Fluorescence Lifetime Imaging Microscopy Techniques Research Articles

Glutathione-capped gold nanoclusters as near-infrared-emitting efficient contrast agents for confocal fluorescence imaging of tissue-mimicking phantoms.

An innovative research has been conducted focused on demonstrating the ability of novel dual-emissive glutathione-stabilized gold nanoclusters (GSH-AuNCs) to perform bright near-infrared (NIR)-emitting contrast agents inside tissue-mimicking agarose-phantoms via two complementary confocal fluorescence imaging techniques. First, using a new and fast microwave-assisted approach, we synthesized photostable dual-emitting GSH-AuNCs with an average size of 3.2 ± 0.4 nm and NIR emission quantum yield of 9.9%. Steady-state fluorescence measurements coupled with fluorescence lifetime imaging microscopy (FLIM) assays performed on lyophilized GSH-AuNCs revealed that the obtained GSH-AuNCs exhibit PL emissions at 610nm (red PL) and, respectively, 800nm (NIR PL) in both solution and powder solid-state. Time-resolved fluorescence measurements showed that the two PL components are characterized by average lifetimes of 407ns (red PL) and 1821ns (NIR PL), respectively. Additionally, due to a partial overlap between the red PL and the absorption of the NIR PL, an energy transfer between the two coexisting emissive centers was discovered and confirmed via steady-state and time-resolved fluorescence measurements. Furthermore, the FLIM analysis performed on powder GSH-AuNCs under 640nm, an excitation more suitable for bioimaging applications, revealed a homogeneous and photostable NIR PL signal from GSH-AuNCs. Finally, the ability of GSH-AuNCs to operate as reliable NIR-emitting contrast agents inside tissue-mimicking agarose-phantoms was demonstrated here for the first time via complementary FLIM and re-scan confocal fluorescence imaging techniques. In consequence, GSH-AuNCs show great promise for future in vivo imaging applications via confocal fluorescence microscopy.

Real-Time Polymer Viscosity-Catalytic Activity Relationships on the Microscale.

Polymer growth induces physical changes to catalyst microenvironments. Here, these physical changes are quantified in real time and are found to influence microscale chemical catalysis and the polymerization rate. By developing a method to "peer into" optically transparent living-polymer particles, simultaneous imaging of both viscosity changes and chemical activity was achieved for the first time with high spatiotemporal resolution through a combination of fluorescence intensity microscopy and fluorescence lifetime imaging microscopy techniques. Specifically, an increase in microenvironment viscosity led to a corresponding local decrease in the catalytic molecular ruthenium ring-opening metathesis polymerization rate, plausibly by restricting diffusional access to active catalytic centers. Consistent with this diffusional-access model, these viscosity changes were found to be monomer-dependent, showing larger changes in microenvironment viscosity in cross-linked polydicyclopentadiene compared to non-crosslinked polynorbornene. The sensitivity and high spatial resolution of the imaging technique revealed significant variations in microviscosities between different particles and subparticle regions. These revealed spatial heterogeneities would not be observable through alternative ensemble analytical techniques that provide sample-averaged measurements. The observed spatial heterogeneities provide a physical mechanism for variation in catalytic chemical activity on the microscale that may accumulate and lead to nonhomogeneous polymer properties on the bulk scale.

A Novel Quantum Dot-Based pH Probe for Long-Term Fluorescence Lifetime Imaging Microscopy Experiments in Living Cells

The use of two nanoparticles for quantitative pH measurements in live cells by means of fluorescence lifetime imaging microscopy (FLIM) is investigated here. These nanoparticles are based on CdSe/ZnS quantum dots (QDs), functionalized with N-acetylcysteine (CdSe/ZnS-A) and with a small peptide containing D-penicillamine and histidine (CdSe/ZnS-PH). CdSe/ZnS-A has tendency to aggregate and nonlinear pH sensitivity in a complex medium containing salts and macromolecules. On the contrary, CdSe/ZnS-PH shows chemical stability, low toxicity, efficient uptake in C3H10T1/2 cells, and good performance as an FLIM probe. CdSe/ZnS-PH also has key advantages over a recently reported probe based on a CdSe/ZnS QD functionalized with D-penicillamine (longer lifetimes and higher pH-sensitivity). A pH(±2σ) of 6.97 ± 0.14 was determined for C3H10T1/2 cells by FLIM employing this nanoprobe. In addition, the fluorescence lifetime signal remains nearly constant for C3H10T1/2 cells treated with CdSe/ZnS-PH for 24 h. These results show the promising applications of this nanoprobe to monitor the intracellular pH and cell state employing the FLIM technique.

