the unrelaxed state and the fully relaxed state. Second, improved methods of model discrimination and model parameter extraction for more complex spectral relaxation phenomena. The analysis workflow was illustrated with examples from the literature.
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Blumhardt, Philipp, Johannes Stein, Jonas Mücksch, et al. "Photo-Induced Depletion of Binding Sites in DNA-PAINT Microscopy." Molecules 23, no. 12 (2018): 3165. http://dx.doi.org/10.3390/molecules23123165.
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The limited photon budget of fluorescent dyes is the main limitation for localization precision in localization-based super-resolution microscopy. Points accumulation for imaging in nanoscale topography (PAINT)-based techniques use the reversible binding of fluorophores and can sample a single binding site multiple times, thus elegantly circumventing the photon budget limitation. With DNA-based PAINT (DNA-PAINT), resolutions down to a few nanometers have been reached on DNA-origami nanostructures. However, for long acquisition times, we find a photo-induced depletion of binding sites in DNA-PAINT microscopy that ultimately limits the quality of the rendered images. Here we systematically investigate the loss of binding sites in DNA-PAINT imaging and support the observations with measurements of DNA hybridization kinetics via surface-integrated fluorescence correlation spectroscopy (SI-FCS). We do not only show that the depletion of binding sites is clearly photo-induced, but also provide evidence that it is mainly caused by dye-induced generation of reactive oxygen species (ROS). We evaluate two possible strategies to reduce the depletion of binding sites: By addition of oxygen scavenging reagents, and by the positioning of the fluorescent dye at a larger distance from the binding site.
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Voskoboynikova, Natalia, Maria Karlova, Rainer Kurre, et al. "A Three-Dimensional Model of the Yeast Transmembrane Sensor Wsc1 Obtained by SMA-Based Detergent-Free Purification and Transmission Electron Microscopy." Journal of Fungi 7, no. 2 (2021): 118. http://dx.doi.org/10.3390/jof7020118.
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The cell wall sensor Wsc1 belongs to a small family of transmembrane proteins, which are crucial to sustain cell integrity in yeast and other fungi. Wsc1 acts as a mechanosensor of the cell wall integrity (CWI) signal transduction pathway which responds to external stresses. Here we report on the purification of Wsc1 by its trapping in water-soluble polymer-stabilized lipid nanoparticles, obtained with an amphipathic styrene-maleic acid (SMA) copolymer. The latter was employed to transfer tagged sensors from their native yeast membranes into SMA/lipid particles (SMALPs), which allows their purification in a functional state, i.e., avoiding denaturation. The SMALPs composition was characterized by fluorescence correlation spectroscopy, followed by two-dimensional image acquisition from single particle transmission electron microscopy to build a three-dimensional model of the sensor. The latter confirms that Wsc1 consists of a large extracellular domain connected to a smaller intracellular part by a single transmembrane domain, which is embedded within the hydrophobic moiety of the lipid bilayer. The successful extraction of a sensor from the yeast plasma membrane by a detergent-free procedure into a native-like membrane environment provides new prospects for in vitro structural and functional studies of yeast plasma proteins which are likely to be applicable to other fungi, including plant and human pathogens.
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Tudor, Mihaela, Roxana Cristina Popescu, Ionela N. Irimescu, et al. "Enhancing Proton Radiosensitivity of Chondrosarcoma Using Nanoparticle-Based Drug Delivery Approaches: A Comparative Study of High- and Low-Energy Protons." International Journal of Molecular Sciences 25, no. 21 (2024): 11481. http://dx.doi.org/10.3390/ijms252111481.
