KEYWORDS: Molecular interactions, Molecules, Biological imaging, Nanodomains, Time correlated single photon counting, Molecular spectroscopy, Cancer detection, Single molecule spectroscopy, Quantum detection, Lung cancer
Fluorescence lifetime imaging (FLIM) is a sensitive tool to investigate protein-protein interactions in biology and may have some niche applications for early detection of cancer in liquid biospies. The gold-standard for such a technique is time-correlated single photon counting (TCSPC). For imaging, this has largely been exploited in laser scanning microscopy techniques. However, these imaging modalities are limited primarily by their particularly slow acquisition speeds compared to conventional imaging methodologies especially when the sample is sparse. We have developed a simple optical platform to exploit the sparseness of single molecule imaging and apply this to preliminary study of the heterodimer status of EGFR and HER3, in cell secreted vesicles (exosomes). In parallel we perform single EV trapping experiments with an ABEL trap to extend our spectroscopic analysis. We perform both fluorescence lifetime spectroscopy and SMLM on single exosomes in vitro to assess changes in the proclivity of heterodimer formation following Osimertinib treatment in lung cancer cells (H1975), which may elude to a possible functional role in cancer treatment resistance.
The Microscopy Innovation Centre (MIC) at King’s College London, aims to provide advanced and home-built imaging solutions to its users. Such microscopy systems are often not provided by core facilities due to issues including the need for specific expertise, specific sample requirements, and continued maintenance. The MIC provides a home to such imaging platforms and has dedicated staff members to maintain as well as provide them to a wide user base. We present a general overview of the MIC as well as showing some of the work done using our home-built microscopes.
A tension-sensitive biosensor was used to detect changes in applied force across the mechanosensitive focal adhesion protein vinculin. When the biosensor is under tension, two fluorescent proteins separate, decreasing the amount of Förster Resonance Energy Transfer (FRET) observed. By measuring FRET using time-correlated single-photon counting fluorescence lifetime imaging microscopy (TCSPC-FLIM) we observe the loss of FRET, as a direct consequence of an applied intracellular force across the biosensor. Mouse embryonic fibroblasts (MEFs) transfected with a vinculin construct encoding the Tension Sensing Module (TSM), demonstrate how force-transduction changes within maturing adhesions in both fixed and live cells.
We report the development of a novel massively-parallelised high-speed multifocal FLIM platform with the ability to acquire data 1024 times faster than a conventional TCSPC system. We demonstrate the system performanceFRET imaging of the fluorescent protein biosensor PercivalHR in iPSC derived neurons to measure the dynamic concentration of ADP/ATP in live cells. The advantages and performance envelope of the system will be shown and the potential for further applications explored.
Time-domain microfluidic fluorescence lifetime flow cytometry enables observation of fluorescence decay of particles or cells over time using time-correlated single photon counting (TCSPC). This method requires the fluorescence lifetime measured from a limited number of photons and in a short amount of time. In current implementations of the technique, the low throughput of state of the art detectors and lack of real-time statistical analysis of the current technology, the timedomain approaches are usually coupled with off-line analysis which impedes its use in flow cell sorting, tracking and capturing. In this work, we apply a 32×32 CMOS SPAD array (MegaFrame camera) for real-time imaging flow cytometry analysis. This technology is integrated into a 1024-beam multifocal fluorescence microscope and incorporating a microfluidic chip at the sample plane enables imaging of cell flow and identification. Furthermore, the 1.5% native pixel fill-factor of the MegaFrame camera is overcome using beamlet reprojection with <10 μW laser power at 490 nm for each beam. Novel hardware algorithms incorporating the center-of-mass method (CMM) with real-time background subtraction and division are implemented within the firmware, allowing lossless recording of TCSPC events at a 500 kHz frame rate with 1024 histogram bins at 52 ps time resolution. Live calculation of background compensated CMM-based fluorescence lifetime is realized at a user-defined frame rate (typically 0.001 ~ 27 kHz) for each SPAD pixel. The work in this paper considers the application of the SPAD array to confocal fluorescence lifetime imaging of multiple coincident particles flowing within a microfluidic channel. Compared to previous flow systems based on single-point detectors, the multi-beam flow system enables visualization, detection and categorization of multiple groups of cells or particles according to their fluorescence lifetime.
