open-source-high-content-analysis-utilizing-automated-fluorescence

Open Source High Content Analysis Utilizing Automated Fluorescence Lifetime Imaging Microscopy

We present an open source high content analysis instrument utilizing automated fluorescence lifetime imaging (FLIM) for assaying protein interactions ...

Imperial College London

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.

Automated screening of neurite outgrowth.

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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.

Angiogenesis: an improved in vitro biological system and automated image-based workflow to aid identification and characterization of angiogenesis and angiogenic modulators.

L&#39;angiogen&egrave;se est un terme g&eacute;n&eacute;ral qui d&eacute;crit la formation de nouvelles pousses microvaisseaux tubulaires qui sont de la taille des vaisseaux sanguins capillaires. L&#39;angiogen&egrave;se est fondamentale dans les &eacute;tapes cl&eacute;s du d&eacute;veloppement embryonnaire, formation des organes et la r&eacute;paration de la plaie et est aussi impliqu&eacute; dans le d&eacute;veloppement et la progression d&#39;une vari&eacute;t&eacute; de conditions pathologiques, y compris le cancer (croissance de la tumeur et les m&eacute;tastases), maladies cardiovasculaires, la r&eacute;tinopathie diab&eacute;tique, age-related macular degeneration, ath&eacute;roscl&eacute;rose et la polyarthrite rhumato&iuml;de. En raison de ses divers r&ocirc;les dans les principaux processus physiologiques et pathologiques, l&#39;angiogen&egrave;se est un domaine important de la recherche m&eacute;dicale, avec un nombre consid&eacute;rable d&#39;angiog&eacute;nique et actuellement l&#39;objet d&#39;essais cliniques des m&eacute;dicaments anti-angiog&eacute;niques. Rentable et efficace de d&eacute;pistage pour les compos&eacute;s de plomb potentiels est donc primordiale. Cependant, les m&eacute;thodes de d&eacute;pistage varient dans leur pertinence physiologique selon les aspects critiques comment fid&egrave;lement de l&#39;angiogen&egrave;se sont repr&eacute;sent&eacute;s. Dosages d&#39;angiogen&egrave;se in vitro sur les cellules sont des outils importants pour le d&eacute;pistage, qui, dans bien des cas, s&#39;appuient sur l&#39;imagerie microscopie pour d&eacute;terminer les effets de la drogue. Malheureusement, ces &eacute;crans peuvent &ecirc;tre entrav&eacute;es par la biologie mal d&eacute;finie, lente acquisition d&#39;images de mat&eacute;riel manuel ou semi-automatique et analyse des donn&eacute;es lente par le logiciel non d&eacute;di&eacute;es. Cet article d&eacute;crit l&#39;utilisation d&#39;une microplaque 96 puits in vitro de l&#39;angiogen&egrave;se, syst&egrave;me de contr&ocirc;le dans le cadre d&#39;un flux de travail int&eacute;gr&eacute;, comprenant (1) mise en place de la biologie dans un syst&egrave;me tridimensionnel physiologiquement pertinent, (2) acquisition d&#39;une s&eacute;rie de tranches de l&#39;image (&quot;stacks&quot;) &agrave; l&#39;aide d&#39;un instrument automatis&eacute; z-sc&egrave;ne, (3) s&#39;effondrer l&#39;image pile s&eacute;rie en ensembles d&#39;images bidimensionnelles, (4) segmenter les objets d&#39;int&eacute;r&ecirc;tet (5) en analysant les mod&egrave;les de segmentation afin d&#39;obtenir des donn&eacute;es statistiquement pertinentes.

Angiogen&egrave;se : un syst&egrave;me biologique in vitro am&eacute;lior&eacute; et des workflow automatis&eacute; bas&eacute; sur l&#39;image pour faciliter l&#39;identification et la caract&eacute;risation de l&#39;angiogen&egrave;se et angiog&eacute;niques modulateurs.

