In JoVE (1)

Other Publications (34)

Articles by Christopher Dunsby in JoVE

 JoVE Biology

Open Source High Content Analysis Utilizing Automated Fluorescence Lifetime Imaging Microscopy

1Photonics Group, Department of Physics, Imperial College London, 2Institute for Chemical Biology, Department of Chemistry, Imperial College London, 3MRC Clinical Sciences Centre, Hammersmith Hospital, 4Chemical Biology Section, Department of Chemistry, Imperial College London, 5Retroscreen Virology Ltd, 6Pfizer Global Research and Development, Pfizer Limited, Sandwich, Kent, UK, 7Centre for Histopathology, Imperial College London


JoVE 55119

Other articles by Christopher Dunsby on PubMed

Single-shot Phase-stepped Wide-field Coherencegated Imaging

Optics Express. Jan, 2003  |  Pubmed ID: 19461712

We present a single-shot wide-field CCD based coherence-gated imaging technique that utilizes spatially separated phase-stepped images and requires only one CCD camera to achieve simultaneous acquisition of four phase-stepped images. This technique provides a relatively low cost system for depth-resolved imaging of dynamic samples. We demonstrate real-time coherence-gated imaging of a moving watch cog, 3D reconstructions of a coin, phase measurements of the surface of a test-chart and depth-resolved imaging in a weakly scattering sample of onion.

Fluorescence Lifetime Imaging Provides Enhanced Contrast when Imaging the Phase-sensitive Dye Di-4-ANEPPDHQ in Model Membranes and Live Cells

Biophysical Journal. Jun, 2006  |  Pubmed ID: 16617080

We apply fluorescence lifetime imaging to the membrane phase-sensing dye di-4-ANEPPDHQ in model membranes and live cells. We show that the 1700 ps lifetime shift between liquid-disordered and liquid-ordered phases offers greater contrast than the 60 nm spectral shift previously reported. Detection of cholesterol-rich membrane microdomains is confirmed by observation of the temperature dependence of membrane order and by cholesterol depletion using methyl-beta-cyclodextrin.

Tissue Characterization Using Dimensionality Reduction and Fluorescence Imaging

Medical Image Computing and Computer-assisted Intervention : MICCAI ... International Conference on Medical Image Computing and Computer-Assisted Intervention. 2006  |  Pubmed ID: 17354820

Multidimensional fluorescence imaging is a powerful molecular imaging modality that is emerging as an important tool in the study of biological tissues. Due to the large volume of multi-spectral data associated with the technique, it is often difficult to find the best combination of parameters to maximize the contrast between different tissue types. This paper presents a novel framework for the characterization of tissue compositions based on the use of time resolved fluorescence imaging without the explicit modeling of the decays. The composition is characterized through soft clustering based on manifold embedding for reducing the dimensionality of the datasets and obtaining a consistent differentiation scheme for determining intrinsic constituents of the tissue. The proposed technique has the benefit of being fully automatic, which could have significant advantages for automated histopathology and increasing the speed of intraoperative decisions. Validation of the technique is carried out with both phantom data and tissue samples of the human pancreas.

Rapid Hyperspectral Fluorescence Lifetime Imaging

Microscopy Research and Technique. May, 2007  |  Pubmed ID: 17366615

We report a rapid hyperspectral fluorescence lifetime imaging (FLIM) instrument that exploits high-speed FLIM technology in a line-scanning microscope. We demonstrate the acquisition of whole-field optically sectioned hyperspectral fluorescence lifetime image stacks (with 32 spectral bins) in less than 40 s and illustrate its application to unstained biological tissue.

Excitation-resolved Hyperspectral Fluorescence Lifetime Imaging Using a UV-extended Supercontinuum Source

Optics Letters. Dec, 2007  |  Pubmed ID: 18059949

We present a time-gated, optically sectioned, hyperspectral fluorescence lifetime imaging (FLIM) microscope incorporating a tunable supercontinuum excitation source extending into the UV. The system is capable of resolving the excitation spectrum, emission spectrum, and fluorescence decays in an optically sectioned image.