Light sheet microscopy has become an indispensable tool for fast, low phototoxicity volumetric imaging of biological samples; predominantly providing structural or analyte concentration data in its standard format. Fluorescence lifetime imaging microscopy (FLIM) provides functional contrast, but often at limited acquisition speeds and with complex implementation.
We have developed a digitally scanned light sheet microscope for fast 2-colour volumetric imaging with imaging performed in the frequency domain at 20MHz using the PCO.FLIM camera. The camera enables rapid acquisition of two phases simultaneously at 0 and 180 degrees and with a phase shift relative to the modulated square-wave excitation. Whilst this frequency domain method has been well known for decades, application to light-sheet imaging is novel and provides straightforward functional read-out of fluorescence resonance energy transfer for protein interaction studies deep inside intact specimens such as Zebrafish. We demonstrate application of fluorescence lifetime contrast using the fluorescent protein biosensors in both live Zebrafish and organoids in digitally scanned light sheet FLIM. We apply signal processing techniques to improve data analysis and visualization and discuss this alongside practical application of real-time volumetric imaging of live biological specimens.
Given the complexity of biological systems, it is necessary to go beyond ensemble measurements and attain information at the single molecule level to accurately probe molecular properties. Single molecule imaging can examine real-time conformational dynamics [1], which is often the underlying cause of heterogeneity in molecular distributions in terms of dipole orientations, spectra, or intramolecular distances, in both stable and unstable systems.
A combination of polarisation-resolved detection and 2-colour alternating laser excitation (ALEX) allows quantification of the anisotropy and stoichiometry of the fluorophores present [2]. As a result, it is possible to accurately quantify energy transfer (e.g. FRET). This technique provides a rapid approach for probing the fluorophore’s environment in terms of viscosity, interactions between molecules, and ligand-substrate binding.
Here we present an optimized TIRF microscope in conjunction with ALEX and steady state fluorescence anisotropy detection [3] for single molecule imaging. Validation and determination of the limits of the technique will be by measurement of isolated fluorescent proteins. Preliminary data of single molecules with a fluorescence anisotropy read-out will be presented and future prospects discussed.
1. Santoso Y, et al. (2010) Conformational transitions in DNA polymerase I revealed by single-molecule FRET. PNAS 107: 715–720.
2. Kapanidis A, et al. (2004) Fluorescence-aided molecule sorting: Analysis of structure and interactions by alternating-laser excitation of single molecules PNAS.
101 8936-8941.
3. Devauges V, et al. (2014) Steady-state acceptor fluorescence anisotropy imaging under evanescent excitation for visualisation of FRET at the plasma membrane.
PLoS One 9: e110695.
We present a digital architecture for fast acquisition of time correlated single photon counting (TCSPC) timestamps from
32×32 CMOS SPAD array. Custom firmware was written to select 64 pixels out of 1024 available for fast transfer of
TCSPC timestamps. Our 64 channel TCSPC is capable of acquiring up to 10 million TCSPC timestamps per second over
a USB2 link. We describe the TCSPC camera (Megaframe), camera interface to the PC and the microscope setup. We
characterize the Megaframe camera for fluorescence lifetime imaging (FLIM) including instrument response function,
time resolution and variability of both across the array. We show a fluorescence lifetime image of a plant specimen
(Convallaria majalis) from a custom-built multifocal multiphoton microscope. The image was acquired in 20 seconds
(with average timestamp acquisition rate of 4.7 million counts per second).