Angiogenese ist ein allgemeiner Begriff, beschreibt die Bildung von neuen schlauchf&ouml;rmige Microvessel Sprossen, die die Gr&ouml;&szlig;e der Kapillare Blutgef&auml;&szlig;e. Angiogenese ist fundamental in entscheidenden Phasen der Embryonalentwicklung, Orgel-Bildung und Wunde Reparatur und engagiert sich auch in der Entwicklung und Progression von einer Vielzahl von Erkrankungen, Krebs (Tumorwachstum und Metastasierung) Herz-Kreislauf-Erkrankungen, Diabetische Retinopathie, altersbedingter Makula-Degeneration, Atherosklerose und rheumatoider Arthritis. Aufgrund seiner vielf&auml;ltigen Rollen in wichtigen physiologischer und pathologischer Prozesse Angiogenese ist ein wichtiger Bereich der medizinischen Forschung, mit einer betr&auml;chtlichen Anzahl von Angiogenese und Anti-angiogenen Drogen derzeit in klinische Studien. Kosteng&uuml;nstige und effiziente Screening f&uuml;r potenzielle Bleiverbindungen ist daher von gr&ouml;&szlig;ter Bedeutung. Screening-Methoden unterscheiden sich jedoch in ihrer physiologischen Bedeutung je nachdem wie gewissenhaft kritische Aspekte der Angiogenese vertreten sind. In-vitro Angiogenese zellbasierte Assays sind wichtige Instrumente f&uuml;r die Pr&uuml;fung, die in vielen F&auml;llen verlassen sich auf imaging Mikroskopie um Drogen Wirkungen festzustellen. Leider solche Bildschirme k&ouml;nnen durch schlecht definierter Biologie behindert werden, Bilderfassung durch manuelle oder semiautomated Hardware und langsame Datenanalyse nicht dedizierte Software verlangsamen. Dieser Artikel beschreibt die Verwendung von einer 96-Well-Mikroplatten in-vitro Angiogenese screening System als Teil einer integrierten Workflows, bestehend aus (1) die Einrichtung der Biologie in einem dreidimensionalen physiologisch relevanten System (2) Erwerb einer Reihe von Bildsegmente (&quot;Stacks&quot;) mit automatisierten Z-B&uuml;hne Instrument, in Gruppen von zweidimensionalen Bildern, (4) segmentieren Sehensw&uuml;rdigkeiten Serie Stapeln (3) reduzieren das Bild, und (5) analysieren die Segmentierung-Muster um statistisch relevante Daten zu erhalten.

Angiogenese: ein verbesserter in-vitro-biologische System und automatisierten Image-basierte Workflow zur Identifizierung und Charakterisierung von Angiogenese und angiogenen Modulatoren Unterst&uuml;tzung.

L&#39;angiogenesi &egrave; un termine generale che descrive la formazione di nuovi germogli microvessel tube-like che sono le dimensioni dei vasi sanguigni capillari. L&#39;angiogenesi &egrave; fondamentale nelle fasi di sviluppo embrionale, formazione organo e la riparazione di ferite ed &egrave; anche coinvolto nello sviluppo e nella progressione di una variet&agrave; di condizioni patologiche, tra cui il cancro (crescita del tumore e metastasi), malattie cardiovascolari, retinopatia diabetica, degenerazione maculare legata all&#39;et&agrave;, aterosclerosi e l&#39;artrite reumatoide. A causa del suoi diversi ruoli chiave dei processi fisiologici e patologici, l&#39;angiogenesi &egrave; un&#39;importante area di ricerca medica, con un numero considerevole di angiogenico e anti-angiogenic farmaci attualmente in fase di sperimentazione clinica. Screening efficienti per potenziali composti del piombo &egrave; pertanto di primaria importanza. Tuttavia, le metodologie di screening variano nella loro rilevanza fisiologica a seconda di come fedelmente criticit&agrave; dell&#39;angiogenesi sono rappresentati. L&#39;angiogenesi in vitro Cell-based assays sono strumenti importanti per lo screening, che in molti casi si basano su imaging microscopia per accertare gli effetti della droga. Purtroppo, tali schermi pu&ograve; essere ostacolata dalla biologia mal definito, lenta acquisizione immagine da hardware manuale o semiautomatica e analisi dei dati lento dal software non dedicato. Questo articolo descrive l&#39;uso di una micropiastra 96 pozzetti in vitro angiogenesi sistema di screening come parte di un flusso di lavoro integrato, composto da (1) impostare la biologia in un sistema tridimensionale fisiologicamente rilevante, (2) acquisendo una serie di fette di immagine (&quot;stack&quot;) utilizzando uno strumento automatizzato z-fase, (3) compressione immagine stack serie nel set di immagini bidimensionali, (4) segmentazione di oggetti di interessee (5) analizzando i modelli di segmentazione per ottenere dati statisticamente rilevanti.

