High resolution fluorescence lifetime maps from minimal photon counts

In the present work, we report the imaging of Au nanostars nanoparticles (AuNSt) and their multifunctional applications in biomedical research and theranostics applications. Their optical and spectroscopic properties are considered for the multimodal imaging purpose. The AuNSt are prepared by the seed-meditated method and characterized for use as an agent for bio-imaging. To demonstrate imaging with AuNSt, penetration and localization in different biological models such as cancer cell culture (A549 lung carcinoma cell), 3D tissue model (multicellular tumor spheroid on the base of human oral squamous carcinoma cell, SAS) and murine skin tissue are studied. AuNSt were visualized using fluorescence lifetime imaging (FLIM) at two-photon excitation with a pulse duration 140 fs, repetition rate 80 MHz and 780 nm wavelength femtosecond laser. Strong emission of AuNSt at two-photon excitation in the near infrared range and fluorescence lifetime less than 0.5 ns were observed. It allows using AuNSt as a fluorescent marker at two-photon fluorescence microscopy and lifetime imaging (FLIM). It was shown that AuNSt can be observed inside a thick sample (tissue and its model). This is the first demonstration using AuNSt as an imaging agent for FLIM at two-photon excitation in biosystems. Increased scattering of near-infrared light upon excitation of AuNSt surface plasmon oscillation was also observed and rendered using a possible contrast agent for optical coherence tomography (OCT). AuNSt detection in a biological system using FLIM is compared with OCT on the model of AuNSt penetrating into animal skin. The AuNSt application for multimodal imaging is discussed.

Probing of local molecular environment in cells is of significant value in creating a fundamental understanding of cellular processes and molecular profiles of diseases, as well as studying drug cell interactions. In order to investigate the dynamically changing in subcellular environment during RNA synthesis, we applied two-photon excited fluorescence lifetime imaging microscopy (FLIM) method to monitor the green fluorescent protein (GFP) fused nuclear protein ASF/SF2. The fluorescence lifetime of fluorophore is known to be in inverse correlation with a local refractive index, and thus fluorescence lifetimes of GFP fusions provide real-time information of the molecular environment of ASF/SF2- GFP. The FLIM results showed continuous and significant fluctuations of fluorescence lifetimes of the fluorescent protein fusions in live HeLa cells under physiological conditions. The fluctuations of fluorescence lifetime values indicated the variations of activities of RNA polymerases. Moreover, treatment with pharmacological drugs inhibiting RNA polymerase activities led to irreversible decreases of fluorescence lifetime values. In summary, our study of FLIM imaging of GFP fusion proteins has provided a sensitive and real-time method to investigate RNA synthesis in live cell nuclei.

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.

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.

Cytoplasmic viscosity is a crucial parameter in determining rates of diffusion-limited reactions. Changes in viscosity are associated with several diseases, whilst nuclear viscosity determines gene integrity, regulation and expression. Yet how drugs including DNA-damaging agents affect viscosity is unknown. We demonstrate the use of a platinum complex, Pt[L]Cl, that localizes efficiently mostly in the nucleus as a probe for nuclear viscosity. The phosphorescence lifetime of Pt[L]Cl is sensitive to viscosity and provides an excellent tool to investigate the impact of DNA damage. We show using Fluorescence Lifetime Imaging (FLIM) that the lifetime of both green and red fluorescent proteins (FP) are also sensitive to changes in cellular viscosity and refractive index. However, Pt[L]Cl proved to be a more sensitive viscosity probe, by virtue of microsecond phosphorescence lifetime versus nanosecond fluorescence lifetime of FP, hence greater sensitivity to bimolecular reactions. DNA damage was inflicted by either a two-photon excitation, one-photon excitation microbeam and X-rays. DNA damage of live cells causes significant increase in the lifetime of either Pt[L]Cl (HeLa cells, 12.5–14.1 µs) or intracellularly expressed mCherry (HEK293 cells, 1.54–1.67 ns), but a decrease in fluorescence lifetime of GFP from 2.65 to 2.29 ns (in V15B cells). These values represent a viscosity change from 8.59 to 20.56 cP as well as significant changes in the refractive index (RI), according to independent calibration. Interestingly DNA damage localized to a submicron region following a laser microbeam induction showed a whole cell viscosity change, with those in the nucleus being greater than the cytoplasm. We also found evidence of a by-stander effect, whereby adjacent un-irradiated cells also showed nuclear viscosity change. Finally, an increase in viscosity following DNA damage was also observed in bacterial cells with an over-expressed mNeonGreen FP, evidenced by the change in its lifetime from 2.8 to 2.4 ns.

