Development of low-cost instrumentation for single point autofluorescence lifetime measurements

Autofluorescence lifetime measurements, which can provide label-free readouts in biological tissues, contrasting e.g. different types and states of tissue matrix components and different cellular metabolites, may have significant clinical potential for diagnosis and to provide surgical guidance. However, the cost of the instrumentation typically used currently presents a barrier to wider implementation. We describe a low-cost single point time-resolved autofluorescence instrument, exploiting modulated laser diodes for excitation and FPGA-based circuitry for detection, together with a custom constant fraction discriminator. Its temporal accuracy is compared against a “gold-standard” instrument incorporating commercial TCSPC circuitry by resolving the fluorescence decays of reference fluorophores presenting single and double exponential decay profiles. To illustrate the potential to read out intrinsic contrast in tissue, we present preliminary measurements of autofluorescence lifetime measurements of biological tissues ex vivo. We believe that the lower cost of this instrument could enhance the potential of autofluorescence lifetime metrology for clinical deployment and commercial development

Tunable fibre-coupled multiphoton microscopy with a negative curvature fibre

Negative curvature fibre (NCF) guides light in its core by inhibiting the coupling of core and cladding modes. In this work, an NCF was designed and fabricated to transmit ultrashort optical pulses for multiphoton microscopy with low group velocity dispersion (GVD) at 800 nm. Its attenuation was measured to be <0.3 dB m(-1) over the range 600-850 nm and the GVD was -180 ± 70 fs(2)  m(-1) at 800 nm. Using an average fibre output power of ∼20 mW and pulse repetition rate of 80 MHz, the NCF enabled pulses with a duration of <200 fs to be transmitted through a length of 1.5 m of fibre over a tuning range of 180 nm without the need for dispersion compensation. In a 4 m fibre, temporal and spectral pulse widths were maintained to within 10% of low power values up to the maximum fibre output power achievable with the laser system used of 278 mW at 700 nm, 808 mW at 800 nm and 420 mW at 860 nm. When coupled to a multiphoton microscope, it enabled imaging of ex vivo tissue using excitation wavelengths from 740 nm to 860 nm without any need for adjustments to the set-up

easySTORM: a robust, lower-cost approach to localisation and TIRF microscopy

TIRF and STORM microscopy are super-resolving fluorescence imaging modalities for which current implementations on standard microscopes can present significant complexity and cost. We present a straightforward and low-cost approach to implement STORM and TIRF taking advantage of multimode optical fibres and multimode diode lasers to provide the required excitation light. Combined with open source software and relatively simple protocols to prepare samples for STORM, including the use of Vectashield for non-TIRF imaging, this approach enables TIRF and STORM imaging of cells labelled with appropriate dyes or expressing suitable fluorescent proteins to become widely accessible at low cost

Correction Approach for Delta Function Convolution Model Fitting of Fluorescence Decay Data in the Case of a Monoexponential Reference Fluorophore

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 (τ(R)(−1) – τ(−1))(−1), where τ(R) 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

In vivo label-free mapping of the effect of a photosystem II inhibiting herbicide in plants using chlorophyll fluorescence lifetime

Background In order to better understand and improve the mode of action of agrochemicals, it is useful to be able to visualize their uptake and distribution in vivo, non-invasively and, ideally, in the field. Here we explore the potential of plant autofluorescence (specifically chlorophyll fluorescence) to provide a readout of herbicide action across the scales utilising multiphoton-excited fluorescence lifetime imaging, wide-field single-photon excited fluorescence lifetime imaging and single point fluorescence lifetime measurements via a fibre-optic probe. Results Our studies indicate that changes in chlorophyll fluorescence lifetime can be utilised as an indirect readout of a photosystem II inhibiting herbicide activity in living plant leaves at three different scales: cellular (~μm), single point (~1 mm2) and macroscopic (~8 × 6 mm2 of a leaf). Multiphoton excited fluorescence lifetime imaging of Triticum aestivum leaves indicated that there is an increase in the spatially averaged chlorophyll fluorescence lifetime of leaves treated with Flagon EC—a photosystem II inhibiting herbicide. The untreated leaf exhibited an average lifetime of 560 ± 30 ps while the leaf imaged 2 h post treatment exhibited an increased lifetime of 2000 ± 440 ps in different fields of view. The results from in vivo wide-field single-photon excited fluorescence lifetime imaging excited at 440 nm indicated an increase in chlorophyll fluorescence lifetime from 521 ps in an untreated leaf to 1000 ps, just 3 min after treating the same leaf with Flagon EC, and to 2150 ps after 27 min. In vivo single point fluorescence lifetime measurements demonstrated a similar increase in chlorophyll fluorescence lifetime. Untreated leaf presented a fluorescence lifetime of 435 ps in the 440 nm excited chlorophyll channel, CH4 (620–710 nm). In the first 5 min after treatment, mean fluorescence lifetime is observed to have increased to 1 ns and then to 1.3 ns after 60 min. For all these in vivo plant autofluorescence lifetime measurements, the plants were not dark-adapted. Conclusions We demonstrate that the local impact of a photosystem II herbicide on living plant leaves can be conveniently mapped in space and time via changes in autofluorescence lifetime, which we attribute to changes in chlorophyll fluorescence. Using portable fibre-optic probe instrumentation originally designed for label-free biomedical applications, this capability could be deployed outside the laboratory for monitoring the distribution of herbicides in growing plants

Adaptive multiphoton endomicroscopy through a dynamically deformed multicore optical fiber using proximal detection

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

Screening for protein-protein interactions using Förster resonance energy transfer (FRET) and fluorescence lifetime imaging microscopy (FLIM)

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

Visualising apoptosis in live zebrafish using fluorescence lifetime imaging with optical projection tomography to map FRET biosensor activity in space and time

Fluorescence lifetime imaging (FLIM) combined with optical projection tomography (OPT) has the potential to map Förster resonant energy transfer (FRET) readouts in space and time in intact transparent or near transparent live organisms such as zebrafish larvae, thereby providing a means to visualise cell signalling processes in their physiological context. Here the first application of FLIM OPT to read out biological function in live transgenic zebrafish larvae using a genetically expressed FRET biosensor is reported. Apoptosis, or programmed cell death, is mapped in 3-D by imaging the activity of a FRET biosensor that is cleaved by Caspase 3, which is a key effector of apoptosis. Although apoptosis is a naturally occurring process during development, it can also be triggered in a variety of ways, including through gamma irradiation. FLIM OPT is shown here to enable apoptosis to be monitored over time, in live zebrafish larvae via changes in Caspase 3 activation following gamma irradiation at 24 hours post fertilisation. Significant apoptosis was observed at 3.5 hours post irradiation, predominantly in the head region

An automated multiwell plate reading film microscope for live cell autofluorescence lifetime assays

Fluorescence lifetime imaging (FLIM) is increasingly used to read out cellular autofluorescence originating from the coenzyme NADH in the context of investigating cell metabolic state. We present here an automated multiwell plate reading FLIM microscope optimized for UV illumination with the goal of extending high content fluorescence lifetime assays to readouts of metabolism. We demonstrate its application to automated cellular autofluorescence lifetime imaging and discuss the key practical issues associated with its implementation. In particular, we illustrate its capability to read out the NADH-lifetime response of cells to metabolic modulators, thereby illustrating the potential of the instrument for cytotoxicity studies, assays for drug discovery and stratified medicine