This PDF file contains the front matter associated with SPIE Proceedings Volume 12384, including the Title Page, Copyright information, Table of Contents, and Conference Committee information.

We report progress on the development of multiphoton excitation methods for the Mesolens, with the aim of increasing the field of view of multiphoton imaging without stitching and tiling or stochastic sampling methods. We give details on our method of upscaling widefield two-photon microscopy to the mesoscale, where we have imaged specimens exceeding 2.5 mm x 2.5 mm in size at a frame speed of 0.2 Hz using a Ti:Sapphire laser for fluorescence excitation and a sensor-shifting camera for fluorescence detection and imaging. We give details on the opportunities and limitation of this technology for multiphoton imaging at the mesoscale, and we conclude with details on our progress to develop a new mesoscale imaging technology that is chromatically corrected from 400-1080nm for both single-photon and multiphoton excitation mesoscopy.

Solar ultraviolet longwave UVA1 exposure of human skin has short-term consequences at cellular and molecular level, leading at long-term to photoaging. Following exposure, reactive oxygen species (ROS) are generated, inducing oxidative stress that might impair cellular metabolic activity. However, the dynamic of UVA1 impact on cellular metabolism remains unknown because of lacking adequate live imaging techniques. Here we assess overtime the UVA1- induced metabolic stress response in reconstructed human skin with multicolor two-photon fluorescence lifetime microscopy (FLIM). Simultaneous imaging of the two endogenous biomarkers nicotinamide adenine dinucleotide (NAD(P)H) and flavin adenine dinucleotide (FAD) by wavelength mixing allows quantifying cellular metabolism in function of NAD(P)⁺/NAD(P)H and FAD/FADH₂ redox ratios We measure NAD(P)H and FAD fluorescence lifetime and fraction of bound coenzymes both in keratinocytes in the epidermis basal layer and in fibroblasts in the dermis superficial layer. After UVA1 exposure, we observe an increase of fraction of bound NAD(P)H and decrease of fraction of bound FAD indicating a metabolic switch from glycolysis to OXPHOS or oxidative stress possibly correlated to ROS generation. NAD(P)H and FAD biomarkers have unique temporal dynamics and sensitivities to skin cell types and UVA1 dose. While FAD biomarker is UVA1 dose-dependent in keratinocytes, NAD(P)H biomarker shows earlier time points modulation in fibroblasts, thus reflecting different skin cells sensitivities to oxidative stress. Finally, we show that a sunscreen including a UVA1 filter MCE prevents UVA1 metabolic stress response from occurring.

Characterizing melanins in situ and determining their 3D z-epidermal distribution is paramount for understanding physiological /pathological processes of melanin neosynthesis, transfer, degradation or modulation with external UV exposure or cosmetic / pharmaceutical products.

Multiphoton fluorescence intensity- and lifetime-based approaches have been shown to afford melanin detection, but how can one quantify melanin in vivo in 3D from multiphoton fluorescence lifetime (FLIM) data, especially since FLIM imaging requires long image acquisition times not compatible with 3D imaging in a clinical setup? We propose an approach combining i) multiphoton FLIM, ii) fast image acquisition times, and iii) a melanin detection method, called pseudo-FLIM, based on slope analysis of autofluorescence intensity decays from temporally binned data.

By comparison to FLIM bi-exponential and phasor analyses of synthetic melanin, melanocytes / keratinocytes coculture and in vivo human skin, we demonstrate that the fast decay shapes of melanin correspond to high slope values, a characteristic one can use to identify melanin in vitro and in vivo. We show that pseudo-FLIM melanin detection is still feasible upon decreasing the image acquisition time, which opens the possibility to quantify melanin from 3D z-stacks. Using parameters of global 3D epidermal melanin density and z-epidermal distribution profile, we provide first insights into the in vivo knowledge of 3D melanin modulations with constitutive pigmentation versus ethnicity, with seasonality over one year and with topical application of retinoic acid or retinol. Applications of pseudo-FLIM-based melanin detection encompass physiological, pathological, or environmental factors-induced pigmentation modulations up to whitening, antiphotoaging, or photoprotection products evaluation.

We applied femtosecond laser multiphoton tomographs, including the novel system MPTcompact with its chiller-free fiber laser positioned inside the measurement head, for multimodal imaging of in vivo human skin of volunteers, that had been exposed to topically applied sunscreens, UV stains, care cosmetics, and pharmaceuticals. Signals are based on two-photon excited and time-resolved fluorescence (FLIM), Second Harmonic Generation (SHG), and confocal reflection. In particular, SHG signals have been detected from silver nanoparticles in mineral sunscreen. Of particular interest was the distribution of the exogenous compounds within the upper epidermis such as the stratum corneum as an efficient skin barrier. The lateral resolution was 300 nm and the time resolution was 250 picoseconds. In addition, the autofluorescence based on NADH and keratin detection was imaged.

Using Becker & Hickl TCSPC FLIM systems with fast TCSPC modules and fast detectors, we found ultra-fast fluorescence decay components in biological material, such as mushroom spores, pollen grains, plant tissue, and malignant melanoma. The component decay times are on the order of 10 to 50 ps, the amplitudes range from about 60% to more than 99%. In general, the shortest lifetimes and the largest amplitudes of the fast component were found in strongly coloured material, such as black mushroom spores and melanoma tissue. However, the lifetime not always correlates with the colour. In particular, a fast decay component was not found in tissue from basal cell papilloma, although it is dark brown. This may open a way to identify melanoma tissue and melanoma cells, and thus provide a new tool to investigate melanoma progression.

The human neurotensin receptor one (NTSR1) is a G protein-coupled receptor. The receptor is activated by a small peptide ligand neurotensin. NTSR1 can be expressed in HEK cells by stable transfection. Previously we used the fluorescent protein markers mRuby3 or mNeonGreen fused to NTSR1 for EMCCD-based Structured Illumination Microscopy (SIM) in living HEK cells. Ligand binding induced conformational changes in NTSR1 which triggered the intracellular signaling processes. Recent single-molecule studies revealed a dynamic monomer/dimer equilibrium of this receptor in artificial lipid bilayers. Here we report on the oligomerization state of human NTSR1 from living cells by trapping them into lipid nanodiscs. Briefly, SMALPs (styrene-maleic acid copolymer lipid nanoparticles) were produced directly from the plasma membranes of living HEK293T FlpIn cells. SMALPs with a diameter of 15 nm were soluble and stable. NTSR1 in SMALPs were analyzed by single-molecule intensity measurements one membrane patch at a time using a custom-built confocal anti-Brownian electrokinetic trap (ABEL trap) microscope. We found oligomerization changes before and after stimulation of the receptor with its ligand neurotensin.

Multiplexed fluorescence detