Wide-field Fluorescence Lifetime Imaging Microscopy Research Articles

High throughput compressive fluorescence lifetime imaging with a silicon photomultiplier detector

Fluorescence lifetime imaging microscopy (FLIM) is a powerful technique for studying biological processes. There exists a growing interest in developing strategies to enhance throughput and reduce acquisition time of FLIM systems, which commonly employ laser scanning excitation and time-correlated single-photon counting (TCSPC) detection. In this work, we propose a wide-field FLIM microscope based on compressive sensing and high photon rate detection (beyond pile-up limit) based on a high-efficiency silicon photomultiplier detector as a single-pixel camera. We experimentally validate the capabilities of this design achieving 20 frames per second FLIM images on free-moving green algae sample.

Quantification of protein-protein interactions and activation dynamics: A new path to predictive biomarkers

Oncogene dysregulation is a driver of neoplasia development and progression. The use of quantitative molecular imaging to quantify oncogene activation will be crucial in developing companion diagnostics which can identify personalised patient regimens. However, the evaluation of oncogene activation does not necessarily correlate with oncoprotein activation. Post-translational modifications, such as phosphorylation, lipidation and methylation, may enhance oncoprotein functionality. It is this functionality that progresses neoplasia and may correlate with patient outcome. Advanced molecular imaging may be used to directly quantify oncoprotein, as opposed to oncogene, activation. Time-resolved Förster Resonance Energy Transfer (TR-FRET) involves the non-radiative transfer of energy from one chromophore to another over distances of 1-10 nm; allowing FRET to be used as a “chemical ruler”. TR-FRET can be utilised to directly elucidate spatial oncoprotein activation in single cells and patient tissues. In single cells, TR-FRET has uncovered the mechanisms by which PKCβ1 is trafficked to the nucleus and cleaved. Additionally it has revealed the mechanism of activation of Akt/PKB, whereby Akt/PKB undergoes a conformational change, allowing the Thr308 site to be phosphorylated by PDK1. Moreover TR-FRET has been utilised to quantify HER2-HER heterodimerisation and Akt/PKB activation states in patient biopsies, where it is shown to be predictive of outcome/relapse. The role of TR-FRET is not solely limited to intracellular signalling events. A study has used TR-FRET to measure intercellular immune-checkpoint receptor-ligand interactions. Within this study it was seen that PD-L1 expression was not indicative of PD-1/PD-L1 interaction states in a range of solid tumours. Crucially, in melanoma and NSCLC, PD-1/PD-L1 interaction was a predictive of an improved patient outcome. PD-L1 expression did not predict patient outcome. Several groups have worked to improve Fluorescence lifetime imaging microscopy (FLIM) acquisition times, including the use of: window-galvanometers; multifocal multiphoton FLIM and parallel pixel excitation coupled with wide-field time-gated FLIM. The development of novel quantitative molecular imaging will be critical in the development of personalised patient therapies in the future.

Fluorescence lifetime DNA-PAINT for multiplexed super-resolution imaging of cells

DNA point accumulation for imaging in nanoscale topography (DNA-PAINT) is a powerful super-resolution technique highly suitable for multi-target (multiplexing) bio-imaging. However, multiplexed imaging of cells is still challenging due to the dense and sticky environment inside a cell. Here, we combine fluorescence lifetime imaging microscopy (FLIM) with DNA-PAINT and use the lifetime information as a multiplexing parameter for targets identification. In contrast to Exchange-PAINT, fluorescence lifetime PAINT (FL-PAINT) can image multiple targets simultaneously and does not require any fluid exchange, thus leaving the sample undisturbed and making the use of flow chambers/microfluidic systems unnecessary. We demonstrate the potential of FL-PAINT by simultaneous imaging of up to three targets in a cell using both wide-field FLIM and 3D time-resolved confocal laser scanning microscopy (CLSM). FL-PAINT can be readily combined with other existing techniques of multiplexed imaging and is therefore a perfect candidate for high-throughput multi-target bio-imaging.