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To overcome chondrosarcoma’s (CHS) high chemo- and radioresistance, we used polyethylene glycol-encapsulated iron oxide nanoparticles (IONPs) for the controlled delivery of the chemotherapeutic doxorubicin (IONPDOX) to amplify the cytotoxicity of proton radiation therapy. Human 2D CHS SW1353 cells were treated with protons (linear energy transfer (LET): 1.6 and 12.6 keV/µm) with and without IONPDOX. Cell survival was assayed using a clonogenic test, and genotoxicity was tested through the formation of micronuclei (MN) and γH2AX foci, respectively. Morphology together with spectral fingerprints of nuclei were measured using enhanced dark-field microscopy (EDFM) assembled with a hyperspectral imaging (HI) module and an axial scanning fluorescence module, as well as scanning electron microscopy (SEM) coupled with energy-dispersive X-Ray spectroscopy (EDX). Cell survival was also determined in 3D SW3153 spheroids following treatment with low-LET protons with/without the IONPDOX compound. IONPDOX increased radiosensitivity following proton irradiation at both LETs in correlation with DNA damage expressed as MN or γH2AX. The IONPDOX–low-LET proton combination caused a more lethal effect compared to IONPDOX–high-LET protons. CHS cell biological alterations were reflected by the modifications in the hyperspectral images and spectral profiles, emphasizing new possible spectroscopic markers of cancer therapy effects. Our findings show that the proposed treatment combination has the potential to improve the management of CHS.
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Kim, Lee, Fujii, et al. "Mitotic Chromosomes in Live Cells Characterized Using High-Speed and Label-Free Optical Diffraction Tomography." Cells 8, no. 11 (2019): 1368. http://dx.doi.org/10.3390/cells8111368.
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The cell nucleus is a three-dimensional, dynamic organelle organized into subnuclear compartments such as chromatin and nucleoli. The structure and function of these compartments are maintained by diffusion and interactions between related factors as well as by dynamic and structural changes. Recent studies using fluorescent microscopic techniques suggest that protein factors can access and are freely mobile in heterochromatin and in mitotic chromosomes, despite their densely packed structure. However, the physicochemical properties of the chromosome during cell division are not fully understood. In the present study, characteristic properties such as the refractive index (RI), volume of the mitotic chromosomes, and diffusion coefficient (D) of fluorescent probes inside the chromosome were quantified using an approach combining label-free optical diffraction tomography with complementary confocal laser-scanning microscopy and fluorescence correlation spectroscopy. Variations in these parameters correlated with osmotic conditions, suggesting that changes in RI are consistent with those of the diffusion coefficient for mitotic chromosomes and cytosol. Serial RI tomography images of chromosomes in live cells during mitosis were compared with three-dimensional confocal micrographs to demonstrate that compaction and decompaction of chromosomes induced by osmotic change were characterized by linked changes in chromosome RI, volume, and the mobilities of fluorescent proteins.
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Fang, Weicheng, Xingxing Cheng, Chang-Jung Sun, Hongfei Xiao, and Jing Wang. "Optimization of the Fabrication of Sustainable Ceramsite Adsorbent from Coal Fly Ash/Waterworks Sludge/Waste Glass for Decolorization of Malachite Green." Adsorption Science & Technology 2023 (February 3, 2023): 1–13. http://dx.doi.org/10.1155/2023/8581697.
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As a traditional dye, malachite green (MG) poses a threat to our environment and health. To decolorize MG, a composite ceramsite adsorbent composed of coal fly ash (CFA), sewage treatment sludge (STS), and waste glass (WG) with a quality ratio of 3 : 3 : 4, respectively, was prepared. The optimal preparation parameters were determined as follows: preheating temperature = 600 ° C , sintering temperature = 1157 ° C , and sintering time = 17 min . Under optimal conditions, scanning electron microscopy (SEM) images show that the X-Com-ceramsite sample exhibits rough features and a porous structure. The obtained X-Com-ceramsite has a good MG decolorization effect (92% decolorization rate with an initial MG concentration of 56.876 mg/L). The q max value of MG can reach up to 37.6 mg/g. The retention degree of MG in the X-Com-ceramsite with a relatively higher pH is stronger, and the adsorption process is spontaneous and endothermic. Synchronous fluorescence, two-dimensional correlation spectroscopy (2D-COS), and Fourier transform infrared spectroscopy (FT-IR) proved that the sensitivity of the C-O/C-O-O functional groups of the carbohydrates on the surface of the X-Com-ceramsite has a higher binding affinity toward MG as the initial concentration of MG changes.
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Kim, Lee, Fujii, Kim, and Pack. "Physicochemical Properties of Nucleoli in Live Cells Analyzed by Label-Free Optical Diffraction Tomography." Cells 8, no. 7 (2019): 699. http://dx.doi.org/10.3390/cells8070699.
