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Segmenting cells within cellular aggregates in 3D is a growing challenge in cell biology due to improvements in capacity and accuracy of microscopy techniques. Here, we describe a pipeline to segment images of cell aggregates in 3D. The pipeline combines neural network segmentations with active meshes. We apply our segmentation method to cultured mouse mammary gland organoids imaged over 24 h with oblique plane microscopy, a high-throughput light-sheet fluorescence microscopy technique. We show that our method can also be applied to images of mouse embryonic stem cells imaged with a spinning disc microscope. We segment individual cells based on nuclei and cell membrane fluorescent markers, and track cells over time. We describe metrics to quantify the quality of the automated segmentation. Our segmentation pipeline involves a Fiji plugin that implements active mesh deformation and allows a user to create training data, automatically obtain segmentation meshes from original image data or neural network prediction, and manually curate segmentation data to identify and correct mistakes. Our active meshes-based approach facilitates segmentation postprocessing, correction, and integration with neural network prediction.
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Núcleo Celular , Redes Neurais de Computação , Animais , Camundongos , Microscopia de Fluorescência/métodos , Processamento de Imagem Assistida por Computador/métodosRESUMO
The technique of remote refocusing is used in optical microscopy to provide rapid axial scanning without mechanically perturbing the sample and in techniques such as oblique plane microscopy that build on remote refocusing to image a tilted plane within the sample. The magnification between the pupils of the primary (O1) and secondary (O2) microscope objectives of the remote-refocusing system has been shown previously by Mohanan and Corbett [J. Microsc.288, 95 (2022)JMICAR0022-272010.1111/jmi.12991] to be crucial in obtaining the broadest possible remote-refocusing range. In this work, we performed an initial alignment of a remote-refocusing system and then studied the effect of axial misalignments of O1 and O2, axial misalignment of the primary tube lens (TL1) relative to the secondary tube lens (TL2), lateral misalignments of TL2, and changes in the focal length of TL2. For each instance of the setup, we measured the mean point spread function F W H M xy of 100 nm fluorescent beads and the normalized bead integrated fluorescence signal, and we calculated the axial and lateral distortion of the system; all of these quantities were mapped over the remote-refocusing range and as a function of lateral image position. This allowed us to estimate the volume over which diffraction-limited performance is achieved and how this changes with the alignment of the system.
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Receptors for the peptide hormones glucagon-like peptide-1 (GLP-1R), glucose-dependent insulinotropic polypeptide (GIPR), and glucagon (GCGR) are important regulators of insulin secretion and energy metabolism. GLP-1R agonists have been successfully deployed for the treatment of type 2 diabetes, but it has been suggested that their efficacy is limited by target receptor desensitization and downregulation due to recruitment of ß-arrestins. Indeed, recently described GLP-1R agonists with reduced ß-arrestin-2 recruitment have delivered promising results in preclinical and clinical studies. We therefore aimed to determine if the same phenomenon could apply to the closely related GIPR and GCGR. In HEK293 cells depleted of both ß-arrestin isoforms the duration of G protein-dependent cAMP/PKA signaling was increased in response to the endogenous ligand for each receptor. Moreover, in wildtype cells, "biased" GLP-1, GCG, and GIP analogs with selective reductions in ß-arrestin-2 recruitment led to reduced receptor endocytosis and increased insulin secretion over a prolonged stimulation period, although the latter effect was only seen at high agonist concentrations. Biased GCG analogs increased the duration of cAMP signaling, but this did not lead to increased glucose output from hepatocytes. Our study provides a rationale for the development of GLP-1R, GIPR, and GCGR agonists with reduced ß-arrestin recruitment, but further work is needed to maximally exploit this strategy for therapeutic purposes.
