481 results found
Lagarto J, Dyer B, Dunsby C, et al., In vivo label-free optical monitoring of structural and metabolic remodeling of myocardium following infarction, Biomedical Optics Express, ISSN: 2156-7085
Cardiac remodeling following myocardial infarction (MI) involves structural and functional alterations in the infarcted and remote viable myocardium that can ultimately lead to heart failure. The underlying mechanisms are not fully understood and, following our previous study of the autofluorescence lifetime and diffuse reflectance signatures of the myocardium in vivo at 16 weeks post MI in rats [Biomed. Opt. Express 6(2), 324 (2015)], we here present data obtained at 1, 2 and 4 weeks post myocardial infarction that help follow the temporal progression of these changes. Our results demonstrate that both structural and metabolic changes in the heart can be monitored from the earliest time points following MI using label-free optical readouts, not only in the region of infarction but also in the remote non-infarcted myocardium. Changes in the autofluorescence intensity and lifetime parameters associated with collagen type I autofluorescence were indicative of progressive collagen deposition in tissue that was most pronounced at earlier time points and in the region of infarction. In addition to significant collagen deposition in infarcted and non-infarcted myocardium, we also report changes in the autofluorescence parameters associated with reduced nicotinamide adenine (phosphate) dinucleotide (NAD(P)H) and flavin adenine dinucleotide (FAD), which we associate with metabolic alterations throughout the heart. Parallel measurements of the diffuse reflectance spectra indicated an increased contribution of reduced cytochrome c. Our findings suggest that combining time-resolved spectrofluorometry and diffuse reflectance spectroscopy could provide a useful means to monitor cardiac function in vivo at the time of surgery.
Munro I, Garcia EAC, Yan M, et al., 2019, Accelerating single molecule localisation microscopy through parallel processing on a high-performance computing cluster, Journal of Microscopy, Vol: 273, Pages: 148-160, ISSN: 1365-2818
Super‐resolved microscopy techniques have revolutionized the ability to study biological structures below the diffraction limit. Single molecule localization microscopy (SMLM) techniques are widely used because they are relatively straightforward to implement and can be realized at relatively low cost, e.g. compared to laser scanning microscopy techniques. However, while the data analysis can be readily undertaken using open source or other software tools, large SMLM data volumes and the complexity of the algorithms used often lead to long image data processing times that can hinder the iterative optimization of experiments. There is increasing interest in high throughput SMLM, but its further development and application is inhibited by the data processing challenges. We present here a widely applicable approach to accelerating SMLM data processing via a parallelized implementation of ThunderSTORM on a high‐performance computing (HPC) cluster and quantify the speed advantage for a four‐node cluster (with 24 cores and 128 GB RAM per node) compared to a high specification (28 cores, 128 GB RAM, SSD‐enabled) desktop workstation. This data processing speed can be readily scaled by accessing more HPC resources. Our approach is not specific to ThunderSTORM and can be adapted for a wide range of SMLM software.
Guo W, Kumar S, Gorlitz F, et al., 2019, Automated fluorescence lifetime imaging high content analysis of Förster resonance energy transfer between endogenously-labeled kinetochore proteins in live budding yeast cells, Slas Technology, Pages: 1-13, ISSN: 2472-6303
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.
To improve the imaging performance of optical projection tomography (OPT) in live samples, we have explored a parallelized implementation of semi-confocal line illumination and detection to discriminate against scattered photons. Slice-illuminated OPT (sl-OPT) improves reconstruction quality in scattering samples by reducing interpixel crosstalk at the cost of increased acquisition time. For in vivo imaging, this can be ameliorated through the use of compressed sensing on angularly undersampled OPT data sets. Here, we demonstrate sl-OPT applied to 3D imaging of bead phantoms and live adult zebrafish.
