Angiogenesis: An Improved <i>In Vitro</i> Biological System and Automated Image-Based Workflow to Aid Identification and Characterization of Angiogenesis and Angiogenic Modulators

Santos, Albert Francis; Zaltsman, Alla B.; Martin, Rhian Clare; Kuzmin, Alexandru; Alexandrov, Yuriy; Roquemore, Elizabeth P.; Jessop, Robert Arnold; Erck, M. G. M. Van; Verheijen, Johan Hendrikus

doi:10.1089/adt.2008.146

Cited by 17 publications

(9 citation statements)

References 51 publications

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“…To further automate the discrimination we then replaced the step of manual segmentation with automatic ROI segmentation by the method of size-tuned non-linear top-hat detection [21]. This procedure identified 2343 BCC and 4034 normal ROIs.…”

Section: Resultsmentioning

confidence: 99%

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

et al. 2012

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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 mm2 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.

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Section: Resultsmentioning

confidence: 99%

“…Automatic image segmentation was performed using size-tuned non-linear top-hat detection that has been described previously [21]. Briefly, this method applies a pixel-wise transformation to the image (formula A2.1 in [21]) that enhances the brightness of a pixel if its close vicinity is also bright and its distant vicinity is dim.…”

Section: Methodsmentioning

confidence: 99%

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

et al. 2012

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“…To identify cells above the background, a size-tuned nonlinear top-hat (nTh) transform [63] was applied to the mTurquoise integrated intensity image. This method applies the pixelwise transformto the integrated intensity image where denotes averaging with a square mask of width .…”

Section: Methodsmentioning

confidence: 99%

Rapid Global Fitting of Large Fluorescence Lifetime Imaging Microscopy Datasets

et al. 2013

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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.

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“…In addition, cells typically display a varying fluorescence protein expression level from cell-tocell and can stretch and change their shape significantly during locomotion. We therefore implemented a size-tuned, nonlinear top-hat function image processing algorithm in Matlab to identify objects within an expected size range [12,13]. The method is based on the ratiometric comparison of the average intensities between a given group of pixels and their surrounding area.…”

Section: Cell Detection and Identificationmentioning

confidence: 99%

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

Alexandrov

Kumar

Andrews

et al. 2015

Biomed. Opt. Express

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Abstract: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.

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Angiogenesis: An Improved In Vitro Biological System and Automated Image-Based Workflow to Aid Identification and Characterization of Angiogenesis and Angiogenic Modulators

Cited by 17 publications

References 51 publications

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

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

Rapid Global Fitting of Large Fluorescence Lifetime Imaging Microscopy Datasets

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

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