Viviane Devauges, Elena Ortiz-Zapater, Christina Efthymiou, Melanie Keppler, Jody Barbeau, Daniel Matthews, James Monypenny of Pitmilly, Paul Barber, Daniel Rolfe, Tony Ng, Simon Ameer-Beg
Photoswitchable and photoactivable proteins Dronpa and PhotoActivable mCherry (PA-mCherry) respectively, were used in order to perform FRET (Förster Resonance Energy Transfer) imaging at the single molecule level, using a FRET standard construct consisting of Dronpa and PA-mCherry separated by seven amino acids expressed in cells. Given Dronpa’s complex photophysical properties and the existence of a preswitched emissive state, irradiation conditions at 491 and 405 nm were optimised. We discuss strategies for observing FRET at the single molecule level with photoactivatable proteins by monitoring modifications in the donor and acceptors emissive states.
Forster/Fluorescence resonant energy transfer (FRET) has become an extremely important technique to explore
biological interactions in cells and tissues. As the non-radiative transfer of energy from the donor to acceptor occurs
typically only within 1-10nm, FRET measurement allows the user to detect localisation events between protein-conjugated
fluorophores. Compared to other techniques, the use of time correlated single photon counting (TCSPC) to
measure fluorescence lifetime (FLIM) has become the gold standard for measuring FRET interactions in cells. The
technique is fundamentally superior to all existing techniques due to its near ideal counting efficiency, inherent low
excitation light flux (reduced photobleaching and toxicity) and time resolution. Unfortunately due to its slow acquisition
time when compared with other techniques, such as Frequency-domain lifetime determination or anisotropy, this makes
it impractical for measuring dynamic protein interactions in cells. The relatively slow acquisition time of TCSPC FLIM-FRET
is simply due to the system usually employing a single-beam scanning approach where each lifetime (and thus
FRET interaction) is determined individually on a voxel by voxel basis. In this paper we will discuss the development a
microscope system which will parallelize TCSPC for FLIM-FRET in a multi-beam multi-detector format. This will
greatly improve the speed at which the system can operate, whilst maintaining both the high temporal resolution and the
high signal-to-noise for which typical TCPSC systems are known for. We demonstrate this idea using spatial light
modulator (SLM) generated beamlets and single photon avalanche detector (SPAD) array. The performance is evaluated
on a plant specimen.
Fluorescence lifetime imaging microscopy (FLIM) is a well established approach for measuring dynamic signalling
events inside living cells, including detection of protein-protein interactions. The improvement in optical penetration of infrared light compared with linear excitation due to Rayleigh scattering and low absorption have provided imaging
depths of up to 1mm in brain tissue but significant image degradation occurs as samples distort (aberrate) the infrared
excitation beam. Multiphoton time-correlated single photon counting (TCSPC) FLIM is a method for obtaining
functional, high resolution images of biological structures. In order to achieve good statistical accuracy TCSPC typically
requires long acquisition times. We report the development of a multifocal multiphoton microscope (MMM), titled
MegaFLI. Beam parallelization performed via a 3D Gerchberg–Saxton (GS) algorithm using a Spatial Light Modulator
(SLM), increases TCSPC count rate proportional to the number of beamlets produced. A weighted 3D GS algorithm is
employed to improve homogeneity. An added benefit is the implementation of flexible and adaptive optical correction.
Adaptive optics performed by means of Zernike polynomials are used to correct for system induced aberrations. Here we present results with significant improvement in throughput obtained using a novel complementary metal-oxide-semiconductor (CMOS) 1024 pixel single-photon avalanche diode (SPAD) array, opening the way to truly high-throughput FLIM.
The Raman spectrum of a fluorescent chromophore typically has many spectral features, which differ markedly between
dyes even if their electronic spectra are similar. This high information content makes it possible to distinguish
biomarkers based on their Raman spectra. Coherent anti-Stokes Raman scattering may therefore allow for the
simultaneous measurement of more biomarkers than is possible with fluorescent imaging, while avoiding bleaching and
sample autofluorescence. We have built a broadband CARS microspectrometer to demonstrate the principle of CARS
multiplexing and investigate the potential to apply the system to studies of biological samples.
