494 results found
Soloviev VY, McGinty J, Stuckey DW, et al., 2011, Förster resonance energy transfer imaging in vivo with approximated radiative transfer equation, Applied Optics, Vol: 50, Pages: 6583-6590
We describe a new light transport model, which was applied to three-dimensional lifetime imaging of Förster resonance energy transfer in mice in vivo. The model is an approximation to the radiative transfer equation and combines light diffusion and ray optics. This approximation is well adopted to wide-field time-gated intensity-based data acquisition. Reconstructed image data are presented and compared with results obtained by using the telegraph equation approximation. The new approach provides improved recovery of absorption and scattering parameters while returning similar values for the fluorescence parameters.
Patalay R, Talbot C, Alexandrov Y, et al., 2011, Non-invasive imaging of skin cancer with fluorescence lifetime imaging using two photon tomography, Optics InfoBase Conference Papers
Multispectral fluorescence lifetime imaging (FLIM) using two photon microscopy as a non-invasive technique for the diagnosis of skin lesions is described. Skin contains fluorophores including elastin, keratin, collagen, FAD and NADH. This endogenous contrast allows tissue to be imaged without the addition of exogenous agents and allows the in vivo state of cells and tissues to be studied. A modified DermaInspect® multiphoton tomography system was used to excite autofluorescence at 760 nm in vivo and on freshly excised ex vivo tissue. This instrument simultaneously acquires fluorescence lifetime images in four spectral channels between 360-655 nm using time-correlated single photon counting and can also provide hyperspectral images. The multispectral fluorescence lifetime images were spatially segmented and binned to determine lifetimes for each cell by fitting to a double exponential lifetime model. A comparative analysis between the cellular lifetimes from different diagnoses demonstrates significant diagnostic potential. © 2011 SPIE-OSA.
Patalay R, Talbot C, Alexandrov Y, et al., 2011, Quantification of cellular autofluorescence of human skin using multiphoton tomography and fluorescence lifetime imaging in two spectral detection channels, BIOMEDICAL OPTICS EXPRESS, Vol: 2, Pages: 3295-3308, ISSN: 2156-7085
Brown ACN, Oddos S, Dobbie IM, et al., 2011, Remodelling of Cortical Actin Where Lytic Granules Dock at Natural Killer Cell Immune Synapses Revealed by Super-Resolution Microscopy, PLOS BIOLOGY, Vol: 9, ISSN: 1544-9173
Benati E, Bellini V, Borsari S, et al., 2011, Quantitative evaluation of healthy epidermis by means of multiphoton microscopy and fluorescence lifetime imaging microscopy, SKIN RESEARCH AND TECHNOLOGY, Vol: 17, Pages: 295-303, ISSN: 0909-752X
Talbot CB, Patalay R, Munro I, et al., 2011, Application of ultrafast gold luminescence to measuring the instrument response function for multispectral multiphoton fluorescence lifetime imaging., Opt Express, Vol: 19, Pages: 13848-13861
When performing multiphoton fluorescence lifetime imaging in multiple spectral emission channels, an instrument response function must be acquired in each channel if accurate measurements of complex fluorescence decays are to be performed. Although this can be achieved using the reference reconvolution technique, it is difficult to identify suitable fluorophores with a mono-exponential fluorescence decay across a broad emission spectrum. We present a solution to this problem by measuring the IRF using the ultrafast luminescence from gold nanorods. We show that ultrafast gold nanorod luminescence allows the IRF to be directly obtained in multiple spectral channels simultaneously across a wide spectral range. We validate this approach by presenting an analysis of multispectral autofluorescence FLIM data obtained from human skin ex vivo.