Lagarto J, Dyer B, Talbot C, et al., 2018, Characterization of NAD(P)H and FAD autofluorescence signatures in a Langendorff isolated-perfused rat heart model, Biomedical Optics Express, Vol: 9, Pages: 4978-4978, ISSN: 2156-7085
Autofluorescence spectroscopy is a promising label-free approach to characterize biological samples with demonstrated potential to report structural and biochemical alterations in tissues in a number of clinical applications. We report a characterization of the ex vivo autofluorescence fingerprint of cardiac tissue, exploiting a Langendorff-perfused isolated rat heart model to induce physiological insults to the heart, with a view to understanding how metabolic alterations affect the autofluorescence signals. Changes in the autofluorescence intensity and lifetime signatures associated with reduced nicotinamide adenine dinucleotide (phosphate) (NAD(P)H) and flavin adenine dinucleotide (FAD) were characterized during oxygen- or glucose-depletion protocols. Results suggest that both NAD(P)H and FAD autofluorescence intensity and lifetime parameters are sensitive to changes in the metabolic state of the heart owing to oxygen deprivation. We also observed changes in NAD(P)H fluorescence intensity and FAD lifetime parameter on reperfusion of oxygen, which might provide information on reperfusion injury, and permanent tissue damage or changes to the tissue during recovery from oxygen deprivation. We found that changes in the autofluorescence signature following glucose-depletion are, in general, less pronounced, and most clearly visible in NAD(P)H related parameters. Overall, the results reported in this investigation can serve as baseline for future investigations of cardiac tissue involving autofluorescence measurements.
Gorlitz F, Guldbrand S, Runcorn T, et al., 2018, easySLM-STED: stimulated emission depletion microscopy with aberration correction, extended field of view and multiple beam scanning, Journal of Biophotonics, ISSN: 1864-063X
We demonstrate a simplified set‐up for STED microscopy with a straightforward alignment procedure that uses a single spatial light modulator (SLM) with collinear incident excitation and depletion beams to provide phase modulation of the beam profiles and correction of optical aberrations. We show that this approach can be used to extend the field of view for STED microscopy by correcting chromatic aberration that otherwise leads to walk‐off between the focused excitation and depletion beams. We further show how this arrangement can be adapted to increase the imaging speed through multibeam excitation and depletion. Fine adjustments to the alignment can be accomplished using the SLM only, conferring the potential for automation.
Sparks H, Kondo H, Hooper S, et al., 2018, Heterogeneity in tumor chromatin-doxorubicin binding revealed by in vivo fluorescence lifetime imaging confocal endomicroscopy, Nature Communications, Vol: 9, ISSN: 2041-1723
We present an approach to quantify drug-target engagement using in vivo fluorescence endomicroscopy, validated with in vitro measurements. Doxorubicin binding to chromatin changes the fluorescence lifetime of histone-GFP fusions that we measure in vivo at single-cell resolution using a confocal laparo/endomicroscope. We measure both intra- and inter-tumor heterogeneity in doxorubicin chromatin engagement in a model of peritoneal metastasis of ovarian cancer, revealing striking variation in the efficacy of doxorubicin-chromatin binding depending on intra-peritoneal or intravenous delivery. Further, we observe significant variations in doxorubicin-chromatin binding between different metastases in the same mouse and between different regions of the same metastasis. The quantitative nature of fluorescence lifetime imaging enables direct comparison of drug-target engagement for different drug delivery routes and between in vitro and in vivo experiments. This uncovers different rates of cell killing for the same level of doxorubicin binding in vitro and in vivo.
