In JoVE (1)

Other Publications (12)

Articles by Yuriy Alexandrov in JoVE

 JoVE Biology

Open Source High Content Analysis Utilizing Automated Fluorescence Lifetime Imaging Microscopy

1Photonics Group, Department of Physics, Imperial College London, 2Institute for Chemical Biology, Department of Chemistry, Imperial College London, 3MRC Clinical Sciences Centre, Hammersmith Hospital, 4Chemical Biology Section, Department of Chemistry, Imperial College London, 5Retroscreen Virology Ltd, 6Pfizer Global Research and Development, Pfizer Limited, Sandwich, Kent, UK, 7Centre for Histopathology, Imperial College London


JoVE 55119

Other articles by Yuriy Alexandrov on PubMed

Automated Screening of Neurite Outgrowth

Journal of Biomolecular Screening. Feb, 2003  |  Pubmed ID: 12854994

Outgrowth of neurites in culture is used for assessing neurotrophic activity. Neurite measurements have been performed very slowly using manual methods or more efficiently with interactive image analysis systems. In contrast, medium-throughput and noninteractive image analysis of neurite screens has not been well described. The authors report the performance of an automated image acquisition and analysis system (IN Cell Analyzer 1000) in the neurite assay. Neuro-2a (N2a) cells were plated in 96-well plates and were exposed to 6 conditions of retinoic acid. Immunofluorescence labeling of the cytoskeleton was used to detect neurites and cell bodies. Acquisition of the images was automatic. The image set was then analyzed by both manual tracing and automated algorithms. On 5 relevant parameters (number of neurites, neurite length, total cell area, number of cells, neurite length per cell), the authors did not observe a difference between the automated analysis and the manual analysis done by tracing. These data suggest that the automated system addresses the same biology as human scorers and with the same measurement precision for treatment effects. However, throughput of the automated system is orders of magnitude higher than with manual methods.

Angiogenesis: an Improved in Vitro Biological System and Automated Image-based Workflow to Aid Identification and Characterization of Angiogenesis and Angiogenic Modulators

Assay and Drug Development Technologies. Oct, 2008  |  Pubmed ID: 19035850

Angiogenesis is a general term describing formation of new tube-like microvessel sprouts that are the size of capillary blood vessels. Angiogenesis is fundamental in key stages of embryonic development, organ formation, and wound repair and is also involved in the development and progression of a variety of pathological conditions, including cancer (tumor growth and metastasis), cardiovascular disease, diabetic retinopathy, age-related macular degeneration, atherosclerosis, and rheumatoid arthritis. Because of its diverse roles in key physiological and pathological processes, angiogenesis is an important area of medical research, with a considerable number of angiogenic and anti-angiogenic drugs currently undergoing clinical trials. Cost-effective and efficient screening for potential lead compounds is therefore of prime importance. However, screening methodologies vary in their physiological relevance depending on how faithfully critical aspects of angiogenesis are represented. Cell-based in vitro angiogenesis assays are important tools for screening, which in many cases rely on imaging microscopy to ascertain drug effects. Unfortunately, such screens can be hampered by poorly defined biology, slow image acquisition by manual or semiautomated hardware, and slow data analysis by non-dedicated software. This article describes use of a 96-well microplate in vitro angiogenesis screening system as part of an integrated workflow, comprising (1) setting up the biology in a three-dimensional physiologically relevant system, (2) acquiring a series of image slices ("stacks") using an automated z-stage instrument, (3) collapsing the image stack series into sets of two-dimensional images, (4) segmenting objects of interest, and (5) analyzing the segmentation patterns in order to obtain statistically relevant data.