Fluorescence lifetime imaging microscopy (FLIM) provides valuable quantitative insights into fluorophores' chemical microenvironment. Due to long computation times and the lack of accessible, open-source real-time analysis toolkits, traditional analysis of FLIM data, particularly with the widely used time-correlated single-photon counting (TCSPC) approach, typically occurs after acquisition. As a result, uncertainties about the quality of FLIM data persist even after collection, frequently necessitating the extension of imaging sessions. Unfortunately, prolonged sessions not only risk missing important biological events but also cause photobleaching and photodamage. We present the first open-source program designed for real-time FLIM analysis during specimen scanning to address these challenges. Our approach combines acquisition with real-time computational and visualization capabilities, allowing us to assess FLIM data quality on the fly. Our open-source real-time FLIM viewer, integrated as a Napari plugin, displays phasor analysis and rapid lifetime determination (RLD) results computed from real-time data transmitted by acquisition software such as the open-source Micro-Manager-based OpenScan package. Our method facilitates early identification of FLIM signatures and data quality assessment by providing preliminary analysis during acquisition. This not only speeds up the imaging process, but it is especially useful when imaging sensitive live biological samples.

Utilizing fluorescent life time imaging microscopy technology for identify carriers of BRCA2 mutation

Chapter thirty-four - Fluorescence Lifetime Imaging Microscopy (FLIM): Instrumentation and Applications