Stimulated Emission Depletion Microscopy with a Supercontinuum Source and Fluorescence Lifetime Imaging

Optics Letters. Jan, 2008  |  Pubmed ID: 18197209

We demonstrate stimulated emission depletion (STED) microscopy implemented in a laser scanning confocal microscope using excitation light derived from supercontinuum generation in a microstructured optical fiber. Images with resolution improvement beyond the far-field diffraction limit in both the lateral and axial directions were acquired by scanning overlapped excitation and depletion beams in two dimensions using the flying spot scanner of a commercially available laser scanning confocal microscope. The spatial properties of the depletion beam were controlled holographically using a programmable spatial light modulator, which can rapidly change between different STED imaging modes and also compensate for aberrations in the optical path. STED fluorescence lifetime imaging microscopy is demonstrated through the use of time-correlated single photon counting.

Three-dimensional Molecular Mapping in a Microfluidic Mixing Device Using Fluorescence Lifetime Imaging

Optics Letters. Aug, 2008  |  Pubmed ID: 18709122

Fluorescence lifetime imaging (FLIM) is used to quantitatively map the concentration of a small molecule in three dimensions in a microfluidic mixing device. The resulting experimental data are compared with computational fluid-dynamics (CFD) simulations. A line-scanning semiconfocal FLIM microscope allows the full mixing profile to be imaged in a single scan with submicrometer resolution over an arbitrary channel length from the point of confluence. Following experimental and CFD optimization, mixing times down to 1.3+/-0.4 ms were achieved with the single-layer microfluidic device.

High-speed High-resolution Imaging of Intercellular Immune Synapses Using Optical Tweezers

Biophysical Journal. Nov, 2008  |  Pubmed ID: 18723590

Imaging in any plane other than horizontal in a microscope typically requires a reconstruction from multiple optical slices that significantly decreases the spatial and temporal resolution that can be achieved. This can limit the precision with which molecular events can be detected, for example, at intercellular contacts. This has been a major issue for the imaging of immune synapses between live cells, which has generally required the reconstruction of en face intercellular synapses, yielding spatial resolution significantly above the diffraction limit and updating at only a few frames per minute. Strategies to address this issue have usually involved using artificial activating substrates such as antibody-coated slides or supported planar lipid bilayers, but synapses with these surrogate stimuli may not wholly resemble immune synapses between two cells. Here, we combine optical tweezers and confocal microscopy to realize generally applicable, high-speed, high-resolution imaging of almost any arbitrary plane of interest. Applied to imaging immune synapses in live-cell conjugates, this has enabled the characterization of complex behavior of highly dynamic clusters of T cell receptors at the T cell/antigen-presenting cell intercellular immune synapse and revealed the presence of numerous, highly dynamic long receptor-rich filopodial structures within inhibitory Natural Killer cell immune synapses.

Multiplexed FRET to Image Multiple Signaling Events in Live Cells

Biophysical Journal. Nov, 2008  |  Pubmed ID: 18757561

We report what to our knowledge is a novel approach for simultaneous imaging of two different Förster resonance energy transfer (FRET) sensors in the same cell with minimal spectral cross talk. Previous methods based on spectral ratiometric imaging of the two FRET sensors have been limited by the availability of suitably bright acceptors for the second FRET pair and the spectral cross talk incurred when measuring in four spectral windows. In contrast to spectral ratiometric imaging, fluorescence lifetime imaging (FLIM) requires measurement of the donor fluorescence only and is independent of emission from the acceptor. By combining FLIM-FRET of the novel red-shifted TagRFP/mPlum FRET pair with spectral ratiometric imaging of an ECFP/Venus pair we were thus able to maximize the spectral separation between our chosen fluorophores while at the same time overcoming the low quantum yield of the far red acceptor mPlum. Using this technique, we could read out a TagRFP/mPlum intermolecular FRET sensor for reporting on small Ras GTP-ase activation in live cells after epidermal growth factor stimulation and an ECFP/Venus Cameleon FRET sensor for monitoring calcium transients within the same cells. The combination of spectral ratiometric imaging of ECFP/Venus and high-speed FLIM-FRET of TagRFP/mPlum can thus increase the spectral bandwidth available and provide robust imaging of multiple FRET sensors within the same cell. Furthermore, since FLIM does not require equal stoichiometries of donor and acceptor, this approach can be used to report on both unimolecular FRET biosensors and protein-protein interactions with the same cell.