Microscopy-based HTS Examines the Mechanism of Stress F-actin Fiber Disruption by Cytochalasin D: Orientation Texture Data Collated with Quantitative Kinetic Modeling

Assay and Drug Development Technologies. Aug, 2009  |  Pubmed ID: 19689205

We report a drug dose-response, end-point study of intracellular filamentous actin (F-actin) by automated fluorescence microscopy, complemented with theoretical kinetic simulation of drug action. We highlight the use of an advanced orientation-sensitive image processing procedure ( transform), specially tailored for the detection of ordered filamentous "patches" in cell images. To examine the extent of stress F-actin disruption caused by the drug, we compare the measured response based on the above transformation with the theoretical data obtained from a quantitative model. We show that the assay data are consistent with the first-order mass action kinetics predicted by a basic reaction model. As a concluding remark, we briefly discuss advantages, perspectives, and challenges of conventional fluorescent microscopy within the context of the quantitative high-throughput screening paradigm.

FLIM FRET Technology for Drug Discovery: Automated Multiwell-plate High-content Analysis, Multiplexed Readouts and Application in Situ

Chemphyschem : a European Journal of Chemical Physics and Physical Chemistry. Feb, 2011  |  Pubmed ID: 21337485

A fluorescence lifetime imaging (FLIM) technology platform intended to read out changes in Förster resonance energy transfer (FRET) efficiency is presented for the study of protein interactions across the drug-discovery pipeline. FLIM provides a robust, inherently ratiometric imaging modality for drug discovery that could allow the same sensor constructs to be translated from automated cell-based assays through small transparent organisms such as zebrafish to mammals. To this end, an automated FLIM multiwell-plate reader is described for high content analysis of fixed and live cells, tomographic FLIM in zebrafish and FLIM FRET of live cells via confocal endomicroscopy. For cell-based assays, an exemplar application reading out protein aggregation using FLIM FRET is presented, and the potential for multiple simultaneous FLIM (FRET) readouts in microscopy is illustrated.

Quantification of Cellular Autofluorescence of Human Skin Using Multiphoton Tomography and Fluorescence Lifetime Imaging in Two Spectral Detection Channels

Biomedical Optics Express. Dec, 2011  |  Pubmed ID: 22162820

We explore the diagnostic potential of imaging endogenous fluorophores using two photon microscopy and fluorescence lifetime imaging (FLIM) in human skin with two spectral detection channels. Freshly excised benign dysplastic nevi (DN) and malignant nodular Basal Cell Carcinomas (nBCCs) were excited at 760 nm. The resulting fluorescence signal was binned manually on a cell by cell basis. This improved the reliability of fitting using a double exponential decay model and allowed the fluorescence signatures from different cell populations within the tissue to be identified and studied. We also performed a direct comparison between different diagnostic groups. A statistically significant difference between the median mean fluorescence lifetime of 2.79 ns versus 2.52 ns (blue channel, 300-500 nm) and 2.08 ns versus 1.33 ns (green channel, 500-640 nm) was found between nBCCs and DN respectively, using the Mann-Whitney U test (p < 0.01). Further differences in the distribution of fluorescence lifetime parameters and inter-patient variability are also discussed.

Multiphoton Multispectral Fluorescence Lifetime Tomography for the Evaluation of Basal Cell Carcinomas

PloS One. 2012  |  Pubmed ID: 22984428

We present the first detailed study using multispectral multiphoton fluorescence lifetime imaging to differentiate basal cell carcinoma cells (BCCs) from normal keratinocytes. Images were acquired from 19 freshly excised BCCs and 27 samples of normal skin (in & ex vivo). Features from fluorescence lifetime images were used to discriminate BCCs with a sensitivity/specificity of 79%/93% respectively. A mosaic of BCC fluorescence lifetime images covering >1 mm(2) is also presented, demonstrating the potential for tumour margin delineation. Using 10,462 manually segmented cells from the image data, we quantify the cellular morphology and spectroscopic differences between BCCs and normal skin for the first time. Statistically significant increases were found in the fluorescence lifetimes of cells from BCCs in all spectral channels, ranging from 19.9% (425-515 nm spectral emission) to 39.8% (620-655 nm emission). A discriminant analysis based diagnostic algorithm allowed the fraction of cells classified as malignant to be calculated for each patient. This yielded a receiver operator characteristic area under the curve for the detection of BCC of 0.83. We have used both morphological and spectroscopic parameters to discriminate BCC from normal skin, and provide a comprehensive base for how this technique could be used for BCC assessment in clinical practice.