Multiphoton microscopy (MPM) is a high resolution (sub-μm) 3D optical imaging technique that has
seen widespread use for microscopy at moderate depth within biological tissue (~1 mm). MPM
combined with Fluorescence lifetime imaging (FLIM) and Fluorescent resonant energy transfer
(FRET) provides the ability to image protein-protein interactions. When applied in-vivo at depth, it will
be a key component to identifying and evaluating drug interaction in tumours.
Unfortunately as one images more deeply into biological tissue, depth is restricted due to the specimen
induced aberrations, which result in deterioration in both the image quality and resolution. Adaptive
optics (AO), a technique first developed for astronomy, has been shown to be successful in overcoming
problems associated with imaging in depth in confocal, multiphoton, CARS and SHG microscopy. The
principle relies on shaping the wavefront with a wavefront modulator to compensate for the distortions
introduced by the biological tissue sample. The success of such a technique relies on being able to
correctly determine the mirror shape required
In this paper we will discuss the development a dedicated MPM FLIM-FRET microscope incorporating
an AO for use in-vivo applications. Using a deformable membrane mirror as a wavefront modulator, a
strategy for implementation will be discussed.
KEYWORDS: Luminescence, Fluorescence resonance energy transfer, Microfluidics, Photons, Signal detection, Proteins, Green fluorescent protein, Cancer, Signal processing, Fluorescence lifetime imaging
Cancer studies require a thorough understanding of how human gene expressions and DNA modifications are translated
at the proteome level. In order to unravel the large and complex interactions between proteins, we have developed a
compact lifetime-based flow cytometer, utilising a commercial microfluidic chip, to screen large non-adherent cell
populations. Fluorescent signals from cells are detected using time correlated single photon counting (TCSPC) in the
burst integrated fluorescence lifetime (BIFL) mode and used to determine the lifetime of each cell. Initially, the system
was tested using 10 μm highly fluorescent beads to determine optical throughput and detection efficiency. The system
was validated with 293T monkey kidney adenocarcinoma cell line transiently transfected with a FRET standard,
consisting of eGPF and mRFP1 fluorescent proteins linked by a19 amino-acid chain. Analysis software was developed to
process detected signals in BIFL mode and chronologically save the transient burst data for each cell in a multi-dimensional
image file.
Fluorescent lifetime imaging microscopy (FLIM) has proven to be a valuable tool in beating the Rayleigh criterion for
light microscopy by measuring Förster resonance energy transfer (FRET) between two fluorophores. Applying
multiphoton FLIM, we previously showed in a human breast cancer cell line that recycling of a membrane receptorgreen
fluorescent protein fusion is enhanced concomitantly with the formation of a receptor:protein kinase C α complex
in the endosomal compartment. We have extended this established technique to probe direct protein-protein interactions
also in vivo. Therefore, we used various expressible fluorescent tags fused to membrane receptor molecules in order to
generate stable two-colour breast carcinoma cell lines via controlled retroviral infection. We used these cell lines for
establishing a xenograft tumour model in immune-compromised Nude mice. Using this animal model in conjunction
with scanning Ti:Sapphire laser-based two-photon excitation, we established deep-tissue multiphoton FLIM in vivo.
For the first time, this novel technique enables us to directly assess donor fluorescence lifetime changes in vivo and we
show the application of this method for intravital imaging of direct protein-protein interactions.
An automated high-content screening microscope has been developed which uses fluorescence anisotropy imaging and fluorescence lifetime microscopy to identify Förster resonant energy transfer between eGFP and mRPF1 in drug screening assays. A wide-field polarization resolved imager is used to simultaneously capture the parallel and perpendicular components of both eGFP and mRFP1 fluorescence emission to provide a high-speed measurement of acceptor depolarization. Donor excited state lifetime measurements performed using laser scanning microscopy is then used to determine the FRET efficiency in a particular assay. A proof-of-principle assay is performed using mutant Jurkat human T-cells to illustrate the process by which FRET is first identified and then quantified by our high-content screening system.