Metabolism is a complex phenomenon in the biological systems, falling into one of two general categories: catabolic and anabolic reactions, or the processes of breaking down and building up, respectively. Changes in cell or tissue metabolic states respond to changes in physiological demands or the onset of pathological conditions. Auto-fluorescent endogenous biomolecules associated with metabolism such as coenzymes nicotinamide adenine dinucleotide (NADH) and flavin adenine dinucleotide (FAD) are key players in the production of adenosine triphosphate (ATP)—the energy currency of life. Thus, changes in the abundance of NADH and FAD can serve as noninvasive biomarkers in cells and tissues. There are other auto-fluorescent endogenous molecules, but none as thoroughly investigated as NADH and FAD by advancements in technology such as intensity and lifetime microscopy. The importance of the coenzymes NADH and FAD in metabolism was fully recognized to the scientific world by the great innovator, the late Prof. Britton Chance (1913–2010). He demonstrated cellular NADH auto-fluorescence in 1958 by fluorescence spectrophotometry 1. He developed a fiber optics based fluorimeter to monitor by NADH, the mitochondrial metabolic state of awake rat brains during 1962 2. He has published over 1,500 research articles. Prof. Chance's passion was in translational research and mentoring diverse student populations—regardless of their gender, ethnicity, nationality, religion, or social status—to influence research communities all over the world. Prof. Chance students regarded him as a role model, great mentor, and dear friend. Prof. Chance has tirelessly promoted scientific cooperation and academic exchanges between East and West 3. The ability to make these visible the endogenous biomarkers introduced by Prof. Chance led to their characterization and pathways. During glycolysis, two molecules of ATP are produced in the cytoplasm. Cytoplasmic NADH is transported to mitochondria by malate–aspartate shuttle 4 where, NADH is oxidized to NAD+ producing malate; malate is transported to mitochondrial matrix, mitochondrial NAD+ is reduced to NADH which is in turn is oxidized to NAD+ by the electron transport chain (ETC) in the respiratory chain in the mitochondrial matrix 4, 5. Mitochondrial NADH is produced by citric acid cycle or Kreb's cycle based on several substrates. The energy stored in mitochondrial NADH is transferred to an electrochemical proton gradient that is essential to drive phosphorylation of ADP to ATP by F0F1 ATP synthase, to produce approximately 30 units of ATP, the energy currency of the cell (see Fig. 1 reproduced from the review by Schaefer et al. [in this issue, 34]). The coenzymes FMN/FAD are derived from riboflavin (vitamin B 2) and are covalently bound to flavoproteins in enzymatic reactions of the Kreb's cycle and ETC which takes place in the mitochondria 4. The ETC complex II of succinate dehydrogenase contains bound FAD cofactor which is reduced to FADH2. FADH2 is then oxidized to FAD by Coenzyme Q 4, 5. Only the oxidized form of the flavins, FpOx (FAD or FMN) are fluorescent whereas, the reduced forms Fp (FADH2 or FMNH2), are nonfluorescent. On the other hand, NAD(P)H is fluorescent whereas, the oxidized form NAD(P)+ is nonfluorescent 5, 6. Three out of the 20 amino acids, Tyrosine (Try or Y), Phenylalanine (Phe or F), and Tryptophan (Trp or W), all aromatic, are autofluorescent absorbing and emitting in the UV region. Tryptophan (Trp), an essential amino acid is of particular interest in the biomedical sciences, their fluorescence intensity and lifetime providing information on protein abundance and is dysregulated in cancer 7, 8. Trp is moderately fluorescent with a quantum yield of 0.1–0.5 and has the highest absorption coefficient of the 3 amino acids, and therefore the highest brightness and the longest emission wavelength of 350 nm 8. Alterations in the tryptophan metabolism are suggested to be a common pathological pathway for clinically distinct neurodegenerative disorders, like Parkinson's, Huntington's, and Alzheimer's diseases 9. Modern technologies such as fluorescence lifetime imaging (FLIM) produce endogenous fluorophore data as readouts of mitochondrial function in their natural environment for investigators in the biological and clinical sciences. The unique characteristics of lifetime are that it is sensitive to environmental changes and insensitive to change in fluorophore concentration or excitation intensity. Lifetime helps investigators to fingerprint the molecular functions under various conditions 10. Lifetime techniques have been used for measuring NADH decays since 1967 by spectrofluorometry 11. FLIM provides both spatial and temporal information of molecules and discriminates the bound from free NADH (ref) and NADH from NAD(P)H 12. In this special Cytometry A issue on Metabolism, 10 well-established authors used multiple imaging techniques to explore metabolism in various directions, by: different cell types in 2D and 3D, single cell analyses and their intra- and inter-cell heterogeneities, response to treatment. While imaging the auto-fluorescent NADH and FAD molecules have come a long way to provide insights into the metabolic states of cells and tissue, Schaefer et al. (in this issue, 34) in their very thorough review discuss the “confounding factors” and potential pitfalls—and how to mitigate them. Lukina et al. (in this issue, 47) and Cao et al. (in this issue, 100) discuss additional correlative biomarkers to NADH in second harmonic generation (SHG) of collagen and the putative FRET interaction of Tryptophan (Trp) and NADH, respectively, both in tumors. Lukina et al. (in this issue, 47) investigated the cellular metabolism collagen in colorectal cancer model. Their results demonstrates that the changes in cellular metabolism and fibrotic stroma organization precede morphological alterations and tumor size reduction, and that indicates that NAD(P)H and collagen can be considered as intrinsic indicators of the response to colorectal cancer treatment. Cao et al. (in this issue, 110) presents data in HeLa cervical cancer cells upon treatment with doxorubicin to show the correlations of Trp-NADH energy transfer (E%) of putative Trp residues on enzymes interacting with NADH during NADH to NAD+ transition to a novel, entirely FLIM-based redox ratio – FLIRR 13. In addition, the NADPH/NADH ratio as per Blacker 12 is established suggesting the changing balance between catabolism and anabolism/ROS defense. Cong et al. (in this issue, 80) applied 2-photon FLIM to characterize the critical factors such as physical and chemical microenvironmental signaling in in vitro 3-dimensional tumor models. Ramanujan (in this issue, 101) used a flow cytometry assay to monitor NAD(p)H oxidation kinetics and metabolic status in human breast cancer cells. He also investigated tumor cell heterogeneity in primary tumor epithelial cells isolated from preclinical animal models. Ma et al. (in this issue, 93) sorted out the leukemia cell lines based on their metabolic state differences between proliferation and quiescent cells using the droplet microfluidic technology and phasor FLIM. This approach may be useful to upstream phenotypic platform to correlate with genomic statistics. Chorvatova et al. (in this issue, 13) provided a detailed analysis of endogenous flavins fluorescence photobleaching arising in living cardiac cells during spectrally resolved confocal imaging. This investigation led to significant modifications of the cell responsiveness to metabolic modulators and its contractility, suggesting functional metabolic alterations in the recorded cells. FLIM based AFI imaging is a sensitive way to monitor the cellular metabolism but there are many issues involved in the complex biological system and that may influence the variability in the lifetime values. Chacko and Eliceiri (in this issue, 56) described in this article that it is important to consider the cellular microenvironment such as confluency, pH, inter-/intra-cellular heterogeneity, and choice of cell line for accurate quantitative FLIM based AFI measurement of cellular metabolism. Altarkistany et al. (in this issue, 70) monitored the shift in the fluorescence decay of NAD(P)H and correlate this shift to changes in metabolism that cells undergo during apoptosis using cytometry based FLIM acquisition with phasor analysis. They have demonstrated free to bound ratios as the ratio of “short-to-long” (S/L) fluorescence lifetime, where S/L was found to consistently decrease with an increase in apoptosis. This special issue on metabolism highlights the challenges involved in the investigation using biomarkers but also provides great opportunities to advance research by using the advanced technologies such as FLIM and data analysis at single cells for tissue/in vivo metabolism mapping using biomarkers such as FLIRR, Tryptophan and SHG for changes in collagen. We would like to thank Dr. Attila Tarnok, Editor-in-Chief and Steven Perez, Associate Managing Editor for their valuable guidance and help during this administration of this special issue.

