Endoscopic tissue characterization by frequency-domain fluorescence lifetime imaging (FD-FLIM)

Frequency domain fluorescence lifetime imaging is a powerful technique that enables the observation of subtle changes in the molecular environment of a fluorescent probe. This technique works by measuring the phase delay between the optical emission and excitation of fluorophores as a function of modulation frequency. However, high-resolution measurements are time consuming, as the excitation modulation frequency must be swept, and faster low-resolution measurements at a single frequency are prone to large errors. Here, we present a low cost optical system for applications in real-time confocal lifetime imaging, which measures the phase vs. frequency spectrum without sweeping. Deemed Lifetime Imaging using Frequency-multiplexed Excitation (LIFE), this technique uses a digitally-synthesized radio frequency comb to drive an acousto-optic deflector, operated in a cat's-eye configuration, to produce a single laser excitation beam modulated at multiple beat frequencies. We demonstrate simultaneous fluorescence lifetime measurements at 10 frequencies over a bandwidth of 48 MHz, enabling high speed frequency domain lifetime analysis of single- and multi-component sample mixtures.

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.

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.

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.

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 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.

.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.

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

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

Sensing ion or ligand concentrations, physico-chemical conditions, and molecular dimerization or conformation change is possible by assays involving fluorescent lifetime imaging. The inherent low throughput of imaging impedes rigorous statistical data analysis on large cell numbers. We address this limitation by developing a fluorescence lifetime-measuring flow cytometer for fast fluorescence lifetime quantification in living or fixed cell populations. The instrument combines a time-correlated single photon counting epifluorescent microscope with microfluidics cell-handling system. The associated computer software performs burst integrated fluorescence lifetime analysis to assign fluorescence lifetime, intensity, and burst duration to each passing cell. The maximum safe throughput of the instrument reaches 3,000 particles per minute. Living cells expressing spectroscopic rulers of varying peptide lengths were distinguishable by Förster resonant energy transfer measured by donor fluorescence lifetime. An epidermal growth factor (EGF)-stimulation assay demonstrated the technique's capacity to selectively quantify EGF receptor phosphorylation in cells, which was impossible by measuring sensitized emission on a standard flow cytometer. Dual-color fluorescence lifetime detection and cell-specific chemical environment sensing were exemplified using di-4-ANEPPDHQ, a lipophilic environmentally sensitive dye that exhibits changes in its fluorescence lifetime as a function of membrane lipid order. To our knowledge, this instrument opens new applications in flow cytometry which were unavailable due to technological limitations of previously reported fluorescent lifetime flow cytometers. The presented technique is sensitive to lifetimes of most popular fluorophores in the 0.5–5 ns range including fluorescent proteins and is capable of detecting multi-exponential fluorescence lifetime decays. This instrument vastly enhances the throughput of experiments involving fluorescence lifetime measurements, thereby providing statistically significant quantitative data for analysis of large cell populations. © 2014 International Society for Advancement of Cytometry

Fiber based fluorescence lifetime imaging has shown great potential for intraoperative diagnosis and guidance of surgical procedures. Here we describe a novel method addressing a significant challenge for the practical implementation of this technique, i.e., the real-time display of the quantified biochemical or functional tissue properties superimposed on the interrogated area. Specifically, an aiming beam (450 nm) generated by a continuous-wave laser beam was merged with the pulsed fluorescence excitation light in a single delivery/collection fiber and then imaged and segmented using a color-based algorithm. We demonstrate that this approach enables continuous delineation of the interrogated location and dynamic augmentation of the acquired frames with the corresponding fluorescence decay parameters. The method was evaluated on a fluorescence phantom and fresh tissue samples. Current results demonstrate that 34 frames per second can be achieved for augmenting videos of 640 × 512 pixels resolution. Also we show that the spatial resolution of the fluorescence lifetime map depends on the tissue optical properties, the scanning speed, and the frame rate. The dice similarity coefficient between the fluorescence phantom and the reconstructed maps was estimated to be as high as 93%. The reported method could become a valuable tool for augmenting the surgeon's field of view with diagnostic information derived from the analysis of fluorescence lifetime data in real-time using handheld, automated, or endoscopic scanning systems. Current method provides also a means for maintaining the tissue light exposure within safety limits. This study provides a framework for using an aiming beam with other point spectroscopy applications.

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.

Fluorescence imaging is being widely explored for intraoperative surgical guidance of head and neck cancers, using exogenous and endogenous contrast. However poor tumor contrast due to non-specific probe accumulation of tnon-targetted as well as receptor targeted probes is a major drawback. Here we show using preclinical and clinical studies that fluorescence lifetime (FLT) imaging can significantly improve the sensitivity and specificity for tumor detection using both indocyanine green (ICG) epidermal growth factor receptor (EGFR) targeted near infrared probes. We also show that FLTs in tissue are highly correlated with receptor expression levels, thereby enabling quantification of receptor quantification in vivo. Ongoing efforts in our group towards the translation of FLT imaging for intraoperative image guidance during surgeries of head and neck lesions, including oral and skin cancer will also be discussed.