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The cell nucleus is three-dimensionally and dynamically organized by nuclear components with high molecular density, such as chromatin and nuclear bodies. The structure and functions of these components are represented by the diffusion and interaction of related factors. Recent studies suggest that the nucleolus can be assessed using various protein probes, as the probes are highly mobile in this organelle, although it is known that they have a densely packed structure. However, physicochemical properties of the nucleolus itself, such as molecular density and volume when cellular conditions are changed, are not yet fully understood. In this study, physical parameters such as the refractive index (RI) and volume of the nucleoli in addition to the diffusion coefficient (D) of fluorescent probe protein inside the nucleolus are quantified and compared by combining label-free optical diffraction tomography (ODT) with confocal laser scanning microscopy (CLSM)-based fluorescence correlation spectroscopy (FCS). 3D evaluation of RI values and corresponding RI images of nucleoli in live HeLa cells successfully demonstrated varying various physiological conditions. Our complimentary method suggests that physical property of the nucleolus in live cell is sensitive to ATP depletion and transcriptional inhibition, while it is insensitive to hyper osmotic pressure when compared with the cytoplasm and nucleoplasm. The result demonstrates that the nucleolus has unique physicochemical properties when compared with other cellular components.
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Breusegem, Sophia Y., Moshe Levi, and Nicholas P. Barry. "Fluorescence Correlation Spectroscopy and Fluorescence Lifetime Imaging Microscopy." Nephron Experimental Nephrology 103, no. 2 (2006): e41-e49. http://dx.doi.org/10.1159/000090615.
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Macháň, Radek, Peter Kapusta, and Martin Hof. "Statistical filtering in fluorescence microscopy and fluorescence correlation spectroscopy." Analytical and Bioanalytical Chemistry 406, no. 20 (2014): 4797–813. http://dx.doi.org/10.1007/s00216-014-7892-7.
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Thompson, N. "Total Internal Reflection with Fluorescence Correlation Spectroscopy." Microscopy and Microanalysis 17, S2 (2011): 38–39. http://dx.doi.org/10.1017/s1431927611001061.
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Arkhipov, Anton, Jana Hüve, Martin Kahms, Reiner Peters, and Klaus Schulten. "Continuous Fluorescence Microphotolysis and Correlation Spectroscopy Using 4Pi Microscopy." Biophysical Journal 93, no. 11 (2007): 4006–17. http://dx.doi.org/10.1529/biophysj.107.107805.
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Watanabe, Takeshi, Yoshinori Iketaki, Takashige Omatsu, Kimihisa Yamamoto, and Masaaki Fujii. "Two-Point Separation in Far-Field Super-Resolution Fluorescence Microscopy Based on Two-Color Fluorescence Dip Spectroscopy, Part I: Experimental Evaluation." Applied Spectroscopy 59, no. 7 (2005): 868–72. http://dx.doi.org/10.1366/0003702054411562.
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The two-point resolution of a novel two-color far-field super-resolution fluorescence microscopy was evaluated by measuring fluorescent beads 100 nm in diameter. This microscopy is based on a combination of two-color fluorescence dip spectroscopy and a phase-modulation technique for a laser beam. By simply introducing two-color laser light, the size of the fluorescent image of a bead was shrunk down to a diameter of 250 nm from the diffraction-limited image with a diameter of 360 nm. For two closely adjacent fluorescent beads with a separation distance of 350 nm, the two-color microscope clearly gave separated fluorescence images, while the conventional one-color fluorescence microscope could not resolve them. It has been proved that our technique breaks Rayleigh's diffraction limit.
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Abbandonato, Gerardo, Katrin Hoffmann, and Ute Resch-Genger. "Determination of quantum yields of semiconductor nanocrystals at the single emitter level via fluorescence correlation spectroscopy." Nanoscale 10, no. 15 (2018): 7147–54. http://dx.doi.org/10.1039/c7nr09332b.
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A microscopy-based method to determine fluorescence quantum yields Φ<sub>F</sub> of dispersed semiconductor nanocrystals at ultralow concentration with fluorescence correlation spectroscopy (FCS) is presented.
