Optimizing machine learning hyperparameters in two-photon FLIM image analysis

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.

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.

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.

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.

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.

.Two-photon fluorescence lifetime imaging microscopy (FLIM) is widely used to capture autofluorescence signals from cellular components to investigate dynamic physiological changes in live cells and tissues. Among these intrinsic fluorophores, nicotinamide adenine dinucleotide (phosphate) (NAD(P)H) and flavin adenine dinucleotide (FAD)—essential coenzymes in cellular respiration—have been used as intrinsic fluorescent biomarkers for metabolic states in cancer and other pathologies. Traditional FLIM imaging for NAD(P)H, FAD, and in particular fluorescence lifetime redox ratio (FLIRR) requires a sequential multiwavelength excitation to avoid spectral bleed-through (SBT). This sequential imaging complicates image acquisition, may introduce motion artifacts, and reduce temporal resolution. Testing several two-photon excitation wavelengths in combination with optimized emission filters, we have proved a FLIM imaging protocol, allowing simultaneous image acquisition with a single 800-nm wavelength excitation for NADH and FAD with negligible SBT. As a first step, standard NADH and FAD single and mixed solutions were tested that mimic biological sample conditions. After these optimization steps, the assay was applied to two prostate cancer live cell lines: African-American (AA) and Caucasian-American (LNCaP), used in our previous publications. FLIRR result shows that, in cells, the 800-nm two-photon excitation wavelength is suitable for NADH and FAD FLIM imaging with negligible SBT. While NAD(P)H signals are decreased, sufficient photons are present for accurate lifetime fitting and FAD signals are measurably increased at lower laser power, compared with the common 890-nm excitation conditions. This single wavelength excitation allows a simplification of NADH and FAD FLIM imaging data analysis, decreasing the total imaging time. It also avoids motion artifacts and increases temporal resolution. This simplified assay will also make it more suitable to be applied in a clinical setting.

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) 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.

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.

Raman fingerprint spectroscopy and fluorescence lifetime imaging are emerging tools for studying metabolic profiles of biological specimens. While Raman fingerprint spectroscopy detects intrinsic molecular vibrations that reflect the molecular composition and chemical environment of a sample, fluorescence lifetime imaging measures changes in the excited-state lifetime of fluorophores that are sensitive to their microenvironments. Here, we present a multimodal imaging platform combining broadband coherent anti-Stokes Raman scattering (BCARS) and two-photon fluorescence lifetime imaging (2p-FLIM) microscopy that can acquire biologically relevant Raman fingerprint spectra and fluorescence lifetime signals in vivo and simultaneously. The tremendous chemical information obtained from spatially co-registered BCARS and 2p-FLIM images allows us to characterize the subtle differences between sub-cellular compartments and verify the potential false-positive results generated by fluorescence imaging alone. This is demonstrated by directly comparing the BCARS, 2p-FLIM, and two-photon excitation fluorescence (TPEF) signals simultaneously obtained from the same dye-stained organelle in the live, intact C. elegans expressing a green fluorescent protein (GFP) marker. In this work, we introduce the BCARS/2p-FLIM/TPEF setup scheme, the image acquisition steps, data processing, and representative results showing that the cross-modality imaging method enables rigorous characterization and in vivo detection at sub-cellular resolution. This protocol provides a framework for simultaneous chemical and fluorescence lifetime imaging to improve the accuracy of biological interpretation in complex living systems.

Cellular metabolic state can serve as a biomarker to indicate the differentiation potential of stem cells into other specialized cell lineages. In this study, two-photon fluorescence lifetime imaging microscopy (2P-FLIM) was applied to determine the fluorescence lifetime and the amounts of the auto-fluorescent metabolic co-factor reduced nicotinamide adenine dinucleotide (NADH) to elucidate the cellular metabolism of human mesenchymal stem cells (hMSCs) in osteogenic and adipogenic differentiation processes. 2P-FLIM provides the free to protein-bound NADH ratio which can serve as the indicator of cellular metabolic state. We measured NADH fluorescence lifetime at 0, 7, and 14 days after hMSCs were induced for either osteogenesis or adipogenesis. In both cases, the average fluorescence lifetime increased significantly at day 14 (P < 0.001), while the ratio of free to protein-bound NADH ratio decreased significantly in 7- days (P < 0.001) and 14-days (P < 0.001). Thus, our results indicated a higher metabolic rate in both osteogenic and adipogenic differentiation processes when compared with undifferentiated hMSCs. This approach may be further utilized to study proliferation efficiency and differentiation potential of stem cells into other specialized cell lineages.

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

BackgroundNonlinear optical (NLO) microscopy techniques have potential to improve the early detection of epithelial ovarian cancer. In this study we showed that multimodal NLO microscopies, including two-photon excitation fluorescence (TPEF), second-harmonic generation (SHG), third-harmonic generation (THG) and fluorescence lifetime imaging microscopy (FLIM) can detect morphological and metabolic changes associated with ovarian cancer progression.Methodology/Principal FindingsWe obtained strong TPEF + SHG + THG signals from fixed samples stained with Hematoxylin & Eosin (H&E) and robust FLIM signal from fixed unstained samples. Particularly, we imaged 34 ovarian biopsies from different patients (median age, 49 years) including 5 normal ovarian tissue, 18 serous tumors and 11 mucinous tumors with the multimodal NLO platform developed in our laboratory. We have been able to distinguish adenomas, borderline, and adenocarcinomas specimens. Using a complete set of scoring methods we found significant differences in the content, distribution and organization of collagen fibrils in the stroma as well as in the morphology and fluorescence lifetime from epithelial ovarian cells.Conclusions/SignificanceNLO microscopes provide complementary information about tissue microstructure, showing distinctive patterns for serous and mucinous ovarian tumors. The results provide a basis to interpret future NLO images of ovarian tissue and lay the foundation for future in vivo optical evaluation of premature ovarian lesions.

Fluorescence lifetime imaging microscopy (FLIM) techniques are widely used in auto-fluorescence imaging to investigate dynamic metabolic states of the cells. Traditional FLIM imaging requires the cells to adhere to the coverslip to collect enough photons for FLIM data processing. Such conditions pose challenges to non-adherent cells, such as acute myeloid leukemia (AML) cells, because of cell motility. We developed a proto-type micro cell trapping array (MCTA) to immobilize cells in picoliter-size wells with location references. The array keeps non-adherent cells in referenced well locations, allows treatment on stage and re-imaging after time for ultimate cell segmentation analysis. Individual wells are analyzed by a pixel-based region-of-interest (ROI) to analyze cellular redox states. This single well trapping and analysis method allows to isolate treatment responses of a small number of cells, compare their range and predict early effect, which may have clinical applications in the context of cancer aggressiveness and treatment outcomes. We expanded the common intensity-based assay for cellular redox state by a Fluorescence Lifetime measurement, NAD(P)H-a2%, serving as an alternative metric. The new assay is flexible and can be applied to other non-adherent cell lines, expanding FLIM applications in both research and the clinic.