Alexandrov Y, Nikolic DS, Dunsby C, et al., 2018, Quantitative time domain analysis of lifetime-based FRET measurements with fluorescent proteins: static random isotropic fluorophore orientation distributions, Journal of Biophotonics, Vol: 11, ISSN: 1864-063X
Förster resonant energy transfer (FRET) measurements are widely used to obtain information about molecular interactions and conformations through the dependence of FRET efficiency on the proximity of donor and acceptor fluorophores. Fluorescence lifetime measurements can provide quantitative analysis of FRET efficiency and interacting population fraction. Many FRET experiments exploit the highly specific labelling of genetically expressed fluorescent proteins, applicable in live cells and organisms. Unfortunately, the typical assumption of fast randomization of fluorophore orientations in the analysis of fluorescence lifetime-based FRET readouts is not valid for fluorescent proteins due to their slow rotational mobility compared to their upper state lifetime. Here, previous analysis of effectively static isotropic distributions of fluorophore dipoles on FRET measurements is incorporated into new software for fitting donor emission decay profiles. Calculated FRET parameters, including molar population fractions, are compared for the analysis of simulated and experimental FRET data under the assumption of static and dynamic fluorophores and the intermediate regimes between fully dynamic and static fluorophores, and mixtures within FRET pairs, is explored. Finally, a method to correct the artefact resulting from fitting the emission from static FRET pairs with isotropic angular distributions to the (incorrect) typically assumed dynamic FRET decay model is presented.
Wisniewski L, Davis S, Lockwood N, et al., 2018, A role for p130Cas in venous sprouting and lymphangiogenesis in the zebrafish, 5th Congress of the ESC-Council-on-Basic-Cardiovascular-Science on Frontiers in Cardio Vascular Biology, Publisher: OXFORD UNIV PRESS, Pages: S90-S90, ISSN: 0008-6363
Rutter GA, Haythorne EA, Georgiadou E, et al., 2018, Pancreatic beta cell-selective deletion of the mitochondrial calcium uniporter (MCU) impairs glucose-stimulated insulin secretion in vitro but not in vivo, Publisher: WILEY, Pages: 42-42, ISSN: 0742-3071
Kim Y, Warren S, Favero F, et al., 2018, Semi-random multicore fibre design for adaptive multiphoton endoscopy, Optics Express, Vol: 26, Pages: 3661-3673, ISSN: 1094-4087
This paper reports the development, modelling and application of a semi-random multicore fibre (MCF) design for adaptive multiphoton endoscopy. The MCF was constructed from 55 sub-units, each comprising 7 single mode cores, in a hexagonally close-packed lattice where each sub-unit had a random angular orientation. The resulting fibre had 385 single mode cores and was double-clad for proximal detection of multiphoton excited fluorescence. The random orientation of each sub-unit in the fibre reduces the symmetry of the positions of the cores in the MCF, reducing the intensity of higher diffracted orders away from the central focal spot formed at the distal tip of the fibre and increasing the maximum size of object that can be imaged. The performance of the MCF was demonstrated by imaging fluorescently labelled beads with both distal and proximal fluorescence detection and pollen grains with distal fluorescence detection. We estimate that the number of independent resolution elements in the final image – measured as the half-maximum area of the two-photon point spread function divided by the area imaged – to be ~3200.
Chen L, Li G, Li Y, et al., 2017, UbasM: An effective balanced optical clearing method for intact biomedical imaging, Scientific Reports, Vol: 7, ISSN: 2045-2322
Optical clearing methods can facilitate deep optical imaging in biological tissue by reducing light scattering and this has enabled accurate three-dimensional signal visualization and quantification of complex biological structures. Unfortunately, existing optical clearing approaches present a compromise between maximizing clearing capability, the preservation of fluorescent protein emission and membrane integrity and the speed of sample processing – with the latter typically requiring weeks for cm scale tissue samples. To address this challenge, we present a new, convenient, aqueous optical clearing agent, termed UbasM: Urea-Based Amino-Sugar Mixture, that rapidly renders fixed tissue samples highly transparent and reliably preserves emission from fluorescent proteins and lipophilic dyes in membrane integrity preserved tissues. UbasM is simple, inexpensive, reproducible and compatible with all labeling methods that we have encountered. It can enable convenient, volumetric imaging of tissue up to the scale of whole adult mouse organs and should be useful for a wide range of light microscopy and tomography techniques applied to biomedical research, especially the study on organism-level systems biology at multiple levels.