We have developed a range of optical biochip devices for conducting live and fixed cell-based assays. The devices
encompass the ability to process an entire assay including fluorescently labelling cells, a microfluidic system to transport
and maintain cells to deliver them to an optical area of the device for measurement, with the possibility of a
incorporating a sorting step in between. On-chip excitation provided by red emitting LED and lasers define the excitation
wavelength of the fluorophore to be incorporated into the assay readout. The challenge for such an integrated
microfluidic optical biochip has been to identify and characterise a longterm fluorescent label suitable for tracking cell
proliferation status in living cells.
Traditional organic fluorophores have inherent disadvantages when considering their use for an on-chip device requiring
longterm cellular tracking. This has led us to utilise inorganic quantum dots (QDots) as fluorophores for on- chip assays.
QDs have unique properties such as photostability, broad absorption and narrow emission spectra and are available in a
range of emission wavelengths including far red. They also have much higher quantum efficiencies than traditional
organic fluorophores thus increasing the possible dynamic range for on-chip detection. Some of the QDots used have the
added advantage of labelling intact cells and being retained and distributed among daughter cells at division, allowing
their detection for up to 6 generations. The use of these QDs off-chip has suggested that they are ideal for live cell, nonperturbing
labelling of division events, whereby over time the QD signal becomes diluted with each generation.
Here we describe the use of quantum dots as live cell tracers for proliferating populations and the potential applications
in drug screening and optical biochip environments.
We present details of the development of a optical biochip, with integrated on-chip laser excitation, for fluorescence
intensity cell based assays. The biochip incorporates an "active surface" for the control and manipulation of fluorescent
species placed directly on the device. The active elements of the biochip are one-dimensional periodic sub-wavelength
corrugations fabricated on a thin gold film. We have made fluorescence intensity measurements of both an organic dye
(Cy5), and immobilized and fluorescently labeled (with 705 nm emitting quantum dots), mammalian tumor cells in
contact with the active surface. Here we show that the presence of the periodic grating can be used to control both the
excitation and fluorescence generation process itself. We demonstrate that the gratings convert evanescent surface optical
modes into well-defined beams of radiation in the far-field and at the surface of the device this produces highly
contrasting regions of fluorescence excitation providing regions of high spatial selectivity.
Cell adhesion and focal complex formation require signalling complexes linking cell adhesion molecules to the cytoskeleton. To understand morphogenetic changes associated with tumour cell spreading, migration and tumour cell metastasis, the molecular mechanisms responsible for the regulation, formation and dissolution at the cell-extracellular matrix (ECM) interface need to be identified. In order to achieve this, an improved axial resolution is desirable. We report on the development of a multi-photon (MP) total internal reflection (TIR) fluorescence lifetime imaging (FLIM)
system that allows the selective excitation of fluorophores, with such an improved axial resolution. Results from initial experiments are presented. High excitation efficiency is achieved by the use of a Nikon 1.45 NA TIRF objective using annular illumination.
Group velocity dispersion (GVD) and pulse front distortion of ultrashort pulses are of critical importance in
efficient multiphoton excitation microscopy. Since measurement of the pulse front distortion due to a lens is not trivial we have developed an imaging interferometric cross-correlator which allows us to measure temporal delays and pulse-widths across the spatial profile of the beam. The instrument consists of a modified Michelson interferometer with a reference arm containing a voice-coil delay stage and an arm which contains the optics under test. The pulse replicas are recombined and incident on a 22×22 lenslet array. The beamlets are focused in a 0.5 mm thick BBO crystal (cut for Type I second harmonic generation), filtered to remove the IR component of the beam and imaged using a 500 fps camera. The GVD and pulse front distortion are extracted from the temporal stack of beamlet images to produce a low resolution spatio-temporal map.