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Raarup, Merete Krog, and Jens Randel Nyengaard. "QUANTITATIVE CONFOCAL LASER SCANNING MICROSCOPY." Image Analysis & Stereology 25, no. 3 (2011): 111. http://dx.doi.org/10.5566/ias.v25.p111-120.
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This paper discusses recent advances in confocal laser scanning microscopy (CLSM) for imaging of 3D structure as well as quantitative characterization of biomolecular interactions and diffusion behaviour by means of one- and two-photon excitation. The use of CLSM for improved stereological length estimation in thick (up to 0.5 mm) tissue is proposed. The techniques of FRET (Fluorescence Resonance Energy Transfer), FLIM (Fluorescence Lifetime Imaging Microscopy), FCS (Fluorescence Correlation Spectroscopy) and FRAP (Fluorescence Recovery After Photobleaching) are introduced and their applicability for quantitative imaging of biomolecular (co-)localization and trafficking in live cells described. The advantage of two-photon versus one-photon excitation in relation to these techniques is discussed.
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Morris, Hannah R., Clifford C. Hoyt, and Patrick J. Treado. "Imaging Spectrometers for Fluorescence and Raman Microscopy: Acousto-Optic and Liquid Crystal Tunable Filters." Applied Spectroscopy 48, no. 7 (1994): 857–66. http://dx.doi.org/10.1366/0003702944029820.
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Acousto-optic tunable filters (AOTF) and liquid crystal tunable filters (LCTF) are evaluated for their suitability as fluorescence microscopy imaging spectrometers. AOTFs are solid-state birefringent crystals that provide an electronically tunable spectral notch passband in response to an applied acoustic field. LCTFs also provide a notch passband that can be controlled by incorporating liquid crystal waveplate retarders within a Lyot birefringent filter. In this paper, spectroscopic performance and imaging quality are contrasted by evaluation of model systems. Studies include transmission imaging of standard resolution targets, multispectral fluorescence emission imaging of tagged polystyrene microspheres, and immunofluorescence imaging of neurotransmitters within rat-brainstem thin sections. In addition, the first use of LCTFs for Raman microscopy is demonstrated. Raman microscopy is a noninvasive spectral imaging technique that can provide chemically significant image contrast complementary to fluorescence microscopy without the use of stains or tags.
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Wu, Jun, Zachary R. Donly, Kevin J. Donly, and Steven Hackmyer. "Demineralization Depth Using QLF and a Novel Image Processing Software." International Journal of Dentistry 2010 (2010): 1–7. http://dx.doi.org/10.1155/2010/958264.
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Quantitative Light-Induced fluorescence (QLF) has been widely used to detect tooth demineralization indicated by fluorescence loss with respect to surrounding sound enamel. The correlation between fluorescence loss and demineralization depth is not fully understood. The purpose of this project was to study this correlation to estimate demineralization depth. Extracted teeth were collected. Artificial caries-like lesions were created and imaged with QLF. Novel image processing software was developed to measure the largest percent of fluorescence loss in the region of interest. All teeth were then sectioned and imaged by polarized light microscopy. The largest depth of demineralization was measured by NIH ImageJ software. The statistical linear regression method was applied to analyze these data. The linear regression model wasY=0.32X+0.17, whereXwas the percent loss of fluorescence andYwas the depth of demineralization. The correlation coefficient was 0.9696. The two-tailed t-test for coefficient was 7.93, indicating theP-value=.0014. TheFtest for the entire model was 62.86, which shows theP-value=.0013. The results indicated statistically significant linear correlation between the percent loss of fluorescence and depth of the enamel demineralization.
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Jones, Debbie L., Michael B. Andrews, Adam N. Swinburne, et al. "Fluorescence spectroscopy and microscopy as tools for monitoring redox transformations of uranium in biological systems." Chemical Science 6, no. 9 (2015): 5133–38. http://dx.doi.org/10.1039/c5sc00661a.
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Müller, Claus B., Kerstin Weiß, Walter Richtering, Anastasia Loman, and Joerg Enderlein. "Calibrating Differential Interference Contrast Microscopy with dual-focus Fluorescence Correlation Spectroscopy." Optics Express 16, no. 6 (2008): 4322. http://dx.doi.org/10.1364/oe.16.004322.