Sherlock B, Warren SC, Alexandrov Y, et al., 2017, In vivo multiphoton microscopy using a handheld scanner with lateral and axial motion compensation, Journal of Biophotonics, Vol: 11, ISSN: 1864-063X
This paper reports a handheld multiphoton fluorescence microscope designed for clinical imaging that incorporates axial motion compensation and lateral image stabilization. Spectral domain optical coherence tomography is employed to track the axial position of the skin surface, and lateral motion compensation is realised by imaging the speckle pattern arising from the optical coherence tomography beam illuminating the sample. Our system is able to correct lateral sample velocities of up to ~65 μm s-1. Combined with the use of negative curvature microstructured optical fibre to deliver tunable ultrafast radiation to the handheld multiphoton scanner without the need of a dispersion compensation unit, this instrument has potential for a range of clinical applications. The system is used to compensate for both lateral and axial motion of the sample when imaging human skin in vivo.
Gorlitz F, Corcoran DS, Garcia Castano EA, et al., 2017, Mapping molecular function to biological nanostructure: combining structured illumination microscopy with fluorescence lifetime imaging (SIM+FLIM), Photonics, Vol: 4, ISSN: 2304-6732
We present a new microscope integrating super-resolved imaging using structured illumination microscopy (SIM) with wide-field optically sectioned fluorescence lifetime imaging (FLIM) to provide optical mapping of molecular function and its correlation with biological nanostructure below the conventional diffraction limit. We illustrate this SIM + FLIM capability to map FRET readouts applied to the aggregation of discoidin domain receptor 1 (DDR1) in Cos 7 cells following ligand stimulation and to the compaction of DNA during the cell cycle.
Noble E, Kumar S, Gorlitz F, et al., 2017, In vivo label-free mapping of the effect of a photosystem II inhibiting herbicide in plants using chlorophyll fluorescence lifetime, Plant Methods, Vol: 13, ISSN: 1746-4811
BackgroundIn order to better understand and improve the mode of action of agrochemicals, it is useful to be able to visualize their uptake and distribution in vivo, non-invasively and, ideally, in the field. Here we explore the potential of plant autofluorescence (specifically chlorophyll fluorescence) to provide a readout of herbicide action across the scales utilising multiphoton-excited fluorescence lifetime imaging, wide-field single-photon excited fluorescence lifetime imaging and single point fluorescence lifetime measurements via a fibre-optic probe.ResultsOur studies indicate that changes in chlorophyll fluorescence lifetime can be utilised as an indirect readout of a photosystem II inhibiting herbicide activity in living plant leaves at three different scales: cellular (~μm), single point (~1 mm2) and macroscopic (~8 × 6 mm2 of a leaf). Multiphoton excited fluorescence lifetime imaging of Triticum aestivum leaves indicated that there is an increase in the spatially averaged chlorophyll fluorescence lifetime of leaves treated with Flagon EC—a photosystem II inhibiting herbicide. The untreated leaf exhibited an average lifetime of 560 ± 30 ps while the leaf imaged 2 h post treatment exhibited an increased lifetime of 2000 ± 440 ps in different fields of view. The results from in vivo wide-field single-photon excited fluorescence lifetime imaging excited at 440 nm indicated an increase in chlorophyll fluorescence lifetime from 521 ps in an untreated leaf to 1000 ps, just 3 min after treating the same leaf with Flagon EC, and to 2150 ps after 27 min. In vivo single point fluorescence lifetime measurements demonstrated a similar increase in chlorophyll fluorescence lifetime. Untreated leaf presented a fluorescence lifetime of 435 ps in the 440 nm excited chlorophyll channel, CH4 (620–710 nm). In the first 5 min after treatment, mean fluorescence lifetime is observed to have increased to 1 ns and then to 1.3 ns after 60 min. For
Lagarto J, Hares JD, Dunsby CW, et al., 2017, Development of low-cost instrumentation for single point autofluorescence lifetime measurements, Journal of Fluorescence, Vol: 27, Pages: 1643-1654, ISSN: 1573-4994
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.