Single-photon peak event detection (SPEED): a computational method for fast photon counting in fluorescence lifetime imaging microscopy.

Fluorescence lifetime imaging microscopy (FLIM) characterizes samples by examining the temporal properties of fluorescence emission, providing useful contrast within samples based on the local physical and biochemical environment of fluorophores. Despite this, FLIM applications have been limited in scope by either poor accuracy or long acquisition times. Here, we present a method for computational single-photon counting of directly sampled time-domain FLIM data that is capable of accurate fluorescence lifetime and intensity measurements while acquiring over 160 Mega-counts-per-second with sub-nanosecond time resolution between consecutive photon counts. We demonstrate that our novel method of Single-photon PEak Event Detection (SPEED) is more accurate than direct pulse sampling and faster than established photon counting FLIM methods. We further show that SPEED can be implemented for imaging and quantifying samples that benefit from higher -throughput and -dynamic range imaging with real-time GPU-accelerated processing and use this capability to examine the NAD(P)H-related metabolic dynamics of apoptosis in human breast cancer cells. Computational methods for photon counting such as SPEED open up more opportunities for fast and accurate FLIM imaging and additionally provide a basis for future innovation into alternative FLIM techniques.

Instant FLIM enables 4D in vivo lifetime imaging of intact and injured zebrafish and mouse brains

Traditional fluorescence microscopy is blind to molecular microenvironment information that is present in a fluorescence lifetime, which can be measured by fluorescence lifetime imaging microscopy (FLIM). However, most existing FLIM techniques are slow to acquire and process lifetime images, difficult to implement, and expensive. Here we present instant FLIM, an analog signal processing method that allows real-time streaming of fluorescence intensity, lifetime, and phasor imaging data through simultaneous image acquisition and instantaneous data processing. Instant FLIM can be easily implemented by upgrading an existing two-photon microscope using cost-effective components and our open-source software. We further improve the functionality, penetration depth, and resolution of instant FLIM using phasor segmentation, adaptive optics, and super-resolution techniques. We demonstrate through-skull intravital 3D FLIM of mouse brains to depths of 300 µm and present the first in vivo 4D FLIM of microglial dynamics in intact and injured zebrafish and mouse brains for up to 12 h.

P‐103: Non‐Destructive FMM Shadow‐Level Evaluation with Fluorescence Lifetime Imaging Microscopy

When OLED materials are depositing on the back‐plane in the evaporation process, they can be mixed in pixels by the FMM shadow. As a result, the luminance is decreased and abnormal display panels are made. Previously, we measured the level of shadow using AFM (atomic force microscopy) technique, but this technique cannot be applied to actual display panel. So we have introduced the FLIM (fluorescence lifetime imaging microscopy) technique to identify the cause of the problem. We can analyze the mixed OLED materials using the lifetime of organic materials at a specific wavelength. Finally, we can define the level of shadow and distinguish the normal and abnormal pixels while the general PL (photoluminescence) method cannot measure the shadow. Therefore, this technique is very useful to confirm the shadow level with non‐destructive and high‐resolution imaging.