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.

Advanced non-invasive fluorescence spectroscopy and imaging for mapping photo-oxidative degradation in acrylonitrile–butadiene–styrene: A study of model samples and of an object from the 1960s

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

Fluorescence lifetime imaging microscopy (FLIM) of the metabolic co-enzyme nicotinamide adenine dinucleotide (phosphate) [NAD(P)H] is a popular method to monitor single-cell metabolism within unperturbed, living 3D systems. However, FLIM of NAD(P)H has not been performed in a light-sheet geometry, which is advantageous for rapid imaging of cells within live 3D samples. We aim to design, validate, and demonstrate a proof-of-concept light-sheet system for NAD(P)H FLIM. A single-photon avalanche diode camera was integrated into a light-sheet microscope to achieve optical sectioning and limit out-of-focus contributions for NAD(P)H FLIM of single cells. An NAD(P)H light-sheet FLIM system was built and validated with fluorescence lifetime standards and with time-course imaging of metabolic perturbations in pancreas cancer cells with 10s integration times. NAD(P)H light-sheet FLIM in vivo was demonstrated with live neutrophil imaging in a larval zebrafish tail wound also with 10s integration times. Finally, the theoretical and practical imaging speeds for NAD(P)H FLIM were compared across laser scanning and light-sheet geometries, indicating a to acquisition speed advantage for the light sheet compared to the laser scanning geometry. FLIM of NAD(P)H is feasible in a light-sheet geometry and is attractive for 3D live cell imaging applications, such as monitoring immune cell metabolism and migration within an organism.

Understanding of cellular regulatory pathways that involve lipid membranes requires the detailed knowledge of their physical state and structure. However, mapping the viscosity and diffusion in the membranes of complex composition is currently a non-trivial technical challenge. We report fluorescence lifetime spectroscopy and imaging (FLIM) of a meso-substituted BODIPY molecular rotor localised in the leaflet of model membranes of various lipid compositions. We prepare large and giant unilamellar vesicles (LUVs and GUVs) containing phosphatidylcholine (PC) lipids and demonstrate that recording the fluorescence lifetime of the rotor allows us to directly detect the viscosity of the membrane leaflet and to monitor the influence of cholesterol on membrane viscosity in binary and ternary lipid mixtures. In phase-separated 1,2-dioleoyl-sn-glycero-3-phosphocholine-cholesterol-sphingomyelin GUVs we visualise individual liquid ordered (Lo) and liquid disordered (Ld) domains using FLIM and assign specific microscopic viscosities to each domain. Our study showcases the power of FLIM with molecular rotors to image microviscosity of heterogeneous microenvironments in complex biological systems, including membrane-localised lipid rafts.