Van de Pette M, Abbas A, Feytout A, et al., 2017, Visualizing changes in Cdkn1c expression links early life adversity to imprint mis-regulation in adults, Cell Reports, Vol: 31, Pages: 1090-1099, ISSN: 2211-1247
Imprinted genes are regulated according to parental origin and can influence embryonic growth and metabolism and confer disease susceptibility.Here we designed sensitive allele-specific reporters to non-invasively monitor imprinted Cdkn1cexpression in mice and showed that expression was modulated by environmental factors encounteredin utero.Acute exposure to chromatin modifyingdrugs resulted in de-repression of paternally inherited (silent) Cdkn1calleles in embryos that was temporary and resolved after birth.In contrast, deprivation of maternal dietary proteinin uteroprovoked permanent de-repression of imprinted Cdkn1cexpression that was sustained into adulthood and occurred through a folate-dependent mechanism of DNA methylation loss.Given the function of imprinted genes in regulating behavior and metabolic processes in adults, these results establish imprinting deregulation as a credible mechanism linking early life adversity to later-life outcomes.Furthermore,Cdkn1c-luciferasemice offer non-invasivetools to identify factors that disrupt epigenetic processes and strategies to limit their long-term impact.
Sparks H, Gorlitz F, Kelly D, et al., 2017, Characterisation of new gated optical image intensifiers for fluorescence lifetime imaging, Review of Scientific Instruments, Vol: 88, ISSN: 1089-7623
We report the characterisation of gated optical image intensifiers for fluorescence lifetime imaging (FLIM), evaluating the performance of several different prototypes that culminate in a new design that provides improved spatial resolution conferred by the addition of a magnetic field to reduce the lateral spread of photoelectrons on their path between the photocathode and microchannel plate, and higher signal to noise ratio conferred by longer time gates. We also present a methodology to compare thesesystems and their capabilities, including the quantitative readouts of Förster resonant energy transfer (FRET).
French PMW, Görlitz F, Kelly D, et al., 2017, Open source high content analysis utilizing automated fluorescence lifetime imaging microscopy, Jove-Journal of Visualized Experiments, Vol: 119, ISSN: 1940-087X
We present an open source high content analysis instrument utilizing automated fluorescence lifetime imaging (FLIM) for assaying protein interactions using Förster resonance energy transfer (FRET) based readouts of fixed or live cells in multiwell plates. This provides a means to screen for cell signaling processes read out using intramolecular FRET biosensors or intermolecular FRET of protein interactions such as oligomerization or heterodimerization, which can be used to identify binding partners. We describe herethe functionality of this automated multiwell plate FLIM instrumentation and present exemplar data from our studies of HIV Gag protein oligomerization and a time course of a FRET biosensor in live cells. A detailed description of the practical implementation is then provided with reference to a list of hardware components and a description of the open source data acquisition software written in μ Manager. The application of FLIMfit, an open source MATLAB-based client for the OMERO platform, to analyze arrays of multiwell plate FLIM data is also presented. The protocols for imaging fixed and live cells are outlined and a demonstration of an automated multiwell plate FLIM experiment using cells expressing fluorescent protein-based FRET constructs is presented. This is complemented by a walk-through of the data analysis for this specific FLIM FRET data set.
Perdios L, Lowe AR, Saladino G, et al., 2017, Conformational transition of FGFR kinase activation revealed by site-specific unnatural amino acid reporter and single molecule FRET, Scientific Reports, Vol: 7, ISSN: 2045-2322
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.