Fast fluorescence lifetime microscopy imaging of any number of discrete irregular regions of interest

Fluorescence lifetime imaging microscopy (FLIM) has been widely used in biomedical research due to its high specificity, high sensitivity and quantification ability in cell microenvironment sensing. The fluorescence lifetime detection method based on time-correlated single photon counting (TCSPC) is one of the most commonly used techniques at present. However, due to the limitation of imaging principles and conditions, this technique has the disadvantages of long data acquisition time and consequently low imaging speed. In this paper, a fast FLIM technique for any number of discrete and irregular regions of interest (ROIs) in biological samples is developed. The technology uses acousto-optic deflectors (AODs) to achieve fast and flexible addressing scanning, optimize the synchronization strategy between AOD and TCSPC, and reconstruct the lifetime image through simple online feature analysis of the ROI shapes. For the case of multiple discrete irregular ROIs in biological samples, it can greatly save the time of data acquisition, thus realizing the fast FLIM imaging of these ROIs, which is benificial to the study of the heterogeneity of biological events in biological system. In particular, the fast fluorescence imaging result for 87 discrete points in the field of view shows that this method can obtain a fluorescence lifetime image in a very short acquisition time (only 52.2 ms) and thus achieving a very fast imaging speed in such a situation. Dynamic FLIM imaging of lysosome probe LysoSensor Green DND-189 in living cells stimulated by ammonium chloride is carried out to monitor the real-time change of pH value in lysosome lumen. The acquisition time for a single fluorescence lifetime image of lysosomes in two ROIs is only 200 ms. The results show that the rapid FLIM technology can be used to dynamically monitor the changes of microenvironment in biological samples, and will play an important role in the microenvironment sensing in living cells.

Controllable growth of Ga2O3 hexagonal prism with pyramid end and monitoring size-dependent electron transfer process in perylene/Ga2O3 conjugate system via FLIM

Hexagonal prisms with hexagonal pyramid end of Ga2O3 microstructures were successfully prepared on gallium arsenide substrate by double cell electrochemical etching method under various growth conditions. The field emission scanning electron microscopy, energy-dispersive X-ray spectroscopy, X-ray photoelectron spectroscopy (XPS), photoluminescence (PL) and fluorescence lifetime imaging microscopy (FLIM) were used to characterize the hexagonal prisms. The EDS analysis confirmed that the prepared prisms were composed dominantly of gallium (Ga) and oxygen (O) with atomic ratio of ~ 2:3. The binding energy of the Ga 2p and Ga 3d XPS peaks revealed that the formation of Ga–O bonding with the highest oxidation state of Ga (Ga3+) and the formed hexagonal microstructures were Ga2O3. The PL spectra at room temperature under excitation at 290 nm exhibit a strong blue emission peak located at about 470 nm. The origin of this PL peak can be attributed to exciton recombination at different intrinsic defect sites such as O vacancies and Ga vacancies. Moreover, FLIM technique has been employed to probe time-resolved photoluminescence properties of individual Ga2O3 prisms with hexagonal tip. FLIM images provide two-dimensional spatial maps of the carrier recombination lifetime which considered the most critical parameter to improve the performance of optoelectronic devices. It has been concluded that the carrier recombination lifetime was strongly sensitive to the size of the hexagonal prism-shaped structures due to surface-related defects. Additionally, a quantitative analysis of a conjugate system prepared by functionalizing hexagonal Ga2O3 prisms with perylene organic dye molecules was reported in this work. The size-dependent electronic coupling between the conduction band level of gallium oxide and excited states of perylene molecules was investigated. We observed that highly efficient electron transfer process occurs for aggregated molecules due to excimer trap states. The grown prism structures seem to be interesting for applications in optoelectronic devices as well as to be used as an effective host medium for organic dye molecules in solar cell applications.

Monitoring the Cellular Delivery of Doxorubicin-Cu Complexes in Cells by Fluorescence Lifetime Imaging Microscopy.

In the prodrug research field, information obtained from traditional end point biochemical assays in drug effect studies could provide neither the dynamic processes nor heterogeneous responses of individual cells. In situ imaging microscopy techniques, especially fluorescence lifetime imaging microscopy (FLIM), could fulfill these requirements. In this work, we used FLIM techniques to observe the entry and release of doxorubicin (Dox)-Cu complexes in live KYSE150 cells. The Dox-Cu complex has weaker fluorescence signals but similar lifetime values as compared to the raw Dox, whose fluorescence could be released by the addition of biothiol compound (such as glutathione). The cell viability results indicated that the Dox-Cu compound has a satisfactory killing effect on KYSE150 cells. The FLIM data showed that free doxorubicin was released from Dox-Cu complexes in cytoplasm of KYSE150 cells and then accumulated in the nucleus. After 90 min administration, the fluorescence lifetime signals reached 1.21 and 1.46 ns in the cytoplasm and nucleus, respectively, reflecting the transformation and transportation of Dox-Cu complexes. In conclusion, this work provides a satisfactory example for the research of prodrug monitored by FLIM techniques, expanding the useful applications of FLIM technique in drug development.

