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.
Imaging viscosity and its spatiotemporal patterns can provide valuable insight into the underlying physical conditions of biochemical reactions and biological processes in cells and tissues. One way to measure viscosity and diffusion is the use of fluorescence recovery after photobleaching (FRAP). We combine FRAP with FLIM and time-resolved fluorescence anisotropy imaging (tr-FAIM), by acquiring time- and polarization-resolved fluorescence images in every frame of a FRAP series. This allows us to simultaneously monitor translational and rotational diffusion. This approach can be applied to measuring diffusion in homogeneous and heterogeneous environments, and in principle also allows the study of homo-FRET. Another way to measure viscosity and diffusion is through specific flexible dyes, e.g. fluorescent molecular rotors, whose fluorescence quantum yield and fluorescence lifetime depend on the viscosity of the environment, in combination with fluorescence lifetime imaging (FLIM). We show that a bodipybased fluorescent molecular rotor targeting mitochondria reports on their viscosity, which changes under physiological stimuli. Both methods can optically measure viscosity and diffusion on the micrometer scale.
We report the simultaneous combination of three powerful techniques in uorescence microscopy: Fluorescence Lifetime Imaging (FLIM), Fluorescence Anisotropy Imaging (FAIM) and Fluorescence Recovery After Photobleaching (FRAP), also called F3 microscopy. An exhaustive calibration of the setup was carried out with several rhodamine 6G (R6G) solutions in water-glycerol and from the combination of the FAIM and FRAP data, the hydrodynamic radius of the dye was directly calculated. The F3 data was analyzed with a home-built MATLAB script, and the setup is currently explored further with Green Fluorescent Protein (GFP). Some molecular dynamic (MD) simulations are currently being run in order to help with the interpretation of the experimental anisotropy data.
Spectrally resolved confocal microscopy and fluorescence lifetime imaging have been used to measure the polarity of lipid-rich regions in living HeLa cells stained with Nile red. The emission peak from the solvatochromic dye in lipid droplets is at a shorter wavelength than other, more polar, stained internal membranes, and this is indicative of a low polarity environment. We estimate that the dielectric constant, ϵ, is around 5 in lipid droplets and 25<ϵ<40 in other lipid-rich regions. Our spectrally resolved fluorescence lifetime imaging microscopy (FLIM) data show that intracellular Nile red exhibits complex, multiexponential fluorescence decays due to emission from a short lifetime locally excited state and a longer lifetime intramolecular charge transfer state. We measure an increase in the average fluorescence lifetime of the dye with increasing emission wavelength, as shown using phasor plots of the FLIM data. We also show using these phasor plots that the shortest lifetime decay components arise from lipid droplets. Thus, fluorescence lifetime is a viable contrast parameter for distinguishing lipid droplets from other stained lipid-rich regions. Finally, we discuss the FLIM of Nile red as a method for simultaneously mapping both polarity and relative viscosity based on fluorescence lifetime measurements.
Meso-substituted boron-dipyrromethene (BODIPY-C12) was used to monitor the viscosity in cells via fluorescence
lifetime imaging (FLIM), and time-resolved fluorescence anisotropy measurements. Our results show that meso-substituted
BODIPY-C12 senses the viscosity in HeLa cells and is insensitive to the surrounding polarity. The
relationship between the fluorescence lifetime and the rotational correlation time of the dye in homogeneous solutions
agree with the combination of the Foerster Hoffmann equation and the Debye-Stokes-Einstein equation.
We present fluorescence lifetime imaging (FLIM) and fluorescence anisotropy imaging along with translational diffusion
measurements of living cells labelled with green fluorescent protein (GFP) recorded in a single experiment. The
experimental set-up allows for time and polarization-resolved fluorescence images to be measured in every frame of a
fluorescence recovery after photobleaching (FRAP) series. We have validated the method using rhodamine 123 in
homogeneous solution prior to measurements of living A431 cells labelled with cdc42-GFP, for which the FRAP
recovery exhibits an immobile fraction and the rotational mobility of the protein is hindered while the fluorescence
lifetime fairly homogeneous across the cell. By eliminating the need for sequential measurements to extract fluorescence
lifetimes and molecular diffusion coefficients we remove artefacts arising from changes in sample morphology and
excessive photobleaching during sequential experiments.
The average fluorescence lifetime of the green fluorescent protein (GFP) in solution is a function of the refractive index of its environment. We report that this is also the case for GFP-tagged proteins in cells. Using time-correlated single-photon counting (TCSPC)–based fluorescence lifetime imaging (FLIM) with a confocal scanning microscope, images of GFP-tagged proteins in cells suspended in different refractive index media are obtained. It is found that the average fluorescence lifetime of GFP decreases on addition of glycerol or sucrose to the media in which the fixed cells are suspended. The inverse GFP lifetime is proportional to the refractive index squared. This is the case for GFP-tagged major histocompatibility complex (MHC) proteins with the GFP located inside the cytoplasm, and also for GPI-anchored GFP that is located outside the cell membrane. The implications of these findings are discussed with regard to total internal reflection fluorescence (TIRF) techniques where the change in refractive index is crucial in producing an evanescent wave to excite fluorophores near a glass interface. Our findings show that the GFP fluorescence lifetime is shortened in TIRF microscopy in comparison to confocal microscopy.
We have used an experimental arrangement comprising two photomultipliers and time-correlated single photon counting
(TCSPC) detection to measure time and polarization-resolved fluorescence decays and images simultaneously.