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Gray, J., S. Lockett, L. Mascio, et al. "Digital imaging microscopy for molecular cytogenetics." Proceedings, annual meeting, Electron Microscopy Society of America 52 (1994): 82–83. http://dx.doi.org/10.1017/s0424820100168141.
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Digital imaging microscopy is an essential tool in molecular cytogenetics. We describe here, hardware and software for sensitive multi-color image acquisition and display, gene mapping, generation ofcopy number karyotypes using comparative genomic hybridization (CGH), and correlation between genotype and histology in three dimensions.(a) QUantitative Image Processing System (QUIPS):Hardware We have developed a QUIPS that is now in routine use. This system is built around a commercial fluorescence microscope equipped with computer-controlled stage, focus and fluorescence excitation filter selection. A high resolution cooled CCD camera and a video frame rate intensified CCD (ICCD) are attached to different ports on the microscope and light is split between them. The real time images of the ICCD are continuously displayed on a monitor and are used for visual scanning and focusing. The high resolution CCD is used for image acquisition. Images from the CCD are digitized at 12 bits per pixel resulting in high dynamic range and high precision. QUIPS employs a fluorescence filter package containing a beam splitter and emission filter that pass light in bands centered at approximately 440 nm (blue), 525 nm (green) and 600 nm (red).
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Zhang, Pengfei, Lingbo Kong, Guiwen Wang, Peter Setlow, and Yong-qing Li. "Monitoring the Wet-Heat Inactivation Dynamics of Single Spores of Bacillus Species by Using Raman Tweezers, Differential Interference Contrast Microscopy, and Nucleic Acid Dye Fluorescence Microscopy." Applied and Environmental Microbiology 77, no. 14 (2011): 4754–69. http://dx.doi.org/10.1128/aem.00194-11.
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ABSTRACTDynamic processes during wet-heat treatment of individual spores ofBacillus cereus,Bacillus megaterium, andBacillus subtilisat 80 to 90°C were investigated using dual-trap Raman spectroscopy, differential interference contrast (DIC) microscopy, and nucleic acid stain (SYTO 16) fluorescence microscopy. During spore wet-heat treatment, while the spores' 1:1 chelate of Ca2+with dipicolinic acid (CaDPA) was released rapidly at a highly variable timeTlag, the levels of spore nucleic acids remained nearly unchanged, and theTlagtimes for individual spores from the same preparation were increased somewhat as spore levels of CaDPA increased. The brightness of the spores' DIC image decreased by ∼50% in parallel with CaDPA release, and there was no spore cortex hydrolysis observed. The lateral diameters of the spores' DIC image and SYTO 16 fluorescence image also decreased in parallel with CaDPA release. The SYTO 16 fluorescence intensity began to increase during wet-heat treatment at a time beforeTlagand reached maximum at a time slightly later thanTrelease. However, the fluorescence intensities of wet-heat-inactivated spores were ∼15-fold lower than those of nutrient-germinated spores, and this low SYTO 16 fluorescence intensity may be due in part to the low permeability of the dormant spores' inner membranes to SYTO 16 and in part to nucleic acid denaturation during the wet-heat treatment.
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James, Jeemol, Jonas Enger, and Marica B. Ericson. "Fluorescence Correlation Spectroscopy Combined with Multiphoton Laser Scanning Microscopy—A Practical Guideline." Applied Sciences 11, no. 5 (2021): 2122. http://dx.doi.org/10.3390/app11052122.