Fluorescence life-time imaging microscopy (FLIM) monitors tumor cell death triggered by photothermal therapy with MoS2 nanosheets

Recently, photothermal therapy (PTT) has been proved to have great potential in tumor therapy. In the last several years, MoS2, as one novel member of nanomaterials, has been applied into PTT due to its excellent photothermal conversion efficacy. In this work, we applied fluorescence lifetime imaging microscopy (FLIM) techniques into monitoring the PPT-triggered cell death under MoS2 nanosheet treatment. Two types of MoS2 nanosheets (single layer nanosheets and few layer nanosheets) were obtained, both of which exhibited presentable photothermal conversion efficacy, leading to high cell death rates of 4T1 cells (mouse breast cancer cells) under PTT. Next, live cell images of 4T1 cells were obtained via directly labeling the mitochondria with Rodamine123, which were then continuously observed with FLIM technique. FLIM data showed that the fluorescence lifetimes of mitochondria targeting dye in cells treated with each type of MoS2 nanosheets significantly increased during PTT treatment. By contrast, the fluorescence lifetime of the same dye in control cells (without nanomaterials) remained constant after laser irradiation. These findings suggest that FLIM can be of great value in monitoring cell death process during PTT of cancer cells, which could provide dynamic data of the cellular microenvironment at single cell level in multiple biomedical applications.

Fast fluorescence lifetime imaging techniques: A review on challenge and development

Fluorescence lifetime imaging microscopy (FLIM) is increasingly used in biomedicine, material science, chemistry, and other related research fields, because of its advantages of high specificity and sensitivity in monitoring cellular microenvironments, studying interaction between proteins, metabolic state, screening drugs and analyzing their efficacy, characterizing novel materials, and diagnosing early cancers. Understandably, there is a large interest in obtaining FLIM data within an acquisition time as short as possible. Consequently, there is currently a technology that advances towards faster and faster FLIM recording. However, the maximum speed of a recording technique is only part of the problem. The acquisition time of a FLIM image is a complex function of many factors. These include the photon rate that can be obtained from the sample, the amount of information a technique extracts from the decay functions, the efficiency at which it determines fluorescence decay parameters from the recorded photons, the demands for the accuracy of these parameters, the number of pixels, and the lateral and axial resolutions that are obtained in biological materials. Starting from a discussion of the parameters which determine the acquisition time, this review will describe existing and emerging FLIM techniques and data analysis algorithms, and analyze their performance and recording speed in biological and biomedical applications.

Fast single-cell biochemistry: theory, open source microscopy and applications

Fluorescence lifetime sensing enables researchers to probe the physicochemical environment of a fluorophore providing a window through which we can observe the complex molecular make-up of the cell. Fluorescence lifetime imaging microscopy (FLIM) quantifies and maps cell biochemistry, a complex ensemble of dynamic processes. Unfortunately, typical high-resolution FLIM systems exhibit rather limited acquisition speeds, often insufficient to capture the time evolution of biochemical processes in living cells. Here, we describe the theoretical background that justifies the developments of high-speed single photon counting systems. We show that systems with low dead-times not only result in faster acquisition throughputs but also improved dynamic range and spatial resolution. We also share the implementation of hardware and software as an open platform, show applications of fast FLIM biochemical imaging on living cells and discuss strategies to balance precision and accuracy in FLIM. The recent innovations and commercialisation of fast time-domain FLIM systems are likely to popularise FLIM within the biomedical community, to impact biomedical research positively and to foster the adoption of other FLIM techniques as well. While supporting and indeed pursuing these developments, with this work we also aim to warn the community about the possible shortcomings of fast single photon counting techniques and to highlight strategies to acquire data of high quality.