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AMP Cíclico/metabolismo , Polipeptídeo Inibidor Gástrico/metabolismo , Peptídeo 1 Semelhante ao Glucagon/metabolismo , Receptor do Peptídeo Semelhante ao Glucagon 1/metabolismo , Incretinas/farmacologia , Receptores dos Hormônios Gastrointestinais/metabolismo , beta-Arrestinas/metabolismo , Animais , Polipeptídeo Inibidor Gástrico/genética , Glucagon/metabolismo , Peptídeo 1 Semelhante ao Glucagon/genética , Receptor do Peptídeo Semelhante ao Glucagon 1/genética , Células HEK293 , Humanos , Secreção de Insulina , Ligantes , Camundongos , Camundongos Endogâmicos C57BL , Receptores dos Hormônios Gastrointestinais/genética , Transdução de Sinais , beta-Arrestinas/genéticaRESUMO
The remote-refocusing approach of Botcherby et al. [Opt. Lett.32, 2007 (2007)10.1364/OL.32.002007] has been applied widely to 2D and 3D fluorescence microscopes to enable rapid refocusing of the optical system without mechanically perturbing the sample. In order for this approach to operate correctly, it requires that the overall magnification of the first two microscope systems matches the ratio of the refractive indices in sample and intermedia image spaces. However, commercially available tube lenses are not always suitable to produce the desired overall magnification. Therefore, a practical approach to produce tube lenses with low expense and diffraction-limited performance is required. Tube lenses can be formed using a pair of stock achromatic doublets, however, selecting appropriate pairs of achromatic doublets from stock optics is a time-consuming process, as many combinations can be considered. In this paper, we present two software packages (Catalogue Generator and Doublet Selector) developed in MATLAB that use the application programming interface (ZOS-API) to the Zemax OpticStudio optical design software to realise an automatic search of stock achromatic doublets to produce microscope tube lenses with a specified focal length, entrance pupil diameter and maximum design field angle. An algorithm to optimise principal plane positions in versions of OpticStudio before 20.2 was also introduced to enable the use of older software versions. To evaluate the performance of Catalogue Generator and Doublet Selector, we used them to generate ten tube lens designs. All of the software-produced tube lenses have a better optical performance than those using manually selected pairs of stock doublets lenses.
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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.
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Endoscópios , Endoscopia/instrumentação , Microscopia de Fluorescência por Excitação Multifotônica , Fibras Ópticas , Desenho de Equipamento , Microesferas , PólenRESUMO
This paper demonstrates multiphoton excited fluorescence imaging 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 all the cores of the PM-MCF in double pass is achieved using a Mach-Zehnder interferometer read out by a scientific CMOS camera operating at 416 Hz. A non-linear least squares fitting procedure is then employed to determine the deformation-induced lateral shift of the excitation spot at the distal tip of the PM-MCF. An experimental validation of this approach is presented that compares the proximally measured deformation-induced lateral shift in focal spot position to an independent distally measured ground truth. The proximal measurement of deformation-induced shift in focal spot position is applied to correct for deformation-induced shifts in focal spot position during raster-scanning multiphoton excited fluorescence imaging.
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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.
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Técnicas Biossensoriais/métodos , Técnicas de Cultura de Células , Imagem Molecular/métodos , Proteínas Quinases/isolamento & purificação , Quinases Proteína-Quinases Ativadas por AMP , Transferência Ressonante de Energia de Fluorescência/métodos , Proteínas de Fluorescência Verde/química , Humanos , Imagem Óptica/métodos , Esferoides Celulares/citologiaRESUMO
A correction is proposed to the Delta function convolution method (DFCM) for fitting a multiexponential decay model to time-resolved fluorescence decay data using a monoexponential reference fluorophore. A theoretical analysis of the discretised DFCM multiexponential decay function shows the presence an extra exponential decay term with the same lifetime as the reference fluorophore that we denote as the residual reference component. This extra decay component arises as a result of the discretised convolution of one of the two terms in the modified model function required by the DFCM. The effect of the residual reference component becomes more pronounced when the fluorescence lifetime of the reference is longer than all of the individual components of the specimen under inspection and when the temporal sampling interval is not negligible compared to the quantity (τR (-1) - τ(-1))(-1), where τR and τ are the fluorescence lifetimes of the reference and the specimen respectively. It is shown that the unwanted residual reference component results in systematic errors when fitting simulated data and that these errors are not present when the proposed correction is applied. The correction is also verified using real data obtained from experiment.
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Corantes Fluorescentes/química , Modelos Teóricos , Espectrometria de Fluorescência/normas , Análise dos Mínimos Quadrados , Dinâmica não Linear , Padrões de ReferênciaRESUMO
Multiplexed imaging of Förster Resonance Energy Transfer (FRET)-based biosensors potentially presents a powerful approach to monitoring the spatio-temporal correlation of signalling pathways within a single live cell. Here, we discuss the potential of homo-FRET based biosensors to facilitate multiplexed imaging. We demonstrate that the homo-FRET between pleckstrin homology domains of Akt (Akt-PH) labelled with mCherry may be used to monitor 3'-phosphoinositide accumulation in live cells and show how global analysis of time resolved fluorescence anisotropy measurements can be used to quantify this accumulation. We further present multiplexed imaging readouts of calcium concentration, using fluorescence lifetime measurements of TN-L15-a CFP/YFP based hetero-FRET calcium biosensor-with 3'-phosphoinositide accumulation.