Enhanced detection of oral dysplasia by structured illumination fluorescence lifetime imaging microscopy

We demonstrate that structured illumination microscopy has the potential to enhance fluorescence lifetime imaging microscopy (FLIM) as an early detection method for oral squamous cell carcinoma. FLIM can be used to monitor or detect changes in the fluorescence lifetime of metabolic cofactors (e.g. NADH and FAD) associated with the onset of carcinogenesis. However, out of focus fluorescence often interferes with this lifetime measurement. Structured illumination fluorescence lifetime imaging (SI-FLIM) addresses this by providing depth-resolved lifetime measurements, and applied to oral mucosa, can localize the collected signal to the epithelium. In this study, the hamster model of oral carcinogenesis was used to evaluate SI-FLIM in premalignant and malignant oral mucosa. Cheek pouches were imaged in vivo and correlated to histopathological diagnoses. The potential of NADH fluorescence signal and lifetime, as measured by widefield FLIM and SI-FLIM, to differentiate dysplasia (pre-malignancy) from normal tissue was evaluated. ROC analysis was carried out with the task of discriminating between normal tissue and mild dysplasia, when changes in fluorescence characteristics are localized to the epithelium only. The results demonstrate that SI-FLIM (AUC = 0.83) is a significantly better (p-value = 0.031) marker for mild dysplasia when compared to widefield FLIM (AUC = 0.63).

Fluorescence lifetime imaging with a megapixel SPAD camera and neural network lifetime estimation

Fluorescence lifetime imaging microscopy (FLIM) is a key technology that provides direct insight into cell metabolism, cell dynamics and protein activity. However, determining the lifetimes of different fluorescent proteins requires the detection of a relatively large number of photons, hence slowing down total acquisition times. Moreover, there are many cases, for example in studies of cell collectives, where wide-field imaging is desired. We report scan-less wide-field FLIM based on a 0.5 MP resolution, time-gated Single Photon Avalanche Diode (SPAD) camera, with acquisition rates up to 1 Hz. Fluorescence lifetime estimation is performed via a pre-trained artificial neural network with 1000-fold improvement in processing times compared to standard least squares fitting techniques. We utilised our system to image HT1080—human fibrosarcoma cell line as well as Convallaria. The results show promise for real-time FLIM and a viable route towards multi-megapixel fluorescence lifetime images, with a proof-of-principle mosaic image shown with 3.6 MP.

Recent progress of fluorescence lifetime imaging microscopy technology and its application

In the past decade, fluorescence lifetime imaging microscopy (FLIM) has been widely used in biomedical research and other fields. As the fluorescence lifetime is unaffected by probe concentration, excitation intensity and photobleaching, the FLIM has the advantages of high specificity, high sensitivity and capability of quantitative measurement in monitoring microenvironment changes and reflecting the intermolecular interactions. Despite decades of technical development, the FLIM technology still faces some challenges in practical applications. For example, its resolution is still difficult to overcome the diffraction limit and the trade-off among imaging speed, image quality and lifetime accuracy needs to be considered. In recent years, a great advance in FLIM and its application has been made due to the rapid development of hardware and software and their integration with other optical technologies. In this review, we first introduce the principle and characteristics of FLIM technology based on time domain and frequency domain. We then summarize the latest progress of FLIM technology:1) imaging speed enhancement based on hardware improvement such as optimized time-correlated single photon counting module, single photon avalanche diode array detector, and acousto-optic deflector scanner; 2) lifetime measurement accuracy improvement by the proposed algorithms such as maximum likelihood estimate, Bayesian analysis and compressed sensing; 3) imaging quality enhancement and spatial resolution improvement by integrating FLIM with other optical technologies such as adaptive optics for correcting the aberration generated in the optical path, special illumination for equipping wide-field FLIM with optical sectioning ability, and super-resolution techniques for exceeding the resolution limit. We then highlight some recent applications in biomedical studies such as signal transduction or plant cell growth, disease diagnosis and treatment in cancers, Alzheimer's disease and skin diseases, assessment for toxicity and treatment efficiency of nanomaterials developed in the past few years. Finally, we present a short discussion on the current challenges and provide an outlook of the future development of enhanced imaging performance for FLIM technology. We hope that our summary on the state-of-the-art FLIM, our commentary on future challenges, and some proposed avenues for further advances will contribute to the development of FLIM technology and its applications in relevant fields.