Fluorescence Lifetime Optical Projection Tomography

Journal of Biophotonics. Oct, 2008  |  Pubmed ID: 19343662

We describe a quantitative fluorescence projection tomography technique which measures the 3-D fluorescence lifetime distribution in optically cleared specimens up 1 cm in diameter. This is achieved by acquiring a series of wide-field time-gated images at different relative time delays with respect to a train of excitation pulses, at a number of projection angles. For each time delay, the 3-D time-gated intensity distribution is reconstructed using a filtered back projection algorithm and the fluorescence lifetime subsequently determined for each reconstructed horizontal plane by iterative fitting to a mono-exponential decay. Due to its inherently ratiometric nature, fluorescence lifetime is robust against intensity based artefacts as well as producing a quantitative measure of the fluorescence signal. We present a 3-D fluorescence lifetime reconstruction of a mouse embryo labelled with an alexa-488 conjugated antibody targeted to the neurofilament, which clearly differentiates between the extrinsic label and the autofluorescence, particularly from the heart and dorsal aorta.

Continuous-flow Polymerase Chain Reaction of Single-copy DNA in Microfluidic Microdroplets

Analytical Chemistry. Jan, 2009  |  Pubmed ID: 19055421

We present a high throughput microfluidic device for continuous-flow polymerase chain reaction (PCR) in water-in-oil droplets of nanoliter volumes. The circular design of this device allows droplets to pass through alternating temperature zones and complete 34 cycles of PCR in only 17 min, avoiding temperature cycling of the entire device. The temperatures for the applied two-temperature PCR protocol can be adjusted according to requirements of template and primers. These temperatures were determined with fluorescence lifetime imaging (FLIM) inside the droplets, exploiting the temperature-dependent fluorescence lifetime of rhodamine B. The successful amplification of an 85 base-pair long template from four different start concentrations was demonstrated. Analysis of the product by gel-electrophoresis, sequencing, and real-time PCR showed that the amplification is specific and the amplification factors of up to 5 x 10(6)-fold are comparable to amplification factors obtained in a benchtop PCR machine. The high efficiency allows amplification from a single molecule of DNA per droplet. This device holds promise for convenient integration with other microfluidic devices and adds a critical missing component to the laboratory-on-a-chip toolkit.

Removal of Background Signals from Fluorescence Thermometry Measurements in PDMS Microchannels Using Fluorescence Lifetime Imaging

Lab on a Chip. Dec, 2009  |  Pubmed ID: 19904413

We report a method for removing unwanted contributions to fluorescence signals from dyes absorbed in polydimethylsiloxane (PDMS) using fluorescence lifetime imaging microscopy (FLIM). By analysing experimental fluorescence decays using a bi-exponential decay model, we are able to discriminate between emission originating from dye molecules in free solution and those absorbed within the PDMS substrate. Simple image processing allows the unwanted background signal to be removed and thus enables a more accurate assessment of temperature. The efficacy of the approach is demonstrated by measuring temperature changes within a droplet-based PCR device.

Response of Rigor Cross-bridges to Stretch Detected by Fluorescence Lifetime Imaging Microscopy of Myosin Essential Light Chain in Skeletal Muscle Fibers

The Journal of Biological Chemistry. Jan, 2011  |  Pubmed ID: 21056977

We applied fluorescence lifetime imaging microscopy to map the microenvironment of the myosin essential light chain (ELC) in permeabilized skeletal muscle fibers. Four ELC mutants containing a single cysteine residue at different positions in the C-terminal half of the protein (ELC-127, ELC-142, ELC-160, and ELC-180) were generated by site-directed mutagenesis, labeled with 7-diethylamino-3-((((2-iodoacetamido)ethyl)amino)carbonyl)coumarin, and introduced into permeabilized rabbit psoas fibers. Binding to the myosin heavy chain was associated with a large conformational change in the ELC. When the fibers were moved from relaxation to rigor, the fluorescence lifetime increased for all label positions. However, when 1% stretch was applied to the rigor fibers, the lifetime decreased for ELC-127 and ELC-180 but did not change for ELC-142 and ELC-160. The differential change of fluorescence lifetime demonstrates the shift in position of the C-terminal domain of ELC with respect to the heavy chain and reveals specific locations in the lever arm region sensitive to the mechanical strain propagating from the actin-binding site to the lever arm.