Go¨rlitz F, Corcoran DS, Sparks H, et al., 2017, Mapping molecular function to biological nanostructure: Combining structured illumination microscopy with fluorescence lifetime imaging
© 2017 OSA. We report the enhancement of spatial resolution and sensitivity of wide-field time-gated imaging and the combination with SIM to map molecular function using FRET to biological nanostructure below the conventional diffraction limit.
Andrews N, Davis S, Hay C, et al., 2017, Functional imaging of live Zebrafish using fluorescence lifetime optical projection tomography, Conference on Imaging, Manipulation, and Analysis of Biomolecules, Cells, and Tissues XV, Publisher: SPIE-INT SOC OPTICAL ENGINEERING, ISSN: 0277-786X
Cortés E, Huidobro PA, Sinclair HG, et al., 2016, Plasmonic nanoprobes for stimulated emission depletion nanoscopy, ACS Nano, Vol: 10, Pages: 10454-10461, ISSN: 1936-0851
Plasmonic nanoparticles influence the absorption and emission processes of nearby emitters due to local enhancements of the illuminating radiation and the photonic density of states. Here, we use the plasmon resonance of metal nanoparticles in order to enhance the stimulated depletion of excited molecules for super-resolved nanoscopy. We demonstrate stimulated emission depletion (STED) nanoscopy with gold nanorods with a long axis of only 26 nm and a width of 8 nm. These particles provide an enhancement of up to 50% of the resolution compared to fluorescent-only probes without plasmonic components irradiated with the same depletion power. The nanoparticle-assisted STED probes reported here represent a ∼2 × 103 reduction in probe volume compared to previously used nanoparticles. Finally, we demonstrate their application toward plasmon-assisted STED cellular imaging at low-depletion powers, and we also discuss their current limitations.
Margineanu A, Chan JJ, Kelly DJ, et al., 2016, Corrigendum: Screening for protein-protein interactions using Förster resonance energy transfer (FRET) and fluorescence lifetime imaging microscopy (FLIM)., Scientific Reports, Vol: 6, ISSN: 2045-2322
Warren SC, Kim Y, Stone JM, et al., 2016, Adaptive multiphoton endomicroscopy through a dynamically deformed multicore optical fiber using proximal detection, Optics Express, Vol: 24, Pages: 21474-21484, ISSN: 1094-4087
This paper demonstrates multiphoton excited fluorescenceimaging through a polarisation maintaining multicore fiber (PM-MCF)while the fiber is dynamically deformed using all-proximal detection.Single-shot proximal measurement of the relative optical path lengths of allthe cores of the PM-MCF in double pass is achieved using a Mach-Zehnderinterferometer read out by a scientific CMOS camera operating at 416 Hz.A non-linear least squares fitting procedure is then employed to determinethe deformation-induced lateral shift of the excitation spot at the distal tip ofthe PM-MCF. An experimental validation of this approach is presented thatcompares the proximally measured deformation-induced lateral shift infocal spot position to an independent distally measured ground truth. Theproximal measurement of deformation-induced shift in focal spot position isapplied to correct for deformation-induced shifts in focal spot positionduring raster-scanning multiphoton excited fluorescence imaging.
Chennell G, Willows RJW, Warren SC, et al., 2016, Imaging of Metabolic Status in 3D Cultures with an Improved AMPK FRET Biosensor for FLIM, Sensors, Vol: 16, ISSN: 1424-8239
We describe an approach to non-invasively map spatiotemporal biochemical and physiological changes in 3D cell culture using Forster Resonance Energy Transfer (FRET) biosensors expressed in tumour spheroids. In particular, we present an improved Adenosine Monophosphate (AMP) Activated Protein Kinase (AMPK) FRET biosensor, mTurquoise2 AMPK Activity Reporter (T2AMPKAR), for fluorescence lifetime imaging (FLIM) readouts that we have evaluated in 2D and 3D cultures. Our results in 2D cell culture indicate that replacing the FRET donor, enhanced Cyan Fluorescent Protein (ECFP), in the original FRET biosensor, AMPK activity reporter (AMPKAR), with mTurquoise2 (mTq2FP), increases the dynamic range of the response to activation of AMPK, as demonstrated using the direct AMPK activator, 991. We demonstrated 3D FLIM of this T2AMPKAR FRET biosensor expressed in tumour spheroids using two-photon excitation.
