Digitally synthesized beat frequency-multiplexed fluorescence lifetime spectroscopy.

Tissue characterization by endoscopic fluorescence imaging of endogenous or exogenous fluorochromes is a promising method for early cancer detection. However, the steady-state fluorescence contrast between healthy tissue and lesions such as early-stage carcinomas is generally poor. The authors propose to improve this contrast by using the additional information contained in the fluorescence lifetime (FLT). The FLT of several fluorochromes is sensitive to their physico-chemical environment.The FLT can be measured by frequency-domain methods. The excitation light from a continuous wave (CW) laser is modulated in amplitude at radio-frequencies by an electro-optic modulator, and delivered to the tissue via an optical fibre. The endoscopie site is imaged by an endoscope on to an optical device. The gain of the fluorescence image detector is also modulated at the same frequency for homodyning. The tissue fluorescence image is recorded at several phases between the excitation and the detection modulations during an acquisition cycle. With these images, an image processor calculates the apparent FLT for each pixel and constructs a lifetime image of the endoscopie site. This process is performed at quasi-video frequencies.The influence of various physical parameters (modulation frequency, number of images by cycle, shot noise, tissue optical properties etc.) has been investigated by analytical analysis, simulation methods and experimentation.Preliminary results obtained on human tissues are also presented to illustrate the potentiality of the method.

This work introduces a dual-excitation, multispectral-detection, frequency-domain fluorescence lifetime imaging (FLIM) handheld endoscopy system for comprehensive autofluorescence imaging of the oral mucosa. The excitation wavelengths and spectral detection channels were selected to preferentially image the fluorescence of endogenous fluorophores relevant for oral epithelial cancer detection. The endoscope dimensions and optical specifications enable direct imaging of oral lesions with minimal patient discomfort. Comprehensive imaging experiments using standard fluorophores demonstrate the capabilities of the FLIM endoscope to measure a wide range of fluorescence lifetimes (0.1-12 ns) with high precision (~100 ps). Clinical in vivo label-free metabolic and biochemical endoscopic FLIM imaging was demonstrated by imaging a malignant oral lesion and healthy mucosa in a patient undergoing a tissue biopsy procedure.

This work introduces a dual-excitation, multispectral-detection, frequency-domain fluorescence lifetime imaging (FLIM) handheld endoscopy system for comprehensive autofluorescence imaging of the oral mucosa. The excitation wavelengths and spectral detection channels were selected to preferentially image the fluorescence of endogenous fluorophores relevant for oral epithelial cancer detection. The endoscope dimensions and optical specifications enable direct imaging of oral lesions with minimal patient discomfort. Comprehensive imaging experiments using standard fluorophores demonstrate the capabilities of the FLIM endoscope to measure a wide range of fluorescence lifetimes (0.1-12 ns) with high precision (∼100 ps). Clinical in vivo label-free metabolic and biochemical endoscopic FLIM imaging was demonstrated by imaging a malignant oral lesion and healthy mucosa in a patient undergoing a tissue biopsy procedure.

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.

.Significance: Detailed biochemical and morphological imaging of the plaque burdened coronary arteries holds the promise of improved understanding of atherosclerosis plaque development, ultimately leading to better diagnostics and therapies.Aim: Development of a dual-modality intravascular catheter supporting swept-source optical coherence tomography (OCT) and frequency-domain fluorescence lifetime imaging (FD-FLIM) of endogenous fluorophores with UV excitation.Approach: We instituted a refined approach to endoscope development that combines simulation in a commercial ray tracing program, fabrication, and a measurement method for optimizing ball-lens performance. With this approach, we designed and developed a dual-modality catheter endoscope based on a double-clad fiber supporting OCT through the core and fluorescence collection through the first cladding. We varied the relative percent of UV excitation launched into the core and first cladding to explore the potential resolution improvement for FD-FLIM. The developed catheter endoscope was optically characterized, including measurement of spatial resolution and fluorescent lifetimes of standard fluorophores. Finally, the system was demonstrated on fresh ex vivo human coronary arteries.Results: The developed endoscope was shown to have optical performance similar to predictions derived from the simulation approach. The FLIM resolution can be improved by over a factor of 4 by primarily illuminating through the core rather than the first cladding. However, time-dependent solarization losses need to be considered when choosing the relative percentage. We ultimately chose to illuminate with 7% of the power transmitting through the core. The resulting catheter endoscope had lateral resolution for OCT and lateral resolution for FD-FLIM. Images of ex vivo coronary arteries are consistent with expectations based on histopathology.Conclusions: The results demonstrate that our approach for endoscope simulation produces reliable predictions of endoscope performance. Simulation results guided our development of a multimodal OCT/FD-FLIM catheter imaging system for investigating atherosclerosis in coronary arteries.

We report on the design and characterization of a multipurpose 64 × 32 CMOS single-photon avalanche diode (SPAD) array. The chip is fabricated in a high-voltage 0.35-μm CMOS technology and consists of 2048 pixels, each combining a very low noise (100 cps at 5-V excess bias) 30-μm SPAD, a prompt avalanche sensing circuit, and digital processing electronics. The array not only delivers two-dimensional intensity information through photon counting in either free-running (down to 10-μs integration time) or time-gated mode, but can also perform smart light demodulation with in-pixel background suppression. The latter feature enables phase-resolved imaging for extracting either three-dimensional depth-resolved images or decay lifetime maps, by measuring the phase shift between a modulated excitation light and the reflected photons. Pixel-level memories enable fully parallel processing and global-shutter readout, preventing motion artifacts (e.g., skew, wobble, motion blur) and partial exposure effects. The array is able to acquire very fast optical events at high frame-rate (up to 100 000 fps) and at single-photon level. Low-noise SPADs ensure high dynamic range (up to 110 dB at 100 fps) with peak photon detection efficiency of almost 50% at 410 nm. The SPAD imager provides different operating modes, thus, enabling both time-domain applications, like fluorescence lifetime imaging (FLIM) and fluorescence correlation spectroscopy, as well as frequency-domain FLIM and lock-in 3-D ranging for automotive vision and lidar.