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. Apr, 2011  |  Pubmed ID: 21112870

Mismatch uracil DNA glycosylase (Mug) from Escherichia coli is an initiating enzyme in the base-excision repair pathway. As with other DNA glycosylases, the abasic product is potentially more harmful than the initial lesion. Since Mug is known to bind its product tightly, inhibiting enzyme turnover, understanding how Mug binds DNA is of significance when considering how Mug interacts with downstream enzymes in the base-excision repair pathway. We have demonstrated differential binding modes of Mug between its substrate and abasic DNA product using both band shift and fluorescence anisotropy assays. Mug binds its product cooperatively, and a stoichiometric analysis of DNA binding, catalytic activity and salt-dependence indicates that dimer formation is of functional significance in both catalytic activity and product binding. This is the first report of cooperativity in the uracil DNA glycosylase superfamily of enzymes, and forms the basis of product inhibition in Mug. It therefore provides a new perspective on abasic site protection and the findings are discussed in the context of downstream lesion processing and enzyme communication in the base excision repair pathway.

Membrane Environment Exerts an Important Influence on Rac-mediated Activation of Phospholipase Cγ2

Molecular and Cellular Biology. Mar, 2011  |  Pubmed ID: 21245382

We performed analyses of the molecular mechanisms involved in the regulation of phospholipase Cγ2 (PLCγ2). We identified several regions in the PLCγ-specific array, γSA, that contribute to autoinhibition in the basal state by occlusion of the catalytic domain. While the activation of PLCγ2 by Rac2 requires stable translocation to the membrane, the removal of the domains required for membrane translocation in the context of an enzyme with impaired autoinhibition generated constitutive, highly active PLC in cells. We further tested the possibility that the interaction of PLCγ2 with its activator protein Rac2 was sufficient for activation through the release of autoinhibition. However, we found that Rac2 binding in the absence of lipid surfaces was not able to activate PLCγ2. Together with other observations, these data suggest that an important consequence of Rac2 binding and translocation to the membrane is that membrane proximity, on its own or together with Rac2, has a role in the release of autoinhibition, resulting in interfacial activation.

Quantitative Evaluation of Healthy Epidermis by Means of Multiphoton Microscopy and Fluorescence Lifetime Imaging Microscopy

Skin Research and Technology : Official Journal of International Society for Bioengineering and the Skin (ISBS) [and] International Society for Digital Imaging of Skin (ISDIS) [and] International Society for Skin Imaging (ISSI). Aug, 2011  |  Pubmed ID: 21518012

Multiphoton microscopy (MPM) enables the assessment of unstained living biological tissue with submicron resolution, whereas fluorescence lifetime imaging microscopy (FLIM) generates image contrast between different states of tissue characterized by various fluorescence decay rates. The aim of this study was to compare the healthy skin of young individuals with that of older subjects, as well as to assess the skin at different body sites, by means of MPM and FLIM.

Remodelling of Cortical Actin Where Lytic Granules Dock at Natural Killer Cell Immune Synapses Revealed by Super-resolution Microscopy

PLoS Biology. Sep, 2011  |  Pubmed ID: 21931537

Natural Killer (NK) cells are innate immune cells that secrete lytic granules to directly kill virus-infected or transformed cells across an immune synapse. However, a major gap in understanding this process is in establishing how lytic granules pass through the mesh of cortical actin known to underlie the NK cell membrane. Research has been hampered by the resolution of conventional light microscopy, which is too low to resolve cortical actin during lytic granule secretion. Here we use two high-resolution imaging techniques to probe the synaptic organisation of NK cell receptors and filamentous (F)-actin. A combination of optical tweezers and live cell confocal microscopy reveals that microclusters of NKG2D assemble into a ring-shaped structure at the centre of intercellular synapses, where Vav1 and Grb2 also accumulate. Within this ring-shaped organisation of NK cell proteins, lytic granules accumulate for secretion. Using 3D-structured illumination microscopy (3D-SIM) to gain super-resolution of ~100 nm, cortical actin was detected in a central region of the NK cell synapse irrespective of whether activating or inhibitory signals dominate. Strikingly, the periodicity of the cortical actin mesh increased in specific domains at the synapse when the NK cell was activated. Two-colour super-resolution imaging revealed that lytic granules docked precisely in these domains which were also proximal to where the microtubule-organising centre (MTOC) polarised. Together, these data demonstrate that remodelling of the cortical actin mesh occurs at the central region of the cytolytic NK cell immune synapse. This is likely to occur for other types of cell secretion and also emphasises the importance of emerging super-resolution imaging technology for revealing new biology.