Optically sectioned wide-field fluorescence lifetime imaging microscopy enabled by structured illumination.

In this paper, we demonstrate the ability of structured illumination microscopy to enhance the ability of fluorescence lifetime imaging to resolve fluorescence lifetimes in relatively thick samples that possess distinct but spectrally overlapping fluorescent layers. Structured illumination fluorescent lifetime imaging microscopy (SI-FLIM) is shown to be able to accurately reconstruct lifetime values in homogenous fluorophore samples (POPOP, NADH, and FAD) as well as accurately measure fluorescent lifetime in two layer models that are layered with NADH/FAD over POPOP, where NADH/FAD and POPOP have spectral overlap. Finally, the ability of SI-FLIM was demonstrated in a hamster cheek pouch ex vivo to show that more accurate lifetimes could be measured for each layer of interest in the oral mucosa (epithelium and submucosa).

Instrumentation Design of a High-Speed Fluorescence Lifetime Imaging Microscope Tailored to High-Throughput Screening for Drug Discovery

Fluorescence lifetime imaging microscopy (FLIM) is a powerful imaging modality that can provide unique information related to the biological microenvironment of a sample. For instance, when applied to quantifying Förster resonant energy transfer (FRET), the decay characteristics of the fluorescence can be indicative of the degree of protein-protein interaction occurring at a subcellular level. There is a clear interest at the drug discovery level to harness the potential of FLIM-FRET and other FLIM techniques to better characterize and understand the mechanisms of their drug leads.To properly test and isolate the effectiveness of drug leads under varied conditions and doses, the drug discovery process uses high-content screening (HCS) microscopes to image 100,000 samples per day. FLIM techniques have so far been unable to provide a suitable combination of acquisition speed and resolution to fill this demand. Widefield FLIM techniques suffer from out-of-focus light, which is especially detrimental to fitting the lifetimes of fluorescence decays. Typical raster-scanning confocal approaches can offer excellent spatial and temporal resolution with a photomultiplier tube as a single-point detector, but are inherently slow to collect a full FLIM image.We are designing a system to meet these HCS requirements by combining a multiplexed raster scanning approach with a streak camera and high-speed readout camera. In a streak camera, photons incident on the entrance slit are focused to a photocathode. The photoelectrons generated at the photocathode are accelerated through a vacuum tube, across a perpendicular time-varying electric field, which can deflect the electrons to the left or right based on their arrival time. The resulting readout would consist of a spatial axis and a temporal axis. The slit of a streak camera is an excellent method of multiplexing the fluorescence lifetime capture along its spatial axis. Our streak camera (Optronis SC-10) can be filled with up to 100 resolvable optical fiber channels.A lenslet array is used to generate 10x10 focal points on the sample, which can then be scanned over the sample with an x-y galvo system. Given sufficient excitation power, this offers a 100x improvement on acquisition speed. The galvo scanner also provides a descan of the fluorescent signals on the return path, such that the fluorescence signals can be focused into a fixed 2-dimensional optical fiber array. To accommodate the streak camera slit, the 100 fiber channels are rearranged from 10x10 to 1x100.We will discuss the current performance of the system, and the potential improvements for each component to get closer to the required acquisition speed. In particular, there is interest in investigating the use of solid-state devices to replace the streak camera. Single-photon avalanche diode (SPAD) arrays are an emerging technology that could greatly simplify the instrumentation, reduce the acquisition time, and improve the light collection efficiency. There are also potential improvements in frame rate and light economy concerning our excitation sources, optics and readout camera.