Polarization-resolved measurements can provide information which may be difficult to extract from lifetime
measurements alone. The combination of fluorescence lifetime and time-resolved anisotropy in an imaging modality
with two detectors minimizes the errors arising from bleaching of a sample between consecutive measurements.
Anisotropy measurements can provide evidence of fluorescence resonance energy transfer between chemically identical
fluorophores (homo-FRET). This phenomenon is not detectable in spectral or lifetime changes, yet a lowering of the
anisotropy and a faster anisotropy decay can provide evidence for close proximity (≤ 10 nm) of adjacent fluorophores
including dimerization and oligomerization of molecules. We have used FLIM and fluorescence anisotropy to measure
variations in fluorescence lifetimes and anisotropy of GFP-tagged proteins in cells in immunological synapse samples
and also acquire images of BODIPY-stained carcinoma cells.
We demonstrateWide-Field Time-Correlated Single Photon Counting (WiFi TCSPC) imaging based on an image
intensifier and a high-speed camera running at 30,000 frames per second. The timing of photon events is thus
performed in parallel, simultaneously on every pixel. The system is applied to lanthanide lifetime measurements
and time-resolved imaging of the lanthanide complex Europium Polyoxometalate (Eu POMs). We measure a
lifetime of 2.98 ms for Eu POMs in solid state, which is in excellent agreement with the literature value.
Fluorescence imaging techniques are powerful tools in the biological and biomedical sciences, because they are
minimally invasive and can be applied to live cells and tissues. The fluorescence emission can be characterized not only
by its intensity and position, by also by its fluorescence lifetime, polarization and wavelength. Fluorescence Lifetime
Imaging (FLIM) in particular has emerged as a key technique to image the environment and interaction of specific
proteins in living cells. Using a time-correlated single photon counting (TCSPC)-based FLIM set-up, we find that the
fluorescence lifetime of GFP-tagged proteins in cells is a function of the refractive index of the medium the cells are
suspended in. In addition, combining Fluorescence Recovery After Photobleaching (FRAP) of fluorescently labeled
proteins of different sizes in sol gels with time-resolved fluorescence anisotropy measurements, we demonstrate that we
can measure their lateral and rotational diffusion. This allows us to infer the size and connectivity of the pores in the sol
gel matrix. Moreover, wide-field photon counting imaging, originally developed for astronomical applications, is a
powerful imaging method because of its high sensitivity and excellent signal-to-noise ratio. It has a distinct advantage
over CCD-based imaging due to the ability to time the arrival of individual photons. The potential of time-resolved wide-field
photon counting imaging with a fast CMOS camera applied to luminescence microscopy is demonstrated.
We present a novel time-resolved photon counting imaging technique and its use in multi-dimensional luminescence
spectroscopy. By using an ultrafast camera coupled to an image intensifier on a microscope, we demonstrate
the potential of wide-field time-correlated single photon counting, with a count rate of up to 5 Mhz. This system
has the advantage of allowing the detection of single photons in parallel in every pixel. We measured the
luminescence decay of Europium Polyoxometalate (POM), and observed contrast on lifetime images of Eu-POM
on silver nanocrystals.
This paper reports on the development of micromachined pillar arrays for the filtering of terahertz radiation. These pillar
arrays are fabricated using ultraviolet based processing of thick SU8. This micromachining technique enables the array
patterns, dimensions, and consequently the filter characteristics, to be readily defined. In particular, we demonstrate that
by combining individual filter arrays with either different periods or pillar diameters we can isolate individual pass bands
in the 1 to 2 THz region.
Terahertz (THz) spectroscopy of a biomolecule with spatial resolution below the diffraction limit of the radiation has
been achieved by use of an all-optical, contactless transient mirror technique. A resolution of around 50 &mgr;m is
determined by the use of a test sample of gold strip lines deposited on GaAs, and the differential THz time-domain
spectroscopy (THz-TDS) response of biotin has been measured in both the presence and absence of the transient mirror
at room temperature. These preliminary results demonstrate the potential for use of the technique for the chemical
identification and characterisation of biomolecules in small volumes with the ultimate goal being microscopic imaging of
live cells. The technique may find applications in quality control for semiconductor processing, and in identifying
material imperfections, i.e. small cracks in non-destructive testing. We discuss the limitations of the transient mirror
technique along with several advantages over other related techniques.
We report on the development of a surface micromachined process for the fabrication of coaxial apertures surrounded by periodic grooves. The process uses a combination of copper electroforming and the negative epoxy based resist, SU8, as a thin flexible substrate. The device dimensions are suitable for the implementation of filters at THz frequencies, and measurements show a pass band centred around 1.5 THz. These devices could form the basis of the next generation of THz biosensors.
At the present time the interaction of Terahertz (THz) radiation with random structures is not well understood. Scattering effects are particularly relevant in this spectral regime, where the wavelength, and the size and separation of scattering centres are often commensurable. This phenomenon can both be used to advantage in imaging and sensing, but conversely can have adverse effects on the interpretation of a "fingerprint" spectrum. A new mathematical method, the Phase Distribution Model, is reported here for the calculation of attenuation and scattering of THz radiation in random materials. This uses a Phase Distribution Function to describe the effect of the non-absorbing scatterers within the media. Experimental measurements undertaken using previously published results, data obtained from specially constructed phantoms and from everyday textiles have been compared with the theory. These experimental results encompass both cylindrical and spherical scattering situations. The model has also been compared with exact calculations using the Pendry code.
A new mathematical method, the Phase Distribution Model, is devised for the calculation of attenuation and scattering of THz radiation in random materials. The accuracy of the approximation is tested by comparison with exact calculations and with experimental measurements on textiles and specially constructed phantoms.
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