Ultrasound-mediated Optical Tomography: a Review of Current Methods

Interface Focus. Aug, 2011  |  Pubmed ID: 22866234

Ultrasound-mediated optical tomography (UOT) is a hybrid technique that is able to combine the high penetration depth and high spatial resolution of ultrasound imaging to overcome the limits imposed by optical scattering for deep tissue optical sensing and imaging. It has been proposed as a method to detect blood concentrations, oxygenation and metabolism at depth in tissue for the detection of vascularized tumours or the presence of absorbing or scattering contrast agents. In this paper, the basic principles of the method are outlined and methods for simulating the UOT signal are described. The main detection methods are then summarized with a discussion of the advantages and disadvantages of each. The recent focus on increasing the weak UOT signal through the use of the acoustic radiation force is explained, together with a summary of our results showing sensitivity to the mechanical shear stiffness and optical absorption properties of tissue-mimicking phantoms.

Shear Wave Elasticity Imaging Based on Acoustic Radiation Force and Optical Detection

Ultrasound in Medicine & Biology. Sep, 2012  |  Pubmed ID: 22749816

Tissue elasticity is closely related to the velocity of shear waves within biologic tissue. Shear waves can be generated by an acoustic radiation force and tracked by, e.g., ultrasound or magnetic resonance imaging (MRI) measurements. This has been shown to be able to noninvasively map tissue elasticity in depth and has great potential in a wide range of clinical applications including cancer and cardiovascular diseases. In this study, a highly sensitive optical measurement technique is proposed as an alternative way to track shear waves generated by the acoustic radiation force. A charge coupled device (CCD) camera was used to capture diffuse photons from tissue mimicking phantoms illuminated by a laser source at 532 nm. CCD images were recorded at different delays after the transmission of an ultrasound burst and were processed to obtain the time of flight for the shear wave. A differential measurement scheme involving generation of shear waves at two different positions was used to improve the accuracy and spatial resolution of the system. The results from measurements on both homogeneous and heterogeneous phantoms were compared with measurements from other instruments and demonstrate the feasibility and accuracy of the technique for imaging and quantifying elasticity. The relative error in estimation of shear wave velocity can be as low as 3.3% with a spatial resolution of 2 mm, and increases to 8.8% with a spatial resolution of 1 mm for the medium stiffness phantom. The system is shown to be highly sensitive and is able to track shear waves propagating over several centimetres given the ultrasound excitation amplitude and the phantom material used in this study. It was also found that the reflection of shear waves from boundaries between regions with different elastic properties can cause significant bias in the estimation of elasticity, which also applies to other shear wave tracking techniques. This bias can be reduced at the expense of reduced spatial resolution.

Multiphoton Laser Tomography and Fluorescence Lifetime Imaging of Basal Cell Carcinoma: Morphologic Features for Non-invasive Diagnostics

Experimental Dermatology. Nov, 2012  |  Pubmed ID: 22882324

Multiphoton laser tomography (MPT) combined with fluorescence lifetime imaging (FLIM) is a non-invasive imaging technique, which gives access to the cellular and extracellular morphology of the skin. The aim of our study was to assess the sensitivity and specificity of MPT/FLIM descriptors for basal cell carcinoma (BCC), to improve BCC diagnosis and the identification of tumor margins. In the preliminary study, FLIM images referring to 35 BCCs and 35 healthy skin samples were evaluated for the identification of morphologic descriptors characteristic of BCC. In the main study, the selected parameters were blindly evaluated on a test set comprising 63 BCCs, 63 healthy skin samples and 66 skin lesions. Moreover, FLIM values inside a region of interest were calculated on 98 healthy skin and 98 BCC samples. In the preliminary study, three epidermal descriptors and 7 BCC descriptors were identified. The specificity of the diagnostic criteria versus 'other lesions' was extremely high, indicating that the presence of at least one BCC descriptor makes the diagnosis of 'other lesion' extremely unlikely. FLIM values referring to BCC cells significantly differed from those of healthy skin. In this study, we identified morphological and numerical descriptors enabling the differentiation of BCC from other skin disorders and its distinction from healthy skin in ex vivo samples. In future, MPT/FLIM may be applied to skin lesions to provide direct clinical guidance before biopsy and histological examination and for the identification of tumor margins allowing a complete surgical removal.