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Técnicas Biossensoriais/métodos , Transferência Ressonante de Energia de Fluorescência/métodos , Transdução de Sinais , Animais , Anisotropia , Cálcio/metabolismo , Linhagem Celular , Transferência Ressonante de Energia de Fluorescência/instrumentação , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Camundongos , Fosfatidilinositóis/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismoRESUMO
OBJECTIVE: This study aimed to realise 3-D super-resolution ultrasound imaging transcutaneously with a row-column array which has far fewer independent electronic channels and a wider field of view than typical fully addressed 2-D matrix arrays. The in vivo image quality of the row-column array is generally poor, particularly when imaging non-invasively. This study aimed to develop a suite of image formation and post-processing methods to improve image quality and demonstrate the feasibility of ultrasound localisation microscopy using a row-column array, transcutaneously on a rabbit model and in a human. METHODS: To achieve this, a processing pipeline was developed which included a new type of rolling window image reconstruction, which integrated a row-column array specific coherence-based beamforming technique with acoustic sub-aperture processing. This and other processing steps reduced the 'secondary' lobe artefacts, and noise and increased the effective frame rate, thereby enabling ultrasound localisation images to be produced. RESULTS: Using an in vitro cross tube, it was found that the procedure reduced the percentage of 'false' locations from â¼26% to â¼15% compared to orthogonal plane wave compounding. Additionally, it was found that the noise could be reduced by â¼7 dB and the effective frame rate was increased to over 4000 fps. In vivo, ultrasound localisation microscopy was used to produce images non-invasively of a rabbit kidney and a human thyroid. CONCLUSION: It has been demonstrated that the proposed methods using a row-column array can produce large field of view super-resolution microvascular images in vivo and in a human non-invasively.
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Imageamento Tridimensional , Ultrassonografia , Coelhos , Animais , Humanos , Ultrassonografia/métodos , Imageamento Tridimensional/métodos , Desenho de Equipamento , Imagens de Fantasmas , Pele/diagnóstico por imagem , Estudos de ViabilidadeRESUMO
How cancer cells determine their shape in response to three-dimensional (3D) geometric and mechanical cues is unclear. We develop an approach to quantify the 3D cell shape of over 60,000 melanoma cells in collagen hydrogels using high-throughput stage-scanning oblique plane microscopy (ssOPM). We identify stereotypic and environmentally dependent changes in shape and protrusivity depending on whether a cell is proximal to a flat and rigid surface or is embedded in a soft environment. Environmental sensitivity metrics calculated for small molecules and gene knockdowns identify interactions between the environment and cellular factors that are important for morphogenesis. We show that the Rho guanine nucleotide exchange factor (RhoGEF) TIAM2 contributes to shape determination in environmentally independent ways but that non-muscle myosin II, microtubules, and the RhoGEF FARP1 regulate shape in ways dependent on the microenvironment. Thus, changes in cancer cell shape in response to 3D geometric and mechanical cues are modulated in both an environmentally dependent and independent fashion.
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Forma Celular , Fatores de Troca do Nucleotídeo Guanina , Humanos , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Fatores de Troca do Nucleotídeo Guanina/genética , Linhagem Celular Tumoral , Microtúbulos/metabolismo , Miosina Tipo II/metabolismo , Fatores de Troca de Nucleotídeo Guanina Rho/metabolismo , Fatores de Troca de Nucleotídeo Guanina Rho/genética , Melanoma/patologia , Melanoma/metabolismoRESUMO
With the widespread interest and uptake of super-resolution ultrasound (SRUS) through localization and tracking of microbubbles, also known as ultrasound localization microscopy (ULM), many localization and tracking algorithms have been developed. ULM can image many centimeters into tissue in-vivo and track microvascular flow non-invasively with sub-diffraction resolution. In a significant community effort, we organized a challenge, Ultrasound Localization and TRacking Algorithms for Super-Resolution (ULTRA-SR). The aims of this paper are threefold: to describe the challenge organization, data generation, and winning algorithms; to present the metrics and methods for evaluating challenge entrants; and to report results and findings of the evaluation. Realistic ultrasound datasets containing microvascular flow for different clinical ultrasound frequencies were simulated, using vascular flow physics, acoustic field simulation and nonlinear bubble dynamics simulation. Based on these datasets, 38 submissions from 24 research groups were evaluated against ground truth using an evaluation framework with six metrics, three for localization and three for tracking. In-vivo mouse brain and human lymph node data were also provided, and performance assessed by an expert panel. Winning algorithms are described and discussed. The publicly available data with ground truth and the defined metrics for both localization and tracking present a valuable resource for researchers to benchmark algorithms and software, identify optimized methods/software for their data, and provide insight into the current limits of the field. In conclusion, Ultra-SR challenge has provided benchmarking data and tools as well as direct comparison and insights for a number of the state-of-the art localization and tracking algorithms.