Talbot CB, Patalay R, Munro I, et al., 2011, Application of ultrafast gold luminescence to measuring the instrument response function for multispectral multiphoton fluorescence lifetime imaging, OPTICS EXPRESS, Vol: 19, Pages: 13848-13861, ISSN: 1094-4087
Coda S, Kennedy GT, Thompson A, et al., 2011, FLUORESCENCE LIFETIME IMAGING FOR LABEL-FREE CONTRAST OF GASTROINTESTINAL DISEASES, International School of Physics "Enrico Fermi", Course CLXXXI "Microscopy Applied to Biophotonics"
INTRODUCTION: Autofluorescence (AF) has been used to distinguish between normal and diseased tissue, but its molecular basis is still unclear and making quantitative intensity measurements is challenging. Fluorescence lifetime imaging (FLIM) measures the decay rate of the autofluorescent signal from tissue, providing quantitative AF contrast. FLIM has been recently implemented by our group in three endoscopic instruments consisting of a confocal laser endomicroscope, a wide-field fibre-optic endoscope and a single point fibre-optic probe. FLIM has the potential to report on tissue structure and function in real-time during endoscopy, providing a label-free means to detect the early onset of diseases that cause changes in tissue AF. We are developing these 3 modalities for in vivo clinical application, supported by ex vivo studies on freshly-biopsied/resected GI tissues.AIMS & METHODS: The aim of this work is to translate our FLIM instrumentation from the optical bench to in vivo clinical application. AF from 43 endoscopic samples from different GI sites was excited using a conventional confocal FLIM microscope in the range 405-420nm, which is compatible with our FLIM endoscopes, and which is the range needed to excite a number of important endogenous GI tissue fluorophores such as porphyrins, flavins, collagen and elastin. The samples were collected from patients undergoing endoscopy, transported to the FLIM laboratory to be imaged and then submitted for histopathology. The following disorders were investigated: Barrett’s oesophagus, gastric cancer, ulcerative colitis, Crohn’s disease, adenomatous polyps and colon cancer. The accuracy of FLIM in discriminating dysplastic/cancerous samples from normal tissue has been tested by measuring the Area Under the Curve (AUC).RESULTS: Our preliminary data show that premalignant or neoplastic samples display either shorter or longer fluorescence lifetime than that of normal tissue. Increased lifetime val
McGinty J, Stuckey DW, Soloviev VY, et al., 2011, In vivo fluorescence lifetime tomography of a FRET probe expressed in mouse, Biomedical Optics Express, Vol: 2, Pages: 1907-1917, ISSN: 2156-7085
Förster resonance energy transfer (FRET) is a powerful biological tool for reading out cell signaling processes. In vivo use of FRET is challenging because of the scattering properties of bulk tissue. By combining diffuse fluorescence tomography with fluorescence lifetime imaging (FLIM), implemented using wide-field time-gated detection of fluorescence excited by ultrashort laser pulses in a tomographic imaging system and applying inverse scattering algorithms, we can reconstruct the three dimensional spatial localization of fluorescence quantum efficiency and lifetime. We demonstrate in vivo spatial mapping of FRET between genetically expressed fluorescent proteins in live mice read out using FLIM. Following transfection by electroporation, mouse hind leg muscles were imaged in vivo and the emission of free donor (eGFP) in the presence of free acceptor (mCherry) could be clearly distinguished from the fluorescence of the donor when directly linked to the acceptor in a tandem (eGFP-mCherry) FRET construct.
Coda S, Kennedy G, Thompson A, et al., 2011, FLUORESCENCE LIFETIME IMAGING OF GASTROINTESTINAL CANCERS, European-Society-for-Medical-Oncology (ESMO) 13th World Congress on Gastrointestinal Cancer, Publisher: OXFORD UNIV PRESS, Pages: v65-v66, ISSN: 0923-7534
McGinty J, Taylor HB, Chen L, et al., 2011, In vivo fluorescence lifetime optical projection tomography, Biomedical Optics Express, Vol: 2, Pages: 1340-1350, ISSN: 2156-7085
We demonstrate the application of fluorescence lifetime optical projection tomography (FLIM-OPT) to in vivo imaging of lysC:GFP transgenic zebrafish embryos (Danio rerio). This method has been applied to unambiguously distinguish between the fluorescent protein (GFP) signal in myeloid cells from background autofluorescence based on the fluorescence lifetime. The combination of FLIM, an inherently ratiometric method, in conjunction with OPT results in a quantitative 3-D tomographic technique that could be used as a robust method for in vivo biological and pharmaceutical research, for example as a readout of Förster resonance energy transfer based interactions.