Activity of PLCε Contributes to Chemotaxis of Fibroblasts Towards PDGF

Journal of Cell Science. Dec, 2012  |  Pubmed ID: 22992460

Cell chemotaxis, such as migration of fibroblasts towards growth factors during development and wound healing, requires precise spatial coordination of signalling events. Phosphoinositides and signalling enzymes involved in their generation and hydrolysis have been implicated in regulation of chemotaxis; however, the role and importance of specific components remain poorly understood. Here, we demonstrate that phospholipase C epsilon (PLCε) contributes to fibroblast chemotaxis towards platelet-derived growth factor (PDGF-BB). Using PLCe1 null fibroblasts we show that cells deficient in PLCε have greatly reduced directionality towards PDGF-BB without detrimental effect on their basal ability to migrate. Furthermore, we show that in intact fibroblasts, signalling events, such as activation of Rac, are spatially compromised by the absence of PLCε that affects the ability of cells to enlarge their protrusions in the direction of the chemoattractant. By further application of live cell imaging and the use of FRET-based biosensors, we show that generation of Ins(1,4,5)P(3) and recruitment of PLCε are most pronounced in protrusions responding to the PDGF-BB gradient. Furthermore, the phospholipase C activity of PLCε is critical for its role in chemotaxis, consistent with the importance of Ins(1,4,5)P(3) generation and sustained calcium responses in this process. As PLCε has extensive signalling connectivity, using transgenic fibroblasts we ruled out its activation by direct binding to Ras or Rap GTPases, and suggest instead new unexpected links for PLCε in the context of chemotaxis.

High-resolution Imaging of Basal Cell Carcinoma: a Comparison Between Multiphoton Microscopy with Fluorescence Lifetime Imaging and Reflectance Confocal Microscopy

Skin Research and Technology : Official Journal of International Society for Bioengineering and the Skin (ISBS) [and] International Society for Digital Imaging of Skin (ISDIS) [and] International Society for Skin Imaging (ISSI). Feb, 2013  |  Pubmed ID: 22970856

The aim of this study was to compare morphological aspects of basal cell carcinoma (BCC) as assessed by two different imaging methods: in vivo reflectance confocal microscopy (RCM) and multiphoton tomography with fluorescence lifetime imaging implementation (MPT-FLIM).

Detection of Cartilage Matrix Degradation by Autofluorescence Lifetime

Matrix Biology : Journal of the International Society for Matrix Biology. Jan, 2013  |  Pubmed ID: 23266527

Cartilage is a vital organ to maintain joint function. Upon arthritis, proteolytic enzymes initiate degradation of cartilage extracellular matrix (ECM) resulting in eventual loss of joint function. However, there are only limited ways of non-invasively monitoring early chemical changes in cartilage matrix. Here we report that the autofluorescence decay profiles of cartilage tissue are significantly affected by proteolytic degradation of cartilage ECM and can be characterised by measurements of the autofluorescence lifetime (AFL). A compact multidimensional fluorometer coupled to a fibre-optic probe was developed for single point measurements of AFL and applied to cartilage that was treated with different proteinases. Upon treating cartilage with bacterial collagenase, trypsin or matrix metalloproteinase 1, a significant dose and time dependent decrease of AFL was observed. Our data suggest that AFL of cartilage tissue is a potential non-invasive readout to monitor cartilage matrix integrity that may contribute to future diagnosis of cartilage defects as well as monitoring the efficacy of anti-joint therapeutic agents.

High Resolution Diagnosis of Common Nevi by Multiphoton Laser Tomography and Fluorescence Lifetime Imaging

Skin Research and Technology : Official Journal of International Society for Bioengineering and the Skin (ISBS) [and] International Society for Digital Imaging of Skin (ISDIS) [and] International Society for Skin Imaging (ISSI). May, 2013  |  Pubmed ID: 23279266

Multiphoton Laser Tomography (MPT) has developed as a non-invasive tool that allows real-time observation of the skin with subcellular resolution. MPT is readily combined with time resolved detectors to achieve fluorescence lifetime imaging (FLIM). The aim of our study was to identify morphologic MPT/FLIM descriptors of melanocytic nevi, referring to cellular and architectural features.