Angiogenesi: un migliorato sistema biologico in vitro e automatizzato workflow basato su immagini per facilitare l&#39;identificazione e caratterizzazione di angiogenesi e angiogenic modulatori.

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&#x30DE;&#x30A4;&#x30AF;&#x30ED; &#x30D7;&#x30EC;&#x30FC;&#x30C8;&#x306E;&#x4F7F;&#x7528;&#x306B;&#x3064;&#x3044;&#x3066;&#x8AAC;&#x660E;&#x3057;&#x307E;&#x3059;&#x3002;&#x3001;&#x3068; &#xFF08;5&#xFF09; &#x5206;&#x6790;&#x5206;&#x5272;&#x30D1;&#x30BF;&#x30FC;&#x30F3; &#x30C7;&#x30FC;&#x30BF;&#x3092;&#x7D71;&#x8A08;&#x7684;&#x306B;&#x53D6;&#x5F97;&#x3059;&#x308B;&#x305F;&#x3081;&#x306B;&#x3002;

&#x8840;&#x7BA1;&#x65B0;&#x751F;: in vitro &#x3067;&#x306E;&#x751F;&#x7269;&#x5B66;&#x7684;&#x30B7;&#x30B9;&#x30C6;&#x30E0;&#x306E;&#x6539;&#x826F;&#x3068;&#x8840;&#x7BA1;&#x65B0;&#x751F;&#x3068;&#x8840;&#x7BA1;&#x65B0;&#x751F;&#x306E;&#x5909;&#x8ABF;&#x5668;&#x306E;&#x540C;&#x5B9A;&#x3068;&#x89E3;&#x6790;&#x3092;&#x652F;&#x63F4;&#x3059;&#x308B;&#x305F;&#x3081;&#x306B;&#x81EA;&#x52D5;&#x5316;&#x3055;&#x308C;&#x305F;&#x30A4;&#x30E1;&#x30FC;&#x30B8; &#x30D9;&#x30FC;&#x30B9;&#x306E;&#x30EF;&#x30FC;&#x30AF;&#x30D5;&#x30ED;&#x30FC;&#x3002;

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La angiog&eacute;nesis es un t&eacute;rmino general que describe la formaci&oacute;n de nuevos brotes de tubo microvasos que son del tama&ntilde;o de los vasos sangu&iacute;neos capilares. Angiog&eacute;nesis es fundamental en las etapas clave del desarrollo embrionario, formaci&oacute;n de &oacute;rganos y la reparaci&oacute;n de la herida y tambi&eacute;n participa en el desarrollo y la progresi&oacute;n de una variedad de condiciones patol&oacute;gicas, incluyendo c&aacute;ncer (crecimiento tumoral y la met&aacute;stasis), enfermedades cardiovasculares, retinopat&iacute;a diab&eacute;tica, degeneraci&oacute;n macular senil, aterosclerosis y artritis reumatoide. Debido a sus diversos papeles en claves procesos fisiol&oacute;gicos y patol&oacute;gicos, la angiog&eacute;nesis es un &aacute;rea importante de investigaci&oacute;n m&eacute;dica, con un n&uacute;mero considerable de angiog&eacute;nicos y antiangiog&eacute;nicos drogas actualmente en ensayos cl&iacute;nicos. Proyecci&oacute;n rentable y eficiente para potenciales compuestos de plomo, resulta de vital importancia. Sin embargo, metodolog&iacute;as de investigaci&oacute;n var&iacute;an en su importancia fisiol&oacute;gica dependiendo de los aspectos m&aacute;s importantes como fiel de la angiog&eacute;nesis est&aacute;n representados. Ensayos in vitro Cell-based de la angiog&eacute;nesis son herramientas importantes para la proyecci&oacute;n, que en muchos casos se basan en im&aacute;genes de microscopia para determinar los efectos de la droga. Desafortunadamente, estas pantallas puede verse obstaculizado por la biolog&iacute;a mal definido, lento de adquisici&oacute;n de imagen por hardware manual o semiautom&aacute;tica y an&aacute;lisis de datos lentas por software no dedicados. Este art&iacute;culo describe el uso de una microplaca de 96 pocillos in vitro angiog&eacute;nesis sistema de detecci&oacute;n como parte de un flujo de trabajo integrado, compuesto por (1) establecer la biolog&iacute;a en un sistema tridimensional fisiol&oacute;gicamente relevante, (2) adquirir una serie de segmentos de la imagen (las &quot;pilas&quot;) utilizando un instrumento automatizado z-etapa, (3) colapsando la imagen pila serie en conjuntos de im&aacute;genes bidimensionales, (4) segmentar objetos de inter&eacute;sy (5) el an&aacute;lisis de los patrones de segmentaci&oacute;n para obtener datos estad&iacute;sticamente relevantes.

