Real-time open-source FLIM analysis.

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.

Fluorescence lifetime imaging microscopy (FLIM) adds an additional dimension to fluorescence microscopy by measuring the fluorophore interactions with the microenvironment in addition to all the benefits and power of fluorescence microscopy. Real-time FLIM, however, requires overcoming unique technical challenges to achieve similar imaging speeds as can be achieved through standard in vivo microscopy techniques. This talk will present an “InstantFLIM” system that achieves real-time (acquisition and processing), super-resolution, 3D in vivo multiphoton FLIM by overcoming these limitations. The system is demonstrated in intact-skull mouse and zebrafish brain imaging models, and 3D autofluorescence FLIM of highly scattering plant tissue.

Artificial intelligence (AI)/machine-learning (ML) algorithms have been heavily used in data processing in various biological and clinical applications. Sensitive biological signaling can be monitored using two-photon fluorescence lifetime imaging microscopy (FLIM). Lifetime fitting, processing, and analyzing FLIM data of biological specimens can be a challenging and time-consuming affair. The recently developed Fluorescence Lifetime Redox Ratio (FLIRR) focuses on tracking metabolic changes ‘before-and-after-treatment’ in live cells using only two lifetime parameters. FLIM data produces many data parameters which are all associated with drug response in living cells. To predict drug cellular responses, we have chosen the Becker & Hickl SPCImage software to fit the lifetime images and the resultant data was used in ML analysis. With the objective of achieving even more robust statistical power predicting earliest drug effects, we developed Python software and autoencoder (AE) models to analyze the multiple biophysical FLIM parameters acquired in 2p-FLIM images of drug response in cervical cancer cells. The use of systematic hyperparameter (HP) tuning shows the variation in performance across the different models, enabling the selection of the highest performing model and HPs for repetition. Our results show that our optimized multi-parameter trained AE models can statistically outperform single FLIRR time-course analysis in discriminating earliest metabolic changes following drug treatment.

Fluorescence lifetime imaging microscopy (FLIM) is now routinely used for dynamic measurements of signaling events inside living cells, including detection of protein-protein interactions. An understanding of the basic physics of fluorescence lifetime measurements is required to use this technique. In this protocol, we describe both the time-correlated single photon counting and the frequency-domain methods for FLIM data acquisition and analysis. We describe calibration of both FLIM systems, and demonstrate how they are used to measure the quenched donor fluorescence lifetime that results from Förster resonance energy transfer (FRET). We then show how the FLIM-FRET methods are used to detect the dimerization of the transcription factor CCAAT/enhancer binding protein-α in live mouse pituitary cell nuclei. Notably, the factors required for accurate determination and reproducibility of lifetime measurements are described. With either method, the entire protocol including specimen preparation, imaging and data analysis takes ∼2 d.

A quantitative assessment of wound status in a murine model was developed using phasor plot presentation of fluorescence lifetime imaging microscopy (FLIM) data. The quantitative assessment is based on calculating Bhattacharyya distance between g coordinates of FLIM data phasor plot density distributions of wound and healthy skin. The approach was validated for both diabetic and non-diabetic mice wounds, including during low-dose photodynamic therapy (LDPDT). Analysis revealed a shift in the FLIM data phasor plot g coordinates, suggesting altered metabolic processes involved in wound healing. Bhattacharyya distances in the LDPDT groups were closer to zero compared to the control group, which was not treated by LDPDT. Bhattacharyya distances in the non-diabetic LDPDT groups were closer to zero compared to the diabetic LDPDT groups that is consistent with the literature regarding the positive role of LDPDT in accelerating wound healing and the role of diabetes mellitus in impairing wound healing.