High-resolution Multiphoton Tomography and Fluorescence Lifetime Imaging of UVB-induced Cellular Damage on Cultured Fibroblasts Producing Fibres

Skin Research and Technology : Official Journal of International Society for Bioengineering and the Skin (ISBS) [and] International Society for Digital Imaging of Skin (ISDIS) [and] International Society for Skin Imaging (ISSI). Aug, 2013  |  Pubmed ID: 23590582

Multiphoton tomography (MPT) is suitable to perform both ex vivo and in vivo investigations of living skin and cell cultures with submicron resolution. Fluorescence lifetime imaging (FLIM) generates image contrast between different states of tissue characterized by various fluorescence decay rates. Our purpose was to combine MPT and FLIM to evaluate fibroblasts and collagen fibres produced in vitro.

Multiphoton Laser Tomography and Fluorescence Lifetime Imaging of Melanoma: Morphologic Features and Quantitative Data for Sensitive and Specific Non-invasive Diagnostics

PloS One. 2013  |  Pubmed ID: 23923016

Multiphoton laser tomography (MPT) combined with fluorescence lifetime imaging (FLIM) is a non-invasive imaging technique, based on the study of fluorescence decay times of naturally occurring fluorescent molecules, enabling a non-invasive investigation of the skin with subcellular resolution. The aim of this retrospective observational ex vivo study, was to characterize melanoma both from a morphologic and a quantitative point of view, attaining an improvement in the diagnostic accuracy with respect to dermoscopy. In the training phase, thirty parameters, comprising both cytological descriptors and architectural aspects, were identified. The training set included 6 melanomas with a mean Breslow thickness±S.D. of 0.89±0.48 mm. In the test phase, these parameters were blindly evaluated on a test data set consisting of 25 melanomas, 50 nevi and 50 basal cell carcinomas. Melanomas in the test phase comprised 8 in situ lesions and had a mean thickness±S.D. of 0.77±1.2 mm. Moreover, quantitative FLIM data were calculated for special areas of interest. Melanoma was characterized by the presence of atypical short lifetime cells and architectural disorder, in contrast to nevi presenting typical cells and a regular histoarchitecture. Sensitivity and specificity values for melanoma diagnosis were 100% and 98%, respectively, whereas dermoscopy achieved the same sensitivity, but a lower specificity (82%). Mean fluorescence lifetime values of melanocytic cells did not vary between melanomas and nevi, but significantly differed from those referring to basal cell carcinoma enabling a differential diagnosis based on quantitative data. Data from prospective preoperative trials are needed to confirm if MPT/FLIM could increase diagnostic specificity and thus reduce unnecessary surgical excisions.

Experimental Proof of Concept of Nanoparticle-assisted STED

Nano Letters. Aug, 2014  |  Pubmed ID: 25053232

We imaged core-shell nanoparticles, consisting of a dye-doped silica core covered with a layer of gold, with a stimulated emission depletion, fluorescence lifetime imaging (STED-FLIM) microscope. Because of the field enhancement provided by the localized surface plasmon resonance of the gold shell, we demonstrate a reduction of the STED depletion power required to obtain resolution improvement by a factor of 4. This validates the concept of nanoparticle-assisted STED (NP-STED), where hybrid dye-plasmonic nanoparticles are used as labels for STED in order to decrease the depletion powers required for subwavelength imaging.

Analysis of DNA Binding and Nucleotide Flipping Kinetics Using Two-color Two-photon Fluorescence Lifetime Imaging Microscopy

Analytical Chemistry. Nov, 2014  |  Pubmed ID: 25303623

Uracil DNA glycosylase plays a key role in DNA maintenance via base excision repair. Its role is to bind to DNA, locate unwanted uracil, and remove it using a base flipping mechanism. To date, kinetic analysis of this complex process has been achieved using stopped-flow analysis but, due to limitations in instrumental dead-times, discrimination of the "binding" and "base flipping" steps is compromised. Herein we present a novel approach for analyzing base flipping using a microfluidic mixer and two-color two-photon (2c2p) fluorescence lifetime imaging microscopy (FLIM). We demonstrate that 2c2p FLIM can simultaneously monitor binding and base flipping kinetics within the continuous flow microfluidic mixer, with results showing good agreement with computational fluid dynamics simulations.

