Multiphoton fluorescence lifetime imaging microscopy (FLIM) and super-resolution fluorescence imaging with a supramolecular biopolymer for the controlled tagging of polysaccharides
Haobo GeFernando Cortezon‐TamaritHui-Chen WangAdam C. SedgwickRory L. ArrowsmithVincenzo MirabelloStanley W. BotchwayTony D. JamesSofia I. Pascu
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A new coumarin-appended boronate ester for fluorogenic imaging which binds polysaccharides in solution and in cells.Keywords:
Biopolymer
Fluorescence-lifetime imaging microscopy
Fluorescence-lifetime imaging microscopy
Photoactivated localization microscopy
Two-photon excitation microscopy
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Fluorescence microscopy became an invaluable tool in cell biology in the past 20 years. However, the information that lies in these studies is often corrupted by a cellular fluorescence background known as autofluorescence. Since the unspecific background often overlaps with most commonly used labels in terms of fluorescence spectra and fluorescence lifetime, the use of spectral filters in the emission beampath or timegating in fluorescence lifetime imaging (FLIM) is often no appropriate means for distinction between signal and background. Despite the prevalence of fluorescence techniques only little progress has been reported in techniques that specifically suppress autofluorescence or that clearly discriminate autofluorescence from label fluorescence. Fluorescence intensity decay shape analysis microscopy (FIDSAM) is a novel technique which is based on the image acquisition protocol of FLIM. Whereas FLIM spatially resolved maps the average fluorescence lifetime distribution in a heterogeneous sample such as a cell, FIDSAM enhances the dynamic image contrast by determination of the autofluorescence contribution by comparing the fluorescence decay shape to a reference function. The technique therefore makes use of the key difference between label and autofluorescence, i.e. that for label fluorescence only one emitting species contributes to fluorescence intensity decay curves whereas many different species of minor intensity contribute to autofluorescence. That way, we were able to suppress autofluorescence contributions from chloroplasts in Arabidopsis stoma cells and from cell walls in Arabidopsis hypocotyl cells to background level. Furthermore, we could extend the method to more challenging labels such as the cyan fluorescent protein CFP in human fibroblasts.
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Fluorescence-lifetime imaging microscopy
Photoactivated localization microscopy
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Many topically applied drugs are naturally fluorescent, but quantifying their uptake into skin via fluorescence emission is complicated by both weak fluorescence and often-overwhelming skin autofluorescence. Fluorescence lifetime imaging microscopy (FLIM) has been found capable of identifying and separating the fluorescence of multiple drugs from skin autofluorescence via their different spectral and lifetime properties. This investigation was focused on combining epi-fluorescence microscopy with deep learning for the quantification of naturally fluorescent topically applied drugs. This combination of deep learning and fluorescence imaging may allow for straightforward relative quantification of fluorescent drugs in tissue using only simple, readily available imaging tools.
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We are developing a time-gated fluorescence lifetime imaging microscopy (FLIM) instrument for in vivo metabolic imaging of corneal tissues, based on the fluorescence of metabolic co-factor FAD. Here we report on the first results of this project, namely on ex vivo measurements done with a time correlated single photon counting FLIM instrument, and on the first measurements done with the time-gated microscope prototype. Ex-vivo measurements with rat corneas show that it is possible to image FAD fluorescence from corneal epithelial layer and to obtain information from its fluorescence decay parameters that correlates to their metabolic activity. Measurements with test-targets and fluorescence lifetime standards show that the prototype of the time-gated fluorescence lifetime microscope for in vivo FAD imaging has the precision and the accuracy, as well as the timing and lateral spatial resolutions, required for such measurements.
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Rapid and label-free single-leukemia-cell identification through fluorescence lifetime imaging microscopy (FLIM) in the high-density microfluidic trapping array.
Fluorescence-lifetime imaging microscopy
Single-Cell Analysis
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In the prodrug research field, information obtained from traditional end point biochemical assays in drug effect studies could provide neither the dynamic processes nor heterogeneous responses of individual cells. In situ imaging microscopy techniques, especially fluorescence lifetime imaging microscopy (FLIM), could fulfill these requirements. In this work, we used FLIM techniques to observe the entry and release of doxorubicin (Dox)–Cu complexes in live KYSE150 cells. The Dox–Cu complex has weaker fluorescence signals but similar lifetime values as compared to the raw Dox, whose fluorescence could be released by the addition of biothiol compound (such as glutathione). The cell viability results indicated that the Dox–Cu compound has a satisfactory killing effect on KYSE150 cells. The FLIM data showed that free doxorubicin was released from Dox–Cu complexes in cytoplasm of KYSE150 cells and then accumulated in the nucleus. After 90 min administration, the fluorescence lifetime signals reached 1.21 and 1.46 ns in the cytoplasm and nucleus, respectively, reflecting the transformation and transportation of Dox–Cu complexes. In conclusion, this work provides a satisfactory example for the research of prodrug monitored by FLIM techniques, expanding the useful applications of FLIM technique in drug development.
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Fluorescence-lifetime imaging microscopy
Autofluorescence
Live cell imaging
Fluorescence cross-correlation spectroscopy
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The purpose of this study is to demonstrate the application of multiphoton fluorescence and second harmonic generation (SHG) microscopy for the ex-vivo visualization of human corneal morphological alterations due to infectious processes. The structural alterations of both cellular and collagenous components can be respectively demonstrated using fluorescence and SHG imaging. In addition, pathogens with fluorescence may be identified within turbid specimens. Our results show that multiphoton microscopy is effective for identifying structural alterations due to corneal infections without the need of histological processing. With additional developments, multiphoton microscopy has the potential to be developed into an imaging technique effective in the clinical diagnosis and monitoring of corneal infections.
Fluorescence-lifetime imaging microscopy
Two-photon excitation microscopy
Second-harmonic imaging microscopy
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Abstract An overview on fluorescence microscopy with high spatial, spectral and temporal resolution is given. In addition to 3D microscopy based on confocal, structured or single plane illumination, spectral imaging and fluorescence lifetime imaging microscopy (FLIM) are used to probe the interaction of a fluorescent molecule with its micro‐environment. Variable‐angle total internal reflection fluorescence microscopy (TIRFM) permits selective measurements of cell membranes or cell‐substrate topology in the nanometre scale and is also combined with spectral or time‐resolved detection. In addition to single cells or cell monolayers, 3‐dimensional cell cultures are of increasing importance, since they are more similar to tissue morphology and function. All methods reported are adapted to low dose of illumination, which is regarded as a key parameter to maintain cell viability. Applications include cancer diagnosis and cell tomography under different physiological conditions. (© 2011 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)
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Live cell imaging
Single-Cell Analysis
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Fluorescence-lifetime imaging microscopy
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