Chapter 3 Time domain FLIM: Theory, instrumentation, and data analysis

An optimal methodology for locating and tracking cellulose nanofibers (CNFs) in vitro and in vivo is crucial to evaluate the environmental health and safety properties of these nanomaterials. Here, we report the use of a new boron-dipyrromethene (BODIPY) reactive fluorescent probe, meso-DichlorotriazineEthyl BODIPY (mDTEB), tailor-made for labeling CNFs used in simulated or in vivo ingestion exposure studies. Time-correlated single photon counting (TCSPC) fluorescence lifetime imaging microscopy (FLIM) was used to confirm covalent attachment and purity of mDTEB-labeled CNFs. The photoluminescence properties of mDTEB-labeled CNFs, characterized using fluorescence spectroscopy, include excellent stability over a wide pH range (pH2 to pH10) and high quantum yield, which provides detection at low (μM) concentrations. FLIM analysis also showed that lignin-like impurities present on the CNF reduce the fluorescence of the mDTEB-labeled CNF, via quenching. Therefore, the chemical composition and the methods of CNF production affect subsequent studies. An in vitro triculture, small intestinal, epithelial model was used to assess the toxicity of ingested mDTEB-labeled CNFs. Zebrafish (Danio rerio) were used to assess in vivo environmental toxicity studies. No cytotoxicity was observed for CNFs, or mDTEB-labeled CNFs, either in the triculture cells or in the zebrafish embryos.

Time-gated fluorescence lifetime imaging microscopy (FLIM) is a powerful technique to assess the biochemistry of cells and tissues. When applied to living thick samples, it is hampered by the lack of optical sectioning and the need of acquiring many images for an accurate measurement of fluorescence lifetimes. Here, we report on the use of processing techniques to overcome these limitations, minimizing the acquisition time, while providing optical sectioning. We evaluated the application of the HiLo and the rapid lifetime determination (RLD) techniques for accurate measurement of fluorescence lifetimes with optical sectioning. HiLo provides optical sectioning by combining the high-frequency content from a standard image, obtained with uniform illumination, with the low-frequency content of a second image, acquired using structured illumination. Our results show that HiLo produces optical sectioning on thick samples without degrading the accuracy of the measured lifetimes. We also show that instrument response function (IRF) deconvolution can be applied with the RLD technique on HiLo images, improving greatly the accuracy of the measured lifetimes. These results open the possibility of using the RLD technique with pulsed diode laser sources to determine accurately fluorescence lifetimes in the subnanosecond range on thick multilayer samples, providing that offline processing is allowed.

Protein kinases of the microtubule affinity regulating kinase (MARK)/Par-1 family play important roles in the establishment of cellular polarity, cell cycle control, and intracellular signal transduction. Disturbance of their function is linked to cancer and brain diseases, e.g. lissencephaly and Alzheimer disease. To understand the biological role of MARK family kinases, we searched for specific inhibitors and a biosensor for MARK activity. A screen of the ChemBioNet library containing ~18,000 substances yielded several compounds with inhibitory activity in the low micromolar range and capable of inhibiting MARK activity in cultured cells and primary neurons, as judged by MARK-dependent phosphorylation of microtubule-associated proteins and its consequences for microtubule integrity. Four of the compounds share a 9-oxo-9H-acridin-10-yl structure as a basis that will serve as a lead for optimization of inhibition efficiency. To test these inhibitors, we developed a cellular biosensor for MARK activity based on a MARK target sequence attached to the 14-3-3 scaffold protein and linked to enhanced cyan or teal and yellow fluorescent protein as FRET donor and acceptor pairs. Transfection of the teal/yellow fluorescent protein sensor into neurons and imaging by fluorescence lifetime imaging revealed that MARK was particularly active in the axons and growth cones of differentiating neurons.

Cadmium zinc telluride (CZT) is the material of choice for high-energy room-temperature X-ray and γ-ray detectors. However, the performance of pixelated detectors is greatly influenced by the quality of CZT. Crystal defects and impurities are one source of shallow and deep level traps for charge carriers. Fluorescence lifetime of the recombination of optically excited charges may indicate the presence and type of defects and impurities in CZT. Fluorescence lifetime imaging microscopy (FLIM) is used to examine the excited-state lifetime in CZT fabricated by different growth methods and conditions. The FLIM set-up analyzes luminescence emitted from the sample following photo excitation. Samples were optically excited above band gap with a pulsed laser (590 nm) for raster scanning a 220 × 165 µm2 sample area. In-situ room-temperature photoluminescence (PL) and FLIM were recorded simultaneously. In order to analyze the FLIM data, two dominant charge carrier decay processes (τ1, τ2) were identified. The luminescence signal decays with a rapid lifetime of τ1 ≈ 50–200 ps, and a large variety of long-lifetime components τ2 were found in the range of 225–900 ps. CZT grown by multi-tube physical vapor transport (MTPVT) showed extremely long-lived recombination decay times up to 3.5 ns in the vicinity of the interface at growth start. Further away from this interface, the recombination lifetime was in the typical range of fast transitions similar to those found in detector-grade CZT fabricated by travelling heater method. Crystalline material quality strongly influences FLIM lifetime. Time-resolved transients of MTPVT-grown CZT compared with industry-leading detector grade CZT (dots: measured data; lines: fitted exponential decay curves).