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Multiphoton laser scanning microscopy (MPM) has opened up an optical window into biological tissues; however, imaging is primarily qualitative. Cell morphology and tissue architectures can be clearly visualized but quantitative analysis of actual concentration and fluorophore distribution is indecisive. Fluorescence correlation spectroscopy (FCS) is a highly sensitive photophysical methodology employed to study molecular parameters such as diffusion characteristics on the single molecule level. In combination with laser scanning microscopy, and MPM in particular, FCS has been referred to as a standard and highly useful tool in biomedical research to study diffusion and molecular interaction with subcellular precision. Despite several proof-of-concept reports on the topic, the implementation of MPM-FCS is far from straightforward. This practical guideline aims to clarify the conceptual principles and define experimental operating conditions when implementing MPM-FCS. Validation experiments in Rhodamine solutions were performed on an experimental MPM-FCS platform investigating the effects of objective lens, fluorophore concentration and laser power. An approach based on analysis of time-correlated single photon counting data is presented. It is shown that the requirement of high numerical aperture (NA) objective lenses is a primary limitation that restricts field of view, working distance and concentration range. Within these restrictions the data follows the predicted theory of Poisson distribution. The observed dependence on laser power is understood in the context of perturbation on the effective focal volume. In addition, a novel interpretation of the effect on measured diffusion time is presented. Overall, the challenges and limitations observed in this study reduce the versatility of MPM-FCS targeting biomedical research in complex and deep tissue—being the general strength of MPM in general. However, based on the systematic investigations and fundamental insights this report can serve as a practical guide and inspire future research, potentially overcoming the technical limitations and ultimately allowing MPM-FCS to become a highly useful tool in biomedical research.
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Sánchez, Susana A., and Enrico Gratton. "Lipid−Protein Interactions Revealed by Two-Photon Microscopy and Fluorescence Correlation Spectroscopy." Accounts of Chemical Research 38, no. 6 (2005): 469–77. http://dx.doi.org/10.1021/ar040026l.
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Korlach, J., P. Schwille, W. W. Webb, and G. W. Feigenson. "Characterization of lipid bilayer phases by confocal microscopy and fluorescence correlation spectroscopy." Proceedings of the National Academy of Sciences 96, no. 15 (1999): 8461–66. http://dx.doi.org/10.1073/pnas.96.15.8461.
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Kudrat-E-Zahan, Md, Salih Zeki Yildiz, and Senem Colak Yazici. "Preparation and Characterization of Highly Fluorescent TGA-CdTe Quantum Dot-Hyamine 1622 Additive Composite." Micro and Nanosystems 12, no. 2 (2020): 92–101. http://dx.doi.org/10.2174/1876402912666200225111411.
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Objective: The aim of the present study was to prepare highly luminescent additive composite polymer with hyamine 1622 and Thioglycolic Acid (TGA) coated CdTe Quantum Dots (QDs). Methods: The additive nano-composite was synthesized by the colloid synthesis method for the first time. The properties like particle size, fluorescence efficiency, fluorescence imaging, self-assembling, quantum dots, encapsulation, etc. were characterized by the employing of instrumental techniques such as 1H and 13C NMR, Fourier Transform Infrared spectroscopy (FT-IR), BAB image analysis system spectroscopy, Atomic Force Microscopy (AFM), Transmission Electron Microscopy (TEM) and Energy Dispersive X-Ray Spectroscopy (EDS). Results: CdTe quantum dots were stabilized successfully in the solid phase by hydrophobic conversion with hyamine 1622 as the cationic surfactant. The experimental results show that the prepared composite is ideal for various applications, easily synthesized, safe, and maintain good fluorescence properties. Conclusion: The newly prepared additive nanocomposite having sharp and narrow excitation/ emission properties is expected to be applicable in biomedical/analytical systems.
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Nie, Shuming, Daniel T. Chiu, and Richard N. Zare. "Real-time observation of single molecules by confocal fluorescence microscopy." Proceedings, annual meeting, Electron Microscopy Society of America 53 (August 13, 1995): 60–61. http://dx.doi.org/10.1017/s0424820100136672.
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The ability to detect, identify, and manipulate individual molecules offer exciting possibilities in many fields, including chemical analysis, materials research, and the biological sciences. A particularly powerful approach is to combine the exquisite sensitivity of laser-induced fluorescence and the spatial localization and imaging capabilities of diffraction-limited or near-field optical microscopes. Unlike scanning tunneling microscopy (STM) and atomic force microscopy (AFM), which lack molecular specificity, optical spectroscopy and microscopy techniques can be used for real-time monitoring and molecular identification at nanometer dimensions or in ultrasmall volumes.We report the use of confocal fluorescence microscopy coupled with a diffraction-limit laser beam and a high-efficiency photodiode for real-time detection of single fluorescent molecules in solution at room temperature. Rigler and Eigen have also demonstrated single-molecule detection with a confocal microscope and fluorescence correlation spectroscopy. The probe (or sampling) volume is effectively an elongated cylinder, with its radius being determined by optical diffraction and length by spherical aberration.