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Algoritmos , Encéfalo , Processamento de Imagem Assistida por Computador , Ultrassonografia , Ultrassonografia/métodos , Camundongos , Animais , Humanos , Processamento de Imagem Assistida por Computador/métodos , Encéfalo/diagnóstico por imagem , Linfonodos/diagnóstico por imagem , MicrobolhasRESUMO
Introduction: Reduced synchrony of calcium release and t-tubule structure organization in individual cardiomyocytes has been linked to loss of contractile strength and arrhythmia. Compared to confocal scanning techniques widely used for imaging calcium dynamics in cardiac muscle cells, light-sheet fluorescence microscopy enables fast acquisition of a 2D plane in the sample with low phototoxicity. Methods: A custom light-sheet fluorescence microscope was used to achieve dual-channel 2D timelapse imaging of calcium and the sarcolemma, enabling calcium sparks and transients in left and right ventricle cardiomyocytes to be correlated with the cell microstructure. Imaging electrically stimulated dual-labelled cardiomyocytes immobilized with para-nitroblebbistatin, a non-phototoxic, low fluorescence contraction uncoupler, with sub-micron resolution at 395 fps over a 38 µm × 170 µm FOV allowed characterization of calcium spark morphology and 2D mapping of the calcium transient time-to-half-maximum across the cell. Results: Blinded analysis of the data revealed sparks with greater amplitude in left ventricle myocytes. The time for the calcium transient to reach half-maximum amplitude in the central part of the cell was found to be, on average, 2 ms shorter than at the cell ends. Sparks co-localized with t-tubules were found to have significantly longer duration, larger area and spark mass than those further away from t-tubules. Conclusion: The high spatiotemporal resolution of the microscope and automated image-analysis enabled detailed 2D mapping and quantification of calcium dynamics of n = 60 myocytes, with the findings demonstrating multi-level spatial variation of calcium dynamics across the cell, supporting the dependence of synchrony and characteristics of calcium release on the underlying t-tubule structure.
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The newly generated software plugin MultiFRET allows for real-time measurements of multiplexed fluorescent biosensors in a near high-throughput fashion. Here we describe a detailed protocol for setup and use of this software for any purpose requiring instant feedback during fluorescence measurement experiments. We further describe its non-primary features including beam splitter misalignment correction, custom calculations through input of simple equations typed in a .txt format, customizable Excel output, and offline bulk analysis of image stacks. Finally, we supply a usage example of a cAMP measurement in cultured rat neonatal cardiomyocytes.
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Técnicas Biossensoriais , Transferência Ressonante de Energia de Fluorescência , Animais , Técnicas Biossensoriais/métodos , Transferência Ressonante de Energia de Fluorescência/métodos , Miócitos Cardíacos , Ratos , SoftwareRESUMO
Ultrasound-modulated optical tomography (UOT) combines optical contrast with ultrasound spatial resolution and has great potential for soft tissue functional imaging. One current problem with this technique is the weak optical modulation signal, primarily due to strong optical scattering in diffuse media and minimal acoustically induced modulation. The acoustic radiation force (ARF) can create large particle displacements in tissue and has been shown to be able to improve optical modulation signals. However, shear wave propagation induced by the ARF can be a significant source of nonlocal optical modulation which may reduce UOT spatial resolution and contrast. In this paper, the time evolution of shear waves was examined on tissue mimicking-phantoms exposed to 5 MHz ultrasound and 532 nm optical radiation and measured with a CCD camera. It has been demonstrated that by generating an ARF with an acoustic burst and adjusting both the timing and the exposure time of the CCD measurement, optical contrast and spatial resolution can be improved by ~110% and ~40% respectively when using the ARF rather than 5 MHz ultrasound alone. Furthermore, it has been demonstrated that this technique simultaneously detects both optical and mechanical contrast in the medium and the optical and mechanical contrast can be distinguished by adjusting the CCD exposure time.