Automated Fluorescence Lifetime Imaging Plate Reader and Its Application to Förster Resonant Energy Transfer Readout of Gag Protein Aggregation

Journal of Biophotonics. May, 2013  |  Pubmed ID: 23184449

Fluorescence lifetime measurements can provide quantitative readouts of local fluorophore environment and can be applied to biomolecular interactions via Förster resonant energy transfer (FRET). Fluorescence lifetime imaging (FLIM) can therefore provide a high content analysis (HCA) modality to map protein-protein interactions (PPIs) with applications in drug discovery, systems biology and basic research. We present here an automated multiwell plate reader able to perform rapid unsupervised optically sectioned FLIM of fixed and live biological samples and illustrate its potential to assay PPIs through application to Gag protein aggregation during the HIV life cycle. We demonstrate both hetero-FRET and homo-FRET readouts of protein aggregation and report the first quantitative evaluation of a FLIM HCA assay by generating dose response curves through addition of an inhibitor of Gag myristoylation. Z' factors exceeding 0.6 are realised for this FLIM FRET assay.

Rapid Global Fitting of Large Fluorescence Lifetime Imaging Microscopy Datasets

PloS One. 2013  |  Pubmed ID: 23940626

Fluorescence lifetime imaging (FLIM) is widely applied to obtain quantitative information from fluorescence signals, particularly using Förster Resonant Energy Transfer (FRET) measurements to map, for example, protein-protein interactions. Extracting FRET efficiencies or population fractions typically entails fitting data to complex fluorescence decay models but such experiments are frequently photon constrained, particularly for live cell or in vivo imaging, and this leads to unacceptable errors when analysing data on a pixel-wise basis. Lifetimes and population fractions may, however, be more robustly extracted using global analysis to simultaneously fit the fluorescence decay data of all pixels in an image or dataset to a multi-exponential model under the assumption that the lifetime components are invariant across the image (dataset). This approach is often considered to be prohibitively slow and/or computationally expensive but we present here a computationally efficient global analysis algorithm for the analysis of time-correlated single photon counting (TCSPC) or time-gated FLIM data based on variable projection. It makes efficient use of both computer processor and memory resources, requiring less than a minute to analyse time series and multiwell plate datasets with hundreds of FLIM images on standard personal computers. This lifetime analysis takes account of repetitive excitation, including fluorescence photons excited by earlier pulses contributing to the fit, and is able to accommodate time-varying backgrounds and instrument response functions. We demonstrate that this global approach allows us to readily fit time-resolved fluorescence data to complex models including a four-exponential model of a FRET system, for which the FRET efficiencies of the two species of a bi-exponential donor are linked, and polarisation-resolved lifetime data, where a fluorescence intensity and bi-exponential anisotropy decay model is applied to the analysis of live cell homo-FRET data. A software package implementing this algorithm, FLIMfit, is available under an open source licence through the Open Microscopy Environment.

Mesoscopic in Vivo 3-D Tracking of Sparse Cell Populations Using Angular Multiplexed Optical Projection Tomography

Biomedical Optics Express. Apr, 2015  |  Pubmed ID: 25909009

We describe an angular multiplexed imaging technique for 3-D in vivo cell tracking of sparse cell distributions and optical projection tomography (OPT) with superior time-lapse resolution and a significantly reduced light dose compared to volumetric time-lapse techniques. We demonstrate that using dual axis OPT, where two images are acquired simultaneously at different projection angles, can enable localization and tracking of features in 3-D with a time resolution equal to the camera frame rate. This is achieved with a 200x reduction in light dose compared to an equivalent volumetric time-lapse single camera OPT acquisition with 200 projection angles. We demonstrate the application of this technique to mapping the 3-D neutrophil migration pattern observed over ~25.5 minutes in a live 2 day post-fertilisation transgenic LysC:GFP zebrafish embryo following a tail wound.