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Cowger, Win, Andrew Gray, Silke H. Christiansen, et al. "Critical Review of Processing and Classification Techniques for Images and Spectra in Microplastic Research." Applied Spectroscopy 74, no. 9 (2020): 989–1010. http://dx.doi.org/10.1177/0003702820929064.
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Microplastic research is a rapidly developing field, with urgent needs for high throughput and automated analysis techniques. We conducted a review covering image analysis from optical microscopy, scanning electron microscopy, fluorescence microscopy, and spectral analysis from Fourier transform infrared (FT-IR) spectroscopy, Raman spectroscopy, pyrolysis gas–chromatography mass–spectrometry, and energy dispersive X-ray spectroscopy. These techniques were commonly used to collect, process, and interpret data from microplastic samples. This review outlined and critiques current approaches for analysis steps in image processing (color, thresholding, particle quantification), spectral processing (background and baseline subtraction, smoothing and noise reduction, data transformation), image classification (reference libraries, morphology, color, and fluorescence intensity), and spectral classification (reference libraries, matching procedures, and best practices for developing in-house reference tools). We highlighted opportunities to advance microplastic data analysis and interpretation by (i) quantifying colors, shapes, sizes, and surface topologies with image analysis software, (ii) identifying threshold values of particle characteristics in images that distinguish plastic particles from other particles, (iii) advancing spectral processing and classification routines, (iv) creating and sharing robust spectral libraries, (v) conducting double blind and negative controls, (vi) sharing raw data and analysis code, and (vii) leveraging readily available data to develop machine learning classification models. We identified analytical needs that we could fill and developed supplementary information for a reference library of plastic images and spectra, a tutorial for basic image analysis, and a code to download images from peer reviewed literature. Our major findings were that research on microplastics was progressing toward the use of multiple analytical methods and increasingly incorporating chemical classification. We suggest that new and repurposed methods need to be developed for high throughput screening using a diversity of approaches and highlight machine learning as one potential avenue toward this capability.
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Gupta, Anjali, Thomas Korte, Andreas Herrmann, and Thorsten Wohland. "Plasma membrane asymmetry of lipid organization: fluorescence lifetime microscopy and correlation spectroscopy analysis." Journal of Lipid Research 61, no. 2 (2019): 252–66. http://dx.doi.org/10.1194/jlr.d119000364.
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A fundamental feature of the eukaryotic cell membrane is the asymmetric arrangement of lipids in its two leaflets. A cell invests significant energy to maintain this asymmetry and uses it to regulate important biological processes, such as apoptosis and vesiculation. The dynamic coupling of the inner or cytoplasmic and outer or exofacial leaflets is a challenging open question in membrane biology. Here, we combined fluorescence lifetime imaging microscopy (FLIM) with imaging total internal reflection fluorescence correlation spectroscopy (ITIR-FCS) to differentiate the dynamics and organization of the two leaflets of live mammalian cells. We characterized the biophysical properties of fluorescent analogs of phosphatidylcholine, sphingomyelin, and phosphatidylserine in the plasma membrane of two mammalian cell lines (CHO-K1 and RBL-2H3). Because of their specific transverse membrane distribution, these probes allowed leaflet-specific investigation of the plasma membrane. We compared the results of the two methods having different temporal and spatial resolution. Fluorescence lifetimes of fluorescent lipid analogs were in ranges characteristic for the liquid ordered phase in the outer leaflet and for the liquid disordered phase in the inner leaflet. The observation of a more fluid inner leaflet was supported by free diffusion in the inner leaflet, with high average diffusion coefficients. The liquid ordered phase in the outer leaflet was accompanied by slower diffusion and diffusion with intermittent transient trapping. Our results show that the combination of FLIM and ITIR-FCS with specific fluorescent lipid analogs is a powerful tool for investigating lateral and transbilayer characteristics of plasma membrane in live cell lines.