Real-time visualization of two-photon fluorescence lifetime imaging microscopy using a wavelength-tunable femtosecond pulsed laser.

A fluorescence lifetime imaging microscopy (FLIM) integrated with two-photon excitation technique was developed. A wavelength-tunable femtosecond pulsed laser with nominal pulse repetition rate of 76-MHz was used to acquire FLIM images with a high pixel rate of 3.91 MHz by processing the pulsed two-photon fluorescence signal. Analog mean-delay (AMD) method was adopted to accelerate the lifetime measurement process and to visualize lifetime map in real-time. As a result, rapid tomographic visualization of both structural and chemical properties of the tissues was possible with longer depth penetration and lower photo-damage compared to the conventional single-photon FLIM techniques.

Use of Fluorescence Lifetime Imaging Microscopy (FLIM) as a Timer of Cell Cycle S Phase.

Incorporation of thymidine analogues in replicating DNA, coupled with antibody and fluorophore staining, allows analysis of cell proliferation, but is currently limited to monolayer cultures, fixed cells and end-point assays. We describe a simple microscopy imaging method for live real-time analysis of cell proliferation, S phase progression over several division cycles, effects of anti-proliferative drugs and other applications. It is based on the prominent (~ 1.7-fold) quenching of fluorescence lifetime of a common cell-permeable nuclear stain, Hoechst 33342 upon the incorporation of 5-bromo-2’-deoxyuridine (BrdU) in genomic DNA and detection by fluorescence lifetime imaging microscopy (FLIM). We show that quantitative and accurate FLIM technique allows high-content, multi-parametric dynamic analyses, far superior to the intensity-based imaging. We demonstrate its uses with monolayer cell cultures, complex 3D tissue models of tumor cell spheroids and intestinal organoids, and in physiological study with metformin treatment.

Reference-independent wide fieldfluorescence lifetime measurements using Frequency-Domain (FD) technique based on phase and amplitude crossing point.

Fluorescence lifetime imaging microscopy (FLIM) is an essential tool in many scientific fields such as biology and medicine thanks to the known advantages of the fluorescence lifetime (FLT) over the classical fluorescence intensity (FI). However, the frequency domain (FD) FLIM technique suffers from its strong dependence on the reference and its compliance to the sample. In this paper, we suggest a new way to calculate the FLT by using the crossing point (CRPO) between the modulation and phase FLTs measured over several light emitting diode (LED) DC currents values instead of either method alone. This new technique was validated by measuring homogeneous substances with known FLT, where the CRPO appears to be the optimal measuring point. Furthermore, the CRPO method was applied in heterogeneous samples. It was found that the CRPO in known mixed solutions is the weighted average of the used solutions. While measuring B16 and lymphocyte cells, the CRPO of the DAPI compound in single FLT regions was measured at 3.5±0.06ns and at 2.83±0.07ns, respectively, both of which match previous reports and multi-frequency analyses. This paper suggests the CRPO as a new method to extract the FLT in problematic cases such as high MCP gains and heterogeneous environments. In traditional FD FLIM measurements, the variation in phase angle and modulation are measured. By measuring over varying DC currents, another variation is detected in the FLT determined through the phase and modulation methods, with the CRPO indicating the true FLT.

Application of the Diver Detection Method to Multiphoton Microscopy and Flim

In two-photon microscopy of turbid media, such as biological tissues, achievable imaging depth depends on ability of detection system to detect weak emission signals induced in deep tissue layers. In the conventional epi-detection method photons are collected from a relatively narrow sample area and acceptance angle, so most of emitted photons remain undetected. The DIVER (Deep Imaging Via Emission Recovery) method utilizes wide photocathode area detector and matching refractive index in the optical path from the sample surface to photocathode; that allows efficient collection of photons from wide area of a turbid sample at almost any angle. Using this method, the imaging depth was increased by factor of 4-6 compared to conventional epi-detection. In addition, the DIVER method offers superior sensitivity in SHG imaging and allows detection of short wavelength emission, up to 320nm. This, in turn, allows THG and three-photon imaging using conventional Ti:Sa laser excitation wavelengths. Coupling with the FLIMBox enables Fluorescence Lifetime Imaging Microscopy (FLIM) in depth and acquiring 3D-FLIM images of tissue samples. Using combination of SHG and FLIM techniques we were able to separate various types of collagens (I to V) in tissue samples. These techniques also allowed us to detect kidney fibrosis at early stages in UUO mouse model. THG imaging is particularly useful for lipid droplets imaging and was used in imaging of adipose tissues and mouse livers in diabetes studies. The special UV-detection capability of the DIVER was also applied to study three-photon excitation FCS of unlabeled protein oligomers.