Visualising Apoptosis in Live Zebrafish Using Fluorescence Lifetime Imaging with Optical Projection Tomography to Map FRET Biosensor Activity in Space and Time

Journal of Biophotonics. Apr, 2016  |  Pubmed ID: 26753623

Fluorescence lifetime imaging (FLIM) combined with optical projection tomography (OPT) has the potential to map Förster resonant energy transfer (FRET) readouts in space and time in intact transparent or near transparent live organisms such as zebrafish larvae, thereby providing a means to visualise cell signalling processes in their physiological context. Here the first application of FLIM OPT to read out biological function in live transgenic zebrafish larvae using a genetically expressed FRET biosensor is reported. Apoptosis, or programmed cell death, is mapped in 3-D by imaging the activity of a FRET biosensor that is cleaved by Caspase 3, which is a key effector of apoptosis. Although apoptosis is a naturally occurring process during development, it can also be triggered in a variety of ways, including through gamma irradiation. FLIM OPT is shown here to enable apoptosis to be monitored over time, in live zebrafish larvae via changes in Caspase 3 activation following gamma irradiation at 24 hours post fertilisation. Significant apoptosis was observed at 3.5 hours post irradiation, predominantly in the head region.

Quantitative in Vivo Optical Tomography of Cancer Progression & Vasculature Development in Adult Zebrafish

Oncotarget. Jul, 2016  |  Pubmed ID: 27259259

We describe a novel approach to study tumour progression and vasculature development in vivo via global 3-D fluorescence imaging of live non-pigmented adult zebrafish utilising angularly multiplexed optical projection tomography with compressive sensing (CS-OPT). This "mesoscopic" imaging method bridges a gap between established ~μm resolution 3-D fluorescence microscopy techniques and ~mm-resolved whole body planar imaging and diffuse tomography. Implementing angular multiplexing with CS-OPT, we demonstrate the in vivo global imaging of an inducible fluorescently labelled genetic model of liver cancer in adult non-pigmented zebrafish that also present fluorescently labelled vasculature. In this disease model, addition of a chemical inducer (doxycycline) drives expression of eGFP tagged oncogenic K-RASV12 in the liver of immune competent animals. We show that our novel in vivo global imaging methodology enables non-invasive quantitative imaging of the development of tumour and vasculature throughout the progression of the disease, which we have validated against established methods of pathology including immunohistochemistry. We have also demonstrated its potential for longitudinal imaging through a study of vascular development in the same zebrafish from early embryo to adulthood. We believe that this instrument, together with its associated analysis and data management tools, constitute a new platform for in vivo cancer studies and drug discovery in zebrafish disease models.

Conformational Transition of FGFR Kinase Activation Revealed by Site-specific Unnatural Amino Acid Reporter and Single Molecule FRET

Scientific Reports. Jan, 2017  |  Pubmed ID: 28045057

Protein kinases share significant structural similarity; however, structural features alone are insufficient to explain their diverse functions. Thus, bridging the gap between static structure and function requires a more detailed understanding of their dynamic properties. For example, kinase activation may occur via a switch-like mechanism or by shifting a dynamic equilibrium between inactive and active states. Here, we utilize a combination of FRET and molecular dynamics (MD) simulations to probe the activation mechanism of the kinase domain of Fibroblast Growth Factor Receptor (FGFR). Using genetically-encoded, site-specific incorporation of unnatural amino acids in regions essential for activation, followed by specific labeling with fluorescent moieties, we generated a novel class of FRET-based reporter to monitor conformational differences corresponding to states sampled by non phosphorylated/inactive and phosphorylated/active forms of the kinase. Single molecule FRET analysis in vitro, combined with MD simulations, shows that for FGFR kinase, there are populations of inactive and active states separated by a high free energy barrier resulting in switch-like activation. Compared to recent studies, these findings support diversity in features of kinases that impact on their activation mechanisms. The properties of these FRET-based constructs will also allow further studies of kinase dynamics as well as applications in vivo.

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