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Zhang, Zhimin, Shaocong Liu, Liang Xu, et al. "Super-resolution microscopy based on parallel detection." Journal of Innovative Optical Health Sciences 12, no. 06 (2019): 1950023. http://dx.doi.org/10.1142/s1793545819500238.
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Image scanning microscopy based on pixel reassignment can improve the confocal resolution limit without losing the image signal-to-noise ratio (SNR) greatly [C. J. R. Sheppard, “Super-resolution in confocal imaging,” Optik 80(2) 53–54 (1988). C. B. Müller, E. Jörg, “Image scanning microscopy, “Phys. Rev. Lett. 104(19) 198101 (2010). C. J. R. Sheppard, S. B. Mehta, R. Heintzmann, “Superresolution by image scanning microscopy using pixel reassignment,” Opt. Lett. 38(15) 2889–2892 (2013)]. Here, we use a tailor-made optical fiber and 19 avalanche photodiodes (APDs) as parallel detectors to upgrade our existing confocal microscopy, termed as parallel-detection super-resolution (PDSR) microscopy. In order to obtain the correct shift value, we use the normalized 2D cross correlation to calculate the shifting value of each image. We characterized our system performance by imaging fluorescence beads and applied this system to observing the 3D structure of biological specimen.
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Marriott, Gerard, Shu Mao, Tomoyo Sakata, et al. "Optical lock-in detection imaging microscopy for contrast-enhanced imaging in living cells." Proceedings of the National Academy of Sciences 105, no. 46 (2008): 17789–94. http://dx.doi.org/10.1073/pnas.0808882105.
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One of the limitations on imaging fluorescent proteins within living cells is that they are usually present in small numbers and need to be detected over a large background. We have developed the means to isolate specific fluorescence signals from background by using lock-in detection of the modulated fluorescence of a class of optical probe termed “optical switches.” This optical lock-in detection (OLID) approach involves modulating the fluorescence emission of the probe through deterministic, optical control of its fluorescent and nonfluorescent states, and subsequently applying a lock-in detection method to isolate the modulated signal of interest from nonmodulated background signals. Cross-correlation analysis provides a measure of correlation between the total fluorescence emission within single pixels of an image detected over several cycles of optical switching and a reference waveform detected within the same image over the same switching cycles. This approach to imaging provides a means to selectively detect the emission from optical switch probes among a larger population of conventional fluorescent probes and is compatible with conventional microscopes. OLID using nitrospirobenzopyran-based probes and the genetically encoded Dronpa fluorescent protein are shown to generate high-contrast images of specific structures and proteins in labeled cells in cultured and explanted neurons and in live Xenopus embryos and zebrafish larvae.
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Huang, Jiahao, Peter X. Chen, and Shawn Wettig. "Fluorescence-based techniques to assess the miscibility and physical stability of a drug–lipid complex." Canadian Journal of Chemistry 97, no. 6 (2019): 496–503. http://dx.doi.org/10.1139/cjc-2018-0404.
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The objective of this study was to evaluate the feasibility of using fluorescence-based techniques to assess the miscibility and physical stability of a drug–lipid complex pharmaceutical dosage form under a solvent-free condition. An indomethacin–phospholipid complex (IDM–DPC) was used as model complex for this study. The miscibility of indomethacin within the phospholipid was assessed by fluorescence spectroscopy, fluorescence microscopy, and infrared spectroscopy. The miscibility limit of the complex system was determined by fluorescence to be 20%–30% drug loading content, showing good correlation with infrared spectroscopy. The physical stability of the IDM–DPC stored at 40 °C was evaluated by fluorescence microscopy. Indomethacin formulated in the lipid complex with an indomethacin loading not more than 30% remained in an amorphous state within a period of 21 days, whereas the samples with a drug loading over 30% started to crystallize earlier with increasing drug content. IDM–DPC having higher miscibilities were found to be more resistant to recrystallization under heating, thus having better physical stability. Fluorescence-based techniques showed convenience and promise in characterizing drug–lipid miscibility and predicting storage stability under a solvent-free condition.