Fluorescence Lifetime Imaging (FLIM) in life sciences based on ultrashort laser scanning microscopy and time-correlated single photon counting (TCSPC) started 30 years ago in Jena/East-Germany. One decade later, first two-photon FLIM images of a human finger were taken with a lab microscope based on a tunable femtosecond Ti:sapphire laser. In 2002/2003, first clinical non-invasive two-photon FLIM studies on patients with dermatological disorders were performed using a novel multiphoton tomograph. Current in vivo two-photon FLIM studies on human subjects are based on TCSPC and focus on (i) patients with skin inflammation and skin cancer as well as brain tumors, (ii) cosmetic research on volunteers to evaluate anti-ageing cremes, (iii) pharmaceutical research on volunteers to gain information on in situ pharmacokinetics, and (iv) space medicine to study non-invasively skin modifications on astronauts during long-term space flights. Two-photon FLIM studies on volunteers and patients are performed with multiphoton FLIM tomographs using near infrared femtosecond laser technology that provide rapid non-invasive and label-free intratissue autofluorescence biopsies with picosecond temporal resolution.

Fluorescence lifetime imaging microscopy (FLIM) has been widely used in the field of biological research because of its high specificity, sensitivity, and quantitative ability in the sensing cellular microenvironment. The most commonly used FLIM technology is based on time-correlated single photon counting (TCSPC). Although the TCSPC method has the highest temporal resolution, the data acquisition time is usually long, and the imaging speed is slow. In this work, we proposed a fast FLIM technology for fluorescence lifetime tracking and imaging of single moving particles, named single particle tracking FLIM (SPT-FLIM). We used feedback-controlled addressing scanning and Mosaic FLIM mode imaging to reduce the number of scanned pixels and the data readout time, respectively. Moreover, we developed a compressed sensing analysis algorithm based on alternating descent conditional gradient (ADCG) for low-photon-count data. We applied the ADCG-FLIM algorithm on both simulated and experimental datasets to evaluate its performance. The results showed that ADCG-FLIM could achieve reliable lifetime estimation with high accuracy and precision in the case of a photon count less than 100. By reducing the photon count requirement for each pixel from, typically, 1000 to 100, the acquisition time for a single frame lifetime image could be significantly shortened, and the imaging speed could be improved to a great extent. On this basis, we obtained lifetime trajectories of moving fluorescent beads using the SPT-FLIM technique. Overall, our work offers a powerful tool for fluorescence lifetime tracking and imaging of single moving particles, which will promote the application of TCSPC-FLIM in biological research.

We intend to develop an efficient method of measuring respiratory function of the cornea. With this purpose, we resorted to fluorescence lifetime imaging microscopy (FLIM) to monitor the metabolic co-factor flavin adenine dinucleotide (FAD). FAD and nicotinamide adenine dinucleotide (NADH) are co-factors of the electron transport chain. Therefore alterations in amount of these molecules reflect alterations in the metabolism. For assessing the potential of FLIM for metabolic imaging of the cornea, we performed a series of experiments using a time-correlated single photon counting (TCSPC) fluorescence lifetime microscope (Picoquant to MicroTime 100 coupled to an Olympus BX51 Microscope). In this technique, the acquired signal is the convolution between the instrument response function (IRF) and the fluorescence signal from the sample. IRF was acquired using an Erythrosin B solution. In this work we show that it is possible to acquire fluorescence lifetime images of rat and bovine corneas using FAD autofluorescence.

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.

Fluorescence lifetime imaging microscopy (FLIM) of reduced nicotinamide adenine dinucleotide and reduced nicotinamide adenine dinucleotide phosphate, together termed NAD(P)H, is often used to analyze and characterize the metabolic state of biological samples. The use of photon counting in FLIM of NAD(P)H often leads to long accumulation times which average out sub-second dynamics. This challenge is overcome with the presented two-photon fast FLIM system that uses an analog detection system and fast digitizer, enabling imaging up to 20 frames per second. Furthermore, GPU processing is used for real-time phasor analysis and visualization of collected data.