A Flexible Wide-field FLIM Endoscope Utilising Blue Excitation Light for Label-free Contrast of Tissue

Journal of Biophotonics. Jan, 2015  |  Pubmed ID: 24573953

Fluorescence lifetime imaging (FLIM) has previously been shown to provide contrast between normal and diseased tissue. Here we present progress towards clinical and preclinical FLIM endoscopy of tissue autofluorescence, demonstrating a flexible wide-field endoscope that utilised a low average power blue picosecond laser diode excitation source and was able to acquire ∼mm-scale spatial maps of autofluorescence lifetimes from fresh ex vivo diseased human larynx biopsies in ∼8 seconds using an average excitation power of ∼0.5 mW at the specimen. To illustrate its potential for FLIM at higher acquisition rates, a higher power mode-locked frequency doubled Ti:Sapphire laser was used to demonstrate FLIM of ex vivo mouse bowel at up to 2.5 Hz using 10 mW of average excitation power at the specimen.

In Vivo Acoustic Super-resolution and Super-resolved Velocity Mapping Using Microbubbles

IEEE Transactions on Medical Imaging. Feb, 2015  |  Pubmed ID: 25265604

The structure of microvasculature cannot be resolved using standard clinical ultrasound (US) imaging frequencies due to the fundamental diffraction limit of US waves. In this work, we use a standard clinical US system to perform in vivo sub-diffraction imaging on a CD1, female mouse aged eight weeks by localizing isolated US signals from microbubbles flowing within the ear microvasculature, and compare our results to optical microscopy. Furthermore, we develop a new technique to map blood velocity at super-resolution by tracking individual bubbles through the vasculature. Resolution is improved from a measured lateral and axial resolution of 112 μm and 94 μ m respectively in original US data, to super-resolved images of microvasculature where vessel features as fine as 19 μm are clearly visualized. Velocity maps clearly distinguish opposing flow direction and separated speed distributions in adjacent vessels, thereby enabling further differentiation between vessels otherwise not spatially separated in the image. This technique overcomes the diffraction limit to provide a noninvasive means of imaging the microvasculature at super-resolution, to depths of many centimeters. In the future, this method could noninvasively image pathological or therapeutic changes in the microvasculature at centimeter depths in vivo.

Time-resolved FRET Reports FGFR1 Dimerization and Formation of a Complex with Its Effector PLCγ1

Advances in Biological Regulation. Jan, 2016  |  Pubmed ID: 26482290

In vitro and in vivo imaging of protein tyrosine kinase activity requires minimally invasive, molecularly precise optical probes to provide spatiotemporal mechanistic information of dimerization and complex formation with downstream effectors. We present here a construct with genetically encoded, site-specifically incorporated, bioorthogonal reporter that can be selectively labelled with exogenous fluorogenic probes to monitor the structure and function of fibroblast growth factor receptor (FGFR). GyrB.FGFR1KD.TC contains a coumermycin-induced artificial dimerizer (GyrB), FGFR1 kinase domain (KD) and a tetracysteine (TC) motif that enables fluorescent labelling with biarsenical dyes FlAsH-EDT2 and ReAsH-EDT2. We generated bimolecular system for time-resolved FRET (TR-FRET) studies, which pairs FlAsH-tagged GyrB.FGFR1KD.TC and N-terminal Src homology 2 (nSH2) domain of phospholipase Cγ (PLCγ), a downstream effector of FGFR1, fused to mTurquoise fluorescent protein (mTFP). We demonstrated phosphorylation-dependent TR-FRET readout of complex formation between mTFP.nSH2 and GyrB.FGFR1KD.TC. By further application of TR-FRET, we also demonstrated formation of the GyrB.FGFR1KD.TC homodimer by coumermycin-induced dimerization. Herein, we present a spectroscopic FRET approach to facilitate and propagate studies that would provide structural and functional insights for FGFR and other tyrosine kinases.

Screening for Protein-protein Interactions Using Förster Resonance Energy Transfer (FRET) and Fluorescence Lifetime Imaging Microscopy (FLIM)

Scientific Reports. Jun, 2016  |  Pubmed ID: 27339025

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.

Corrigendum: Screening for Protein-protein Interactions Using Förster Resonance Energy Transfer (FRET) and Fluorescence Lifetime Imaging Microscopy (FLIM)

Scientific Reports. Sep, 2016  |  Pubmed ID: 27654516

Conformational Transition of FGFR Kinase Activation Revealed by Site-specific Unnatural Amino Acid Reporter and Single Molecule FRET

Scientific Reports. Jan, 2017  |  Pubmed ID: 28045057

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.

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