An automated multiwell plate reading flim microscope for live cell autofluorescence lifetime assays

Kelly, Douglas J.; Warren, Sean; Kumar, Sunil; Lagarto, João L.; Dyer, Benjamin; Margineanu, Anca; Lam, Eric W.‐F.; Dunsby, Chris; French, Paul M. W.

doi:10.1142/s1793545814500254

Cited by 6 publications

(7 citation statements)

References 45 publications

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“…The automated FLIM FRET assays reported in this work were undertaken with fixed cells, but could readily be applied to live cells for which similar performance is expected, in line with our previous work 62 . We are developing an open hardware approach to FLIM high content analysis and the latest versions of our open source software for data acquisition and analysis, together with and descriptions of hardware components is available on our website at http://www3.imperial.ac.uk/photonics/research/biomedical-imaging/openflimhca .…”

Section: Discussionsupporting

confidence: 74%

Screening for protein-protein interactions using Förster resonance energy transfer (FRET) and fluorescence lifetime imaging microscopy (FLIM)

Margineanu

Chan

Kelly

et al. 2016

Sci Rep

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We present a high content multiwell plate cell-based assay approach to quantify protein interactions directly in cells using Förster resonance energy transfer (FRET) read out by automated fluorescence lifetime imaging (FLIM). Automated FLIM is implemented using wide-field time-gated detection, typically requiring only 10 s per field of view (FOV). Averaging over biological, thermal and shot noise with 100’s to 1000’s of FOV enables unbiased quantitative analysis with high statistical power. Plotting average donor lifetime vs. acceptor/donor intensity ratio clearly identifies protein interactions and fitting to double exponential donor decay models provides estimates of interacting population fractions that, with calibrated donor and acceptor fluorescence intensities, can yield dissociation constants. We demonstrate the application to identify binding partners of MST1 kinase and estimate interaction strength among the members of the RASSF protein family, which have important roles in apoptosis via the Hippo signalling pathway. KD values broadly agree with published biochemical measurements.

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Section: Discussionsupporting

confidence: 74%

Screening for protein-protein interactions using Förster resonance energy transfer (FRET) and fluorescence lifetime imaging microscopy (FLIM)

Margineanu

Chan

Kelly

et al. 2016

Sci Rep

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“…Stilbene-3 is a monoexponential dye with a reported fluorescence lifetime of 1.2 ns in water [ 38 ] and therefore represents a convenient test sample for our system. A solution of 50 μM Stilbene-3 in purified water at room temperature was measured with excitation at 375 nm.…”

Section: Fluorescence Measurements Of Reference Fluorophoresmentioning

confidence: 99%

Development of Low-Cost Instrumentation for Single Point Autofluorescence Lifetime Measurements

et al. 2017

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Autofluorescence lifetime measurements, which can provide label-free readouts in biological tissues, contrasting e.g. different types and states of tissue matrix components and different cellular metabolites, may have significant clinical potential for diagnosis and to provide surgical guidance. However, the cost of the instrumentation typically used currently presents a barrier to wider implementation. We describe a low-cost single point time-resolved autofluorescence instrument, exploiting modulated laser diodes for excitation and FPGA-based circuitry for detection, together with a custom constant fraction discriminator. Its temporal accuracy is compared against a “gold-standard” instrument incorporating commercial TCSPC circuitry by resolving the fluorescence decays of reference fluorophores presenting single and double exponential decay profiles. To illustrate the potential to read out intrinsic contrast in tissue, we present preliminary measurements of autofluorescence lifetime measurements of biological tissues ex vivo. We believe that the lower cost of this instrument could enhance the potential of autofluorescence lifetime metrology for clinical deployment and commercial development.Electronic supplementary materialThe online version of this article (doi:10.1007/s10895-017-2101-7) contains supplementary material, which is available to authorized users.

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“…Lifetime-based readouts are relatively insensitive to fluorophore concentration, to excitation/detection efficiencies, and to scattering and absorption in the sample or instrument, 2 and so are useful for assays in cells and biological tissue. While FLIM can provide intrinsic readouts utilizing endogenous fluorophores, for example, enabling the mapping of cellular metabolic changes 3,4 or stem cell differentiation, 5,6 and has been implemented with HCA, 7 it is most commonly applied to exogenous fluorophores. These include small-molecule dyes that stain specific cellular compartments or are conjugated to antibodies, typically used with fixed samples or fluorescent proteins (FPs) encoded by plasmids, which are often employed for live cell imaging.…”

Section: Introductionmentioning

confidence: 99%

Automated Fluorescence Lifetime Imaging High-Content Analysis of Förster Resonance Energy Transfer between Endogenously Labeled Kinetochore Proteins in Live Budding Yeast Cells

et al. 2019

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We describe an open-source automated multiwell plate fluorescence lifetime imaging (FLIM) methodology to read out Förster resonance energy transfer (FRET) between fluorescent proteins (FPs) labeling endogenous kinetochore proteins (KPs) in live budding yeast cells. The low copy number of many KPs and their small spatial extent present significant challenges for the quantification of donor fluorescence lifetime in the presence of significant cellular autofluorescence and photobleaching. Automated FLIM data acquisition was controlled by µManager and incorporated wide-field time-gated imaging with optical sectioning to reduce background fluorescence. For data analysis, we used custom MATLAB-based software tools to perform kinetochore foci segmentation and local cellular background subtraction and fitted the fluorescence lifetime data using the open-source FLIMfit software. We validated the methodology using endogenous KPs labeled with mTurquoise2 FP and/or yellow FP and measured the donor fluorescence lifetimes for foci comprising 32 kinetochores with KP copy numbers as low as ~2 per kinetochore under an average labeling efficiency of 50%. We observed changes of median donor lifetime ≥250 ps for KPs known to form dimers. Thus, this FLIM high-content analysis platform enables the screening of relatively low-copy-number endogenous protein–protein interactions at spatially confined macromolecular complexes.

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An automated multiwell plate reading flim microscope for live cell autofluorescence lifetime assays

Cited by 6 publications

References 45 publications

Screening for protein-protein interactions using Förster resonance energy transfer (FRET) and fluorescence lifetime imaging microscopy (FLIM)

Screening for protein-protein interactions using Förster resonance energy transfer (FRET) and fluorescence lifetime imaging microscopy (FLIM)

Development of Low-Cost Instrumentation for Single Point Autofluorescence Lifetime Measurements

Automated Fluorescence Lifetime Imaging High-Content Analysis of Förster Resonance Energy Transfer between Endogenously Labeled Kinetochore Proteins in Live Budding Yeast Cells

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