A phase-modulation fluorometer was used for on-line detection of benzo(k)fluoranthene and benzo(b)fluoranthene in a mixture of the two that was isocratically eluted from a high-performance liquid chromatographic column. Three different mobile-phase solvent compositions provided three degrees of chromatographic separation, ranging from complete to almost no separation. Fluorescence lifetimes were calculated from phase and modulation data that were collected at three different excitation modulation frequencies. Heterogeneity analysis was then used to calculate the fluorescence lifetimes and fractional intensity contributions of each component at each point along the chromatograms. The fractional intensity contributions were multiplied by the fluorescence intensities in the steady-state chromatogram of the mixture in order to construct the fluorescence intensity chromatograms of the individual components. Retention times and fluorescence lifetimes of the components obtained from the constructed chromatograms were in good agreement with values obtained from steady-state chromatograms of the individual components. Relative errors of the peak intensities were as high as 23%. Batch experiments were also performed to determine the best results that could be expected for the heterogeneity analysis of mixtures of the two components in the solvent systems that were used in the chromatographic experiments.

Rationale Lung-resident memory T cells (TRM) play a critical role in protecting against respiratory infection. Maintained in a quiescent state, TRM are poised for rapid effector function upon reactivation with cognate antigen. Kronoscan is a clinic-ready, fibre-based, optical imaging system developed by the University of Edinburgh, capable of measuring both fluorescence intensity and lifetime. The aim of this study is to develop a staining protocol to enable the detection of human and non-human primate (NHP) pulmonary TRM populations in situ. Methods Human lungs (deemed non-suitable for transplantation) following ex vivo lung perfusion (EVLP) and NHP lungs (cynomolgus macaque obtained from Charles River Laboratories) were enzymatically digested to achieve a single cell suspension. Cells were stained with cross-reactive antibodies prior to flow cytometric analysis. Fluorescence intensity and lifetime imaging was also performed using Kronoscan on MACs-enriched, antibody-stained, pulmonary T cells. Results Human and NHP TRM were primarily identified using CD69 and CD103 and co-expressed a number of chemokine receptors (CXCR3, CCR10), adhesion molecules (CD49a, CD49d) and the immune checkpoint inhibitor PD-1. Differences in phenotype were observed between CD4+ and CD8+ TRM. TRM could be identified via fluorescence intensity and lifetime imaging using Kronoscan. Conclusions A cross-reactive antibody panel capable of detecting human and NHP pulmonary TRM was successfully developed. Fibre-based fluorescence lifetime imaging may provide a valuable tool for assessing pulmonary TRM populations in situ, reflecting the level of immunity following infection and vaccination.

FLIO is based on Fluorescence Lifetime Imaging Microscopy (FLIM), which is used in basic science to investigate metabolic conditions in cell cultures and tissues. After excitation of fluorophores with a laser light, the fluorophore returns to its ground state by releasing light with a longer wavelength. The time that is needed for the fluorophore to return from the excited state to the ground state can be measured as the fluorescence lifetime in specific emission wavelengths. As fluorescence lifetimes are dependent on pH, ion concentration, and molecular binding or proximity to other energy acceptors, FLIO allows for identification and specification of different metabolic conditions within the tissue.

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.

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.

Chapter Three - Applications of Fluorescence Lifetime Spectroscopy and Imaging to Lipid Domains In Vivo

Fluorescence imaging using near-infrared (NIR) fluorescent contrast agents is increasingly being investigated as intraoperative tool to visualize, in real-time, tissues of interest such as tumors, lymph nodes or nerve bundles. Generally, spectral imaging systems are used that measure the intensity of fluorescent signals. However, to aid in a more specific detection of these fluorescent signals, fluorescence lifetime can be added to the image. The lifetime is independent of the intensity and in addition, multiple tracers emitting around the same wavelengths can still be distinguished based on their difference in lifetime. Imaging lifetimes, however, requires a much more advanced imaging system. None of the currently approved fluorescence guidance systems support fluorescence lifetime and today’s available lifetime imaging technology (TCSPC, ICCD) does not allow imaging the sub-nanosecond lifetimes of NIR dyes with the efficiency needed to reach video frame rates. In this paper, we present a 32×32-pixel proof-of-concept camera based on our novel CAPS-pixel based gated image sensor. This camera is specifically targeted at imaging fluorescence lifetimes at NIR wavelengths with high efficiency for the use in fluorescence-guided surgery and is a first step towards a full camera with video resolution at video frame rates. We describe the camera system and how it is used to image fluorescence lifetimes. Next, we show the lifetime imaging capability by imaging the lifetimes of two different nanosecond visible dyes (fluorescein and acridine orange) in cuvette and two more challenging (sub-)nanosecond NIR dyes (ICG and IRDye800CW). Lastly, we validate the camera by imaging NIR fluorescence phantoms in a mouse.