Deep-UV fluorescence lifetime imaging microscopy

A novel fluorescence lifetime imaging microscopy (FLIM) working with deep UV 240-280 nm wavelength excitations has been developed. UV-FLIM is used for measurement of defect-related fluorescence and its changes upon annealing from femtosecond laser-induced modifications in fused silica. This FLIM technique can be used with microfluidic and biosamples to characterize temporal characteristics of fluorescence upon UV excitation, a capability easily added to a standard microscope-based FLIM. UV-FLIM was tested to show annealing of the defects induced by silica structuring with ultrashort laser pulses. Frequency-domain fluorescence measurements were converted into the time domain to extract long fluorescence lifetimes from defects in silica.

Time-Domain Fluorescence Lifetime Imaging Microscopy: A Quantitative Method to Follow Transient Protein–Protein Interactions in Living Cells

Quantitative analysis in Förster resonance energy transfer (FRET) imaging studies of protein-protein interactions within live cells is still a challenging issue. Many cellular biology applications aim at the determination of the space and time variations of the relative amount of interacting fluorescently tagged proteins occurring in cells. This relevant quantitative parameter can be, at least partially, obtained at a pixel-level resolution by using fluorescence lifetime imaging microscopy (FLIM). Indeed, fluorescence decay analysis of a two-component system (FRET and no FRET donor species), leads to the intrinsic FRET efficiency value (E) and the fraction of the donor-tagged protein that undergoes FRET (fD). To simultaneously obtain fD and E values from a two-exponential fit, data must be acquired with a high number of photons, so that the statistics are robust enough to reduce fitting ambiguities. This is a time-consuming procedure. However, when fast-FLIM acquisitions are used to monitor dynamic changes in protein-protein interactions at high spatial and temporal resolutions in living cells, photon statistics and time resolution are limited. In this case, fitting procedures are unreliable, even for single lifetime donors. We introduce the concept of a minimal fraction of donor molecules involved in FRET (mfD), obtained from the mathematical minimization of fD. Here, we discuss different FLIM techniques and the compromises that must be made between precision and time invested in acquiring FLIM measurements. We show that mfD constitutes an interesting quantitative parameter for fast FLIM because it gives quantitative information about transient interactions in live cells.

Diffusion Reflection and Fluorescence Lifetime Imaging Microscopy Study of Fluorophore-Conjugated Gold Nanoparticles or Nanorods in Solid Phantoms.

In this paper we report the optical properties of fluorescein-conjugated gold nanoparticles (GNPs) in solid phantoms using diffusion reflection (DR) and fluorescence lifetime imaging microscopy (FLIM). The GNPs attached with fluorescein in solution were studied by fluorescence correlation spectroscopy. The intensity decays were recorded to reveal the fluorescence lifetime of fluorescein while in the near-field vicinity of the GNPs. The DR method was used to explore the solid phantoms containing GNPs, indicating the light propagation from the surface of solid phantoms. The resulting DR slopes of the reflected intensity showed the higher the GNP concentration, the bigger the slope. Fluorescence intensity, lifetime, and anisotropy images of solid phantoms were investigated by FLIM. The exploration of optical properties and molecular imaging combined with DR and FLIM methods is a new approach that has not been established until now. The combined DR–FLIM technique is expected to provide discrimination based on unique spectroscopic fingerprints of GNPs that could be utilized for cell imaging. This paper includes a combined study with a variety of methods, which may lead to multimodal imaging for surfaces (by FLIM) and deep penetration (up to cm by the DR) together.

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.