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Óptica e Fotônica , Tomografia Óptica/instrumentação , Ultrassom , Acústica/instrumentação , Meios de Contraste , Desenho de Equipamento , Aumento da Imagem/instrumentação , Aumento da Imagem/métodos , Lasers , Teste de Materiais , Imagens de Fantasmas , Tomografia Óptica/métodosRESUMO
Oblique plane microscopy (OPM) is a light sheet microscopy technique that uses a single high numerical aperture microscope objective to both illuminate a tilted plane within the specimen and to obtain an image of the tilted illuminated plane. In this paper, we present a new OPM configuration that enables both the illumination and detection focal planes to be swept simultaneously and remotely through the sample volume, enabling high speed volumetric imaging. We demonstrate the high speed imaging capabilities of the system by imaging calcium dynamics in cardiac myocytes in 2D at 926 frames per second and in 3D at 21 volumes per second. In the future, higher frame rate CCD cameras will enable volumetric imaging at much greater rates, leading to new capabilities to study dynamic events in cells at high speeds in two and three dimensions.
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Imageamento Tridimensional/métodos , Microscopia de Fluorescência/métodos , Miócitos Cardíacos/citologia , Compostos de Anilina/metabolismo , Animais , Núcleo Celular/metabolismo , Ratos , Fatores de Tempo , Xantenos/metabolismoRESUMO
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.
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Ouro/química , Imageamento Tridimensional/instrumentação , Imageamento Tridimensional/métodos , Luminescência , Microscopia de Fluorescência por Excitação Multifotônica/instrumentação , Microscopia de Fluorescência por Excitação Multifotônica/métodos , Humanos , Técnicas In Vitro , Nanotubos , Espectrometria de Fluorescência , Fatores de TempoRESUMO
We present an approach to laser scanning endomicroscopy that requires no moving parts and can be implemented with no distal scanners or optics, permitting extremely compact endoscopic probes to be developed. Our approach utilizes a spatial light modulator to correct for phase variations across a fiber imaging bundle and to encode for arbitrary wavefronts at the distal end of the fiber bundle. Thus, it is possible to realize both focusing and beam scanning at the output of the fiber bundle with no distal components. We present proof of principle results to illustrate three-dimensional scanning of the focal spot and exemplar images of a United States Air Force resolution test chart.
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Microscopia Confocal/métodos , Imageamento Tridimensional , Luz , Fenômenos ÓpticosRESUMO
We report the supramolecular organization of killer Ig-like receptor (KIR) phosphorylation using a technique applicable to imaging phosphorylation of any green fluorescent protein-tagged receptor at an intercellular contact or immune synapse. Specifically, we use fluorescence lifetime imaging (FLIM) to report Förster resonance energy transfer (FRET) between GFP-tagged KIR2DL1 and a Cy3-tagged generic anti-phosphotyrosine monoclonal antibody. Visualization of KIR phosphorylation in natural killer (NK) cells contacting target cells expressing cognate major histocompatibility complex class I proteins revealed that inhibitory signaling is spatially restricted to the immune synapse. This explains how NK cells respond appropriately when simultaneously surveying susceptible and resistant target cells. More surprising, phosphorylated KIR was confined to microclusters within the aggregate of KIR, contrary to an expected homogeneous distribution of KIR signaling across the immune synapse. Also, yellow fluorescent protein-tagged Lck, a kinase important for KIR phosphorylation, accumulated in a multifocal distribution at inhibitory synapses. Spatial confinement of receptor phosphorylation within the immune synapse may be critical to how activating and inhibitory signals are integrated in NK cells.
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Estruturas da Membrana Celular/metabolismo , Junções Intercelulares/imunologia , Células Matadoras Naturais/metabolismo , Receptores Imunológicos/metabolismo , Transdução de Sinais/imunologia , Linhagem Celular , Estruturas da Membrana Celular/ultraestrutura , Transferência Ressonante de Energia de Fluorescência , Humanos , Junções Intercelulares/ultraestrutura , Células Matadoras Naturais/efeitos dos fármacos , Células Matadoras Naturais/imunologia , Fosforilação , Pirimidinas/farmacologia , Agregação de Receptores/imunologia , Receptores Imunológicos/antagonistas & inibidores , Receptores KIR , Receptores KIR2DL1 , Transdução de Sinais/efeitos dos fármacos , Quinases da Família src/antagonistas & inibidores , Quinases da Família src/metabolismoRESUMO
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.