In situ spectral analysis can be used to understand the targeting and interaction of agents in cellular compartments. A range of novel red excitable fluorescent probes, related to the anthraquinone family of anti-cancer agents, were designed for their DNA affinic properties and their ability to enter and penetrate living cells. We report on the spectral features of these probes, both in solution and bound within intact cells, to identify unique fluorescent signatures that exploit their use in bioassays on optical biochip devices.
The probes demonstrated red shifted emission spectra and increased 2 photon lifetime, with minimal fluorescent enhancement, upon binding to DNA. Spectral confocal laser scanning microscopy revealed complex emission profiles representing the bound (nuclear) and unbound (cytoplasmic) fractions of the DNA probes within live interphase, mitotic and apoptotic cells.
Analysis of the emission peaks encoded the spectra to provide cell compartment recognition and profiles for cells in different cell states. Sampling the entire emission spectra of these probes for cell locating, even in the presence of unbound molecules, provides good signal-to-noise in biochip devices. Furthermore, by sampling the fluorescence output at specific spectral windows we can obtain high spatial information without imaging.
The technological challenge is to integrate these fluorophores and appropriate detection capacity onto an optical biochip platform with microfluidic systems for cell handling.
We report on the development of a simple technique for obtaining time-domain information using dc detection of fluorescence. We show that this is feasible for assays where a change in lifetime of an indicator occurs in reaction to an analyte, in fluorescence resonance energy transfer for example, and could be particularly useful for assays performed in the scaled-down environment of a "lab-on-a-chip". A rate equation model is presented which allows an objective analysis of the relative importance of the key measurement parameters: optical saturation of the fluorophore and excitation pulse characteristics. We present a comparison of the model with a cuvette based analysis of a carbocyanine dye where the excitation source is a 650 nm wavelength, self-pulsing AlGaInP laser diode.
We report on the development of a stroboscopic excitation technique using a self-pulsing laser, and show that it is a feasible method for obtaining fluorescence lifetime information from a biochip format. The self-pulsing lasers described here are versatile devices which have been used for one photon excitation measurements to determine the lifetime of cyanine 5 in water and ethanol. The same devices have been used to develop a technique whereby the emphasis for time-resolution of a lifetime measurement can be transferred to the excitation source from the detector and processing electronics by virtue of the multiple-pulse, variable frequency nature of the laser output.
The spatio-temporal localization of molecular interactions within cells in situ is of great importance in elucidating the key mechanisms in regulation of fundamental process within the cell. Measurements of such near-field localization of protein complexes may be achieved by the detection of fluorescence (or Forster) resonance energy transfer (FRET) between protein-conjugated fluorophores. We demonstrate the applicability of time-correlated single photon counting multiphoton microscopy to the spatio-temporal localization of protein-protein interactions in live and fixed cell populations. Intramolecular interactions between protein hetero-dimers are investigated using green fluorescent protein variants. We present an improved monomeric form of the red fluorescent protein, mRFP1, as the acceptor in biological fluorescence resonance energy transfer (FRET) experiments using the enhanced green fluorescent protein as donor. We find particular advantage in using this fluorophore pair for quantitative measurements of FRET. The technique was exploited to demonstrate a novel receptor-kinase interaction between the chemokine receptor (CXCR4) and protein kinase C (PKC) α in carcinoma cells for both live and fixed cell experiments.
FLIM/FRET is an extremely powerful technique that can microscopically locate nanometre-scale protein-protein interactions within live or fixed cells, both in vitro and in vivo. The key to performing sensitive FRET, via FLIM, besides the use of appropriate fluorophores, is the analysis of the time-resolved data present at each image pixel. The fluorescent transient will, in general, exhibit multi-exponential kinetics: at least two exponential components arise from both the interacting and non-interacting protein. We shall describe a novel method and computer program for the global analysis of time resolved data, either at the single level or through global analysis of grouped pixel data. Kinetic models are fitted using the Marquardt algorithm and iterative convolution of the excitation signal, in a computationally-efficient manner. The fitting accuracy and sensitivity of the algorithm has been tested using modelled data, including the addition of simulated Poisson noise and repetitive excitation pulses which are typical of a TCSPC system. We found that the increased signal to noise ratio offered by both global and invariance fitting is highly desirable. When fitting mono-exponential data, the effects of a ca. 12.5 ns (ca. 80 MHz) repetitive excitation do not preclude the accurate extraction of populations with lifetimes in the range 0.1 to 10 ns, even when these effects are not represented in the fitting algorithm. Indeed, with global or invariance fitting of a 32x32 pixel area, the error in extracted lifetime can be lower than 0.4% for signals with a peak of 500 photon counts or more. In FRET simulations, modelling GFP with a non-interacting lifetime of 2.15 ns, it was possible to accurately detect a 10% interacting population with a lifetime of 0.8 ns.