Angiog&eacute;nesis: un sistema biol&oacute;gico in vitro mejorado y automatizado flujo de trabajo basado en im&aacute;genes para facilitar la identificaci&oacute;n y caracterizaci&oacute;n de moduladores de la angiog&eacute;nesis y angiog&eacute;nicos.

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 (<cos(2)theta> 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.

Microscopy-based HTS examines the mechanism of stress F-actin fiber disruption by cytochalasin D: orientation texture data collated with quantitative kinetic modeling.

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.

FLIM FRET technology for drug discovery: automated multiwell-plate high-content analysis, multiplexed readouts and application in situ.

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.

Quantification of cellular autofluorescence of human skin using multiphoton tomography and fluorescence lifetime imaging in two spectral detection channels.

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.

Multiphoton multispectral fluorescence lifetime tomography for the evaluation of basal cell carcinomas.

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.

Automated fluorescence lifetime imaging plate reader and its application to Förster resonant energy transfer readout of Gag protein aggregation.

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.

Rapid global fitting of large fluorescence lifetime imaging microscopy datasets.

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.

Mesoscopic in vivo 3-D tracking of sparse cell populations using angular multiplexed optical projection tomography.

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.

Visualising apoptosis in live zebrafish using fluorescence lifetime imaging with optical projection tomography to map FRET biosensor activity in space and time.

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.

Quantitative in vivo optical tomography of cancer progression & vasculature development in adult zebrafish.

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.

Article	Year
Automated screening of neurite outgrowth. Journal of biomolecular screening\| PubMed ID: 12854994	2003
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\| PubMed ID: 19035850	2008
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\| PubMed ID: 19689205	2009
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\| PubMed ID: 21337485	2011
Quantification of cellular autofluorescence of human skin using multiphoton tomography and fluorescence lifetime imaging in two spectral detection channels. Biomedical optics express\| PubMed ID: 22162820	2011
Multiphoton multispectral fluorescence lifetime tomography for the evaluation of basal cell carcinomas. PloS one\| PubMed ID: 22984428	2012
Automated fluorescence lifetime imaging plate reader and its application to Förster resonant energy transfer readout of Gag protein aggregation. Journal of biophotonics\| PubMed ID: 23184449	2013
Rapid global fitting of large fluorescence lifetime imaging microscopy datasets. PloS one\| PubMed ID: 23940626	2013
Mesoscopic in vivo 3-D tracking of sparse cell populations using angular multiplexed optical projection tomography. Biomedical optics express\| PubMed ID: 25909009	2015
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\| PubMed ID: 26753623	2016
Quantitative in vivo optical tomography of cancer progression & vasculature development in adult zebrafish. Oncotarget\| PubMed ID: 27259259	2016
Conformational transition of FGFR kinase activation revealed by site-specific unnatural amino acid reporter and single molecule FRET. Scientific reports\| PubMed ID: 28045057	2017

Yuriy Alexandrov

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