Optical refocusing three-dimensional wide-field fluorescence lifetime imaging microscopy

Three-dimensional fluorescence lifetime microscopy is achieved by combining wide-field fluorescence lifetime imaging with a remote optical refocusing method. As required for some applications in dynamic research for physics, chemistry, or biology, it is thereby not necessary to move the sample, i.e., the specimen is not disturbed during measurement. Using a fluorescent microsphere the performance of the system has been tested successfully with respect to three-dimensional fluorescence lifetime microscopy as well as time-resolved fluorescence spectroscopy.

Real-time fluorescence lifetime imaging system with a 32 × 32 013μm CMOS low dark-count single-photon avalanche diode array

A compact real-time fluorescence lifetime imaging microscopy (FLIM) system based on an array of low dark count 0.13microm CMOS single-photon avalanche diodes (SPADs) is demonstrated. Fast background-insensitive fluorescence lifetime determination is achieved by use of a recently proposed algorithm called 'Integration for Extraction Method' (IEM) [J. Opt. Soc. Am. A 25, 1190 (2008)]. Here, IEM is modified for a wider resolvability range and implemented on the FPGA of the new SPAD array imager. We experimentally demonstrate that the dynamic range and accuracy of calculated lifetimes of this new camera is suitable for widefield FLIM applications by imaging a variety of test samples, including various standard fluorophores covering a lifetime range from 1.6ns to 16ns, microfluidic mixing of fluorophore solutions, and living fungal spores of Neurospora Crassa. The calculated lifetimes are in a good agreement with literature values. Real-time fluorescence lifetime imaging is also achieved, by performing parallel 32 x 16 lifetime calculations, realizing a compact and low-cost FLIM camera and promising for bigger detector arrays.

Wide-field photon counting fluorescence lifetime imaging microscopy: application to photosynthesizing systems

Fluorescence lifetime imaging microscopy (FLIM) is a technique that visualizes the excited state kinetics of fluorescence molecules with the spatial resolution of a fluorescence microscope. We present a scanningless implementation of FLIM based on a time- and spacecorrelated single photon counting (TSCSPC) method employing a position-sensitive quadrant anode detector and wide-field illumination. The standard time-correlated photon counting approach leads to picosecond temporal resolution, making it possible to resolve complex fluorescence decays. This allows parallel acquisition of time-resolved images of biological samples under minimally invasive low-excitation conditions (<10 mW/cm(2)). In this way unwanted photochemical reactions induced by high excitation intensities and distorting the decay kinetics are avoided. Comparably low excitation intensities are practically impossible to achieve with a conventional laser scanning microscope, where focusing of the excitation beam into a tight spot is required. Therefore, wide-field FLIM permits to study Photosystem II (PS II) in a way so far not possible with a laser scanning microscope. The potential of the wide-field TSCSPC method is demonstrated by presenting FLIM measurements of the fluorescence dynamics of photosynthetic systems in living cells of the chlorophyll d-containing cyanobacterium Acaryochloris marina.

Innovating lifetime microscopy: a compact and simple tool for life sciences, screening, and diagnostics

Fluorescence lifetime imaging microscopy (FLIM) allows the investigation of the physicochemical environment of fluorochromes and protein-protein interaction mapping by Forster resonance energy transfer (FRET) in living cells. However, simpler and cheaper solutions are required before this powerful analytical technique finds a broader application in the life sciences. Wide-field frequency-domain FLIM represents a solution whose application is currently limited by the need for multichannel-plate image intensifiers. We recently showed the feasibility of using a charge-coupled device/complementory metal-oxide semiconductor (CCD/CMOS) hybrid lock-in imager, originally developed for 3-D vision, as an add-on device for lifetime measurements on existing wide-field microscopes. In the present work, the performance of the setup is validated by comparison with well-established wide-field frequency-domain FLIM measurements. Furthermore, we combine the lock-in imager with solid-state light sources. This results in a simple, inexpensive, and compact FLIM system, operating at a video rate and capable of single-shot acquisition by virtue of the unique parallel retrieval of two phase-dependent images. This novel FLIM setup is used for cellular and FRET imaging, and for high-throughput and fast imaging applications. The all-solid-state design bridges the technological gap that limits the use of FLIM in areas such as drug discovery and medical diagnostics.