Thompson AJ, Paterson C, Neil MAA, et al., 2011, Adaptive phase compensation for ultracompact laser scanning endomicroscopy, OPTICS LETTERS, Vol: 36, Pages: 1707-1709, ISSN: 0146-9592
Grippon S, Zhao Q, Robinson T, et al., 2011, Differential modes of DNA binding by mismatch uracil DNA glycosylase from Escherichia coli: implications for abasic lesion processing and enzyme communication in the base excision repair pathway, Nucleic Acids Research, Vol: 39, Pages: 2593-2603, ISSN: 1362-4962
Mismatch uracil DNA glycosylase (Mug) fromEscherichia coli is an initiating enzyme in thebase-excision repair pathway. As with other DNAglycosylases, the abasic product is potentiallymore harmful than the initial lesion. Since Mug isknown to bind its product tightly, inhibitingenzyme turnover, understanding how Mug bindsDNA is of significance when considering how Muginteracts with downstream enzymes in the baseexcisionrepair pathway. We have demonstrateddifferential binding modes of Mug between its substrateand abasic DNA product using both band shiftand fluorescence anisotropy assays. Mug binds itsproduct cooperatively, and a stoichiometric analysisof DNA binding, catalytic activity and saltdependenceindicates that dimer formation is offunctional significance in both catalytic activity andproduct binding. This is the first report ofcooperativity in the uracil DNA glycosylase superfamilyof enzymes, and forms the basis of productinhibition in Mug. It therefore provides a new perspectiveon abasic site protection and the findingsare discussed in the context of downstream lesionprocessing and enzyme communication in the baseexcision repair pathway.
Everett KL, Buehler A, Bunney TD, et al., 2011, Membrane Environment Exerts an Important Influence on Rac-Mediated Activation of Phospholipase C gamma 2, MOLECULAR AND CELLULAR BIOLOGY, Vol: 31, Pages: 1240-1251, ISSN: 0270-7306
Salehi-Reyhani A, Kaplinsky J, Burgin E, et al., 2011, A first step towards practical single cell proteomics: a microfluidic antibodycapture chip with TIRF detection, Lab Chip, Vol: 11, Pages: 1256-1261
We have developed a generic platform to undertake the analysis of protein copy number from singlecells. The approach described here is ‘all-optical’ whereby single cells are manipulated into separate analysis chambers using an optical trap; single cells are lysed by a shock wave caused by laser-induced microcavitation, and the protein released from a single cell is measured by total internal reflection microscopy as it is bound to micro-printed antibody spots within the device. The platform was tested using GFP transfected cells and the relative precision of the measurement method was determined to be 88%. Single cell measurements were also made on a breast cancer cell line to measure the relative levels of unlabelled human tumour suppressor protein p53 using a chip incorporating an antibody sandwich assay format. These results suggest that this is a viable method for measuring relative protein levels in single cells.
Kumar S, Alibhai D, Margineanu A, et al., 2011, 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, Vol: 12, Pages: 609-626, ISSN: 1439-4235
A fluorescence lifetime imaging (FLIM) technology platform intendedto read out changes in Fçrster resonance energy transfer(FRET) efficiency is presented for the study of protein interactionsacross the drug-discovery pipeline. FLIM provides arobust, inherently ratiometric imaging modality for drug discoverythat could allow the same sensor constructs to betranslated from automated cell-based assays through smalltransparent organisms such as zebrafish to mammals. To thisend, an automated FLIM multiwell-plate reader is described forhigh content analysis of fixed and live cells, tomographic FLIMin zebrafish and FLIM FRET of live cells via confocal endomicroscopy.For cell-based assays, an exemplar application readingout protein aggregation using FLIM FRET is presented, andthe potential for multiple simultaneous FLIM (FRET) readoutsin microscopy is illustrated.
Margineanu A, Laine R, Kumar S, et al., 2011, Multiplexed Time Lapse Fluorescence Lifetime Readouts in an Optically Sectioning Time-Gated Imaging Microscope, 55th Annual Meeting of the Biophysical-Society, Publisher: CELL PRESS, Pages: 183-183, ISSN: 0006-3495
Thompson A, Manning H, Brydegaard M, et al., 2011, Hyperspectral fluorescence lifetime fibre probe spectroscopy for use in the study and diagnosis of osteoarthritis and skin cancer, SPIE Photonics West 2011, Publisher: Society of Photo-optical Instrumentation Engineers (SPIE), ISSN: 1996-756X
We present the application of two fibre-optic-coupled time-resolved spectrofluorometers and a compact steady-state diffuse reflected light/fluorescence spectrometer to in vivo and ex vivo studies of skin cancer and osteoarthritis. In a clinical study of skin cancer, 27 lesions on 25 patients were investigated in vivo before surgical excision of the region measured. Preliminary analysis reveals a statistically significant decrease in the autofluorescence lifetime of basal cell carcinomas compared to neighbouring healthy tissue. A study of autofluorescence signals associated with the onset of osteoarthritis indicates autofluorescence lifetime changes associated with collagen degradation.