Margineanu A, Chan JJ, Kelly DJ, et al., 2016, Screening for protein-protein interactions using Förster resonance energy transfer (FRET) and fluorescence lifetime imaging microscopy (FLIM), Scientific Reports, Vol: 6, ISSN: 2045-2322
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.
Rutter GA, Semplici F, Mondragon A, et al., 2016, Cell type-specific deletion in mice reveals roles for PAS kinase in insulin and glucagon production, Diabetologia, Vol: 59, Pages: 1938-1947, ISSN: 1432-0428
Background and Aims. Per-Arnt-Sim domain containing kinase (PASK) is a nutrient regulated protein kinase previously implicated in the control of insulin gene expression and glucagon secretion. Here, we explore the roles of the kinase in the control of islet hormone release by generating mice deleted selectively for the Pask gene in pancreatic beta or alpha cells. Methods. Floxed alleles of Pask were produced by homologous recombination and animals bred with mice bearing beta (Ins1Cre, PaskBKO), or alpha (PPG-Cre; PaskAKO) cell selective Cre recombinase alleles. Glucose homeostasis and hormone secretion in vivo and in vitro, gene expression, and islet cell mass, were measured using standard techniques.Results. Ins1Cre-based recombination led to efficient beta cell targeted deletion of Pask. Beta cell mass was reduced by 36.5% (p<0.05) compared to controls in PaskBKO mice, as well as in global null Pask mice (38%, p<0.05). PaskBKO mice displayed normal body weight and fasting glycemia, but slightly impaired glucose tolerance, and beta cell proliferation, after maintenance on a high fat diet. Whilst glucose tolerance was unaffected in PaskAKO mice, glucose infusion rates were increased, and glucagon secretion tended to be lower, during hypoglycemic clamps. Though alpha cell mass was increased (21.9%, p<0.05), glucagon release at low glucose was impaired (p<0.05) in PaskAKO islets. Conclusions. The present findings demonstrate cell autonomous roles for PASK in the control of pancreatic endocrine hormone secretion. Differencesbetween the glycemic phenotype of global versus cell type specific null mice suggest important roles for tissue interactions in the control of glycemia by the kinase.
Kumar S, Lockward N, Ramel M-C, et al., 2016, Quantitative in vivo optical tomography of cancer progression & vasculature development in adult zebrafish, Oncotarget, Vol: 7, Pages: 43939-43948, ISSN: 1949-2553
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.
Kim Y, Warren SC, Stone JM, et al., 2016, Adaptive Multiphoton Endomicroscope Incorporating a Polarization-Maintaining Multicore Optical Fibre, IEEE Journal of Selected Topics in Quantum Electronics, Vol: 22, ISSN: 1558-4542
We present a laser scanning multiphoton endomicroscopewith no distal optics or mechanical components that incorporatesa polarization-maintaining (PM) multicore optical fibre todeliver, focus, and scan ultrashort pulsed radiation for two-photonexcited fluorescence imaging. We show theoretically that the use ofa PM multicore fibre in our experimental configuration enhancesthe fluorescence excitation intensity achieved in the focal spot comparedto a non-PM optical fibre with the same geometry and con-firm this by computer simulations based on numerical wavefrontpropagation. In our experimental system, a spatial light modulator(SLM) is utilised to program the phase of the light input to each ofthe cores of the endoscope fibre such that the radiation emergingfrom the distal end of the fibre interferes to provide the focusedscanning excitation beam. We demonstrate that the SLM can enabledynamic phase correction of path-length variations across themulticore optical fibre whilst the fibre is perturbed with an updaterate of 100 Hz.
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