A technique is presented to utilize time-resolved fluorescence from acceptors in donor-acceptro Forster resonance energy transfer (FRET) fluorophore pairs to improve the efficiency and accuracy of FRET lifetime determinations. This technique was applied experimentally and the results presented. This technique could be useful in fluorescence-liftime imaging systems which can measure more than one wavelength channel simultaneously.
KEYWORDS: 3D image processing, Fractal analysis, In vivo imaging, Point spread functions, 3D metrology, Microscopes, Hough transforms, Statistical analysis, Tissues, Cancer
The understanding of tumour angiogenesis and response to vascular-targeted drugs are of increasing interest in cancer research. We present 3D images of the in vivo tumour vasculature captured utilising multi-photon microscopy together with the results of manual and semi-automated delineation of the vascular network using novel in-house-developed software and algorithms. The software presented is aimed at aiding in these investigations and other problems where linear or dendritic structures are to be delineated from 3D data sets. A new algorithm, CHARM, based on a compact Hough transform and the formation of a radial map, has been used to automatically locate vessel centres and measure diameters. The robustness of this algorithm to image smoothing and noise has been investigated. Statistical information characterising the network in terms of vascular parameters as well as more complex analyses, such as fractal dimension, are now possible and examples are presented.
We demonstrate the applicability of time-correlated single photon counting multiphoton microscopy to the spatio-temporal localisation of protein-protein interactions in situ. Examples of new fluorescent protein variants with enhanced properties are given and the development of FRET biosensors for simultaneous measurement of multiple intra- and inter-molecular interactions is illustrated by experimental evidence of an energy transfer cascade via multiple acceptors. The juxtaposition of interacting population and FRET efficiency is elucidated, with a priori knowledge, by multi-exponential analysis.
Using fluorescence resonance energy transfer (FRET) measured by fluorescence lifetime imaging microscopy (FLIM) we have explored the protein-protein interactions between fluorescent protein tagged fusion proteins of the activation pathways of PKC and NFkB. We observe FRET between CFP-IκB and YFP-p65 in unstimulated cells and when treated with TNFα. We also observed a reduction of the fluorescent lifetime of CFP-IκB in the absence of YFP-p65 when TNFα is present.
Recent interest in vascular targeting and anti-angiogenic drug treatments for cancer has stimulated fundamental research regarding the modes of action of these drugs as well as studies of the development and re-modeling of the vascular network following treatment. Multiphoton fluorescence microscopy is employed for in vivo mapping of three-dimensional blood vessel distribution in tumors grown in rodent dorsal skin-flap window chamber preparations. Accurate visualization of the vasculature in three-dimensions allows us to perform dynamic experiments in thick biological specimens in vivo. Examples of in vivo imaging of tumor vasculature are given and compared to normal tissue vasculature. The dynamic responses of blood vessels to treatment with the vascular targeting drug combretastatin A4-P are presented and discussed. The implementation of time-domain imaging by reversed stop-start time-correlated single photon counting (RSS-TCSPC) is discussed as a method for feature extraction in the presence of exogenous and endogenous fluorophores. In particular, the segmentation of the vascular network is demonstrated. Additional contrast, indicative of probe environmental factors, may also be realized. We present examples of in vivo lifetime imaging as a method to elucidate the physiological processes of the tumor microenvironment.
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