Ushakov DS, Caorsi V, Ibanez-Garcia D, et al., 2011, Response of Rigor Cross-bridges to Stretch Detected by Fluorescence Lifetime Imaging Microscopy of Myosin Essential Light Chain in Skeletal Muscle Fibers, JOURNAL OF BIOLOGICAL CHEMISTRY, Vol: 286, Pages: 842-850
Talbot CB, Patalay R, Munro I, et al., 2011, A multispectral FLIM tomograph for in vivo imaging of skin cancer, Conference on Multiphoton Microscopy in the Biomedical Sciences XI, Publisher: SPIE-INT SOC OPTICAL ENGINEERING, ISSN: 0277-786X
Alibhai D, Kumar S, Kelly D, et al., 2011, An automated wide-field, time-gated, optically sectioning, fluorescence lifetime imaging multiwell plate reader for high-content analysis of protein-protein interactions, Conference on Three-Dimensional and Multidimensional Microscopy - Image Acquisition and Processing XVIII, Publisher: SPIE-INT SOC OPTICAL ENGINEERING, ISSN: 0277-786X
Patalay R, Talbot C, Alexandrov Y, et al., 2011, Non-invasive imaging of skin cancer with fluorescence lifetime imaging using two photon tomography, Conference on Clinical and Biomedical Spectroscopy and Imaging II, Publisher: SPIE-INT SOC OPTICAL ENGINEERING, ISSN: 0277-786X
French PMW, 2011, Fluorescence lifetime imaging: FLIM for cell biology, drug discovery and label-free diagnosis
I will present FLIM technology to read out biomolecular interactions across the scales from labelled proteins in solution and in cells through automated plate readers to imaging disease models and endoscopic diagnosis using autofluorescence. © 2011 OSA: BODA/NTM/OMP/OTA.
Lenz MO, Brown ACN, Auksorius E, et al., 2011, A STED-FLIM microscope applied to imaging the Natural Killer cell immune synapse, Conference on Multiphoton Microscopy in the Biomedical Sciences XI, Publisher: SPIE-INT SOC OPTICAL ENGINEERING, ISSN: 0277-786X
McGinty J, Talbot C, Owen D, et al., 2011, Fluorescence Lifetime Imaging Microscopy, Endoscopy and Tomography, Editors: Boas, Pitris, Ramanujam, ISBN: 1420090364
Purbhoo MA, Liu H, Oddos S, et al., 2010, Dynamics of sub-synaptic vesicles and surface microclusters at the T cell immunological synapse, Annual Congress of the British-Society-for-Immunology, Publisher: WILEY-BLACKWELL PUBLISHING, INC, Pages: 175-175, ISSN: 0019-2805
McGinty J, Stuckey D, Laine R, et al., 2010, Time-domain fluorescence lifetime optical projection tomography
We present a platform for measuring the fluorescence lifetime distribution in mesoscopic samples (~0.1-1cm) based on optical projection tomography and time-gated imaging. This is applied to optically cleared embryos expressing a calcium sensing FRET probe. © OSA / BIOMED/DH 2010.
Ibanez-Garcia D, Requejo-Isidro J, Webb MR, et al., 2010, Fluorescence Lifetime Imaging Reveals that the Environment of the ATP Binding Site of Myosin in Muscle Senses Force, BIOPHYSICAL JOURNAL, Vol: 99, Pages: 2163-2169, ISSN: 0006-3495
McGinty J, Galletly NP, Dunsby C, et al., 2010, Wide-field fluorescence lifetime imaging of cancer, BIOMEDICAL OPTICS EXPRESS, Vol: 1, Pages: 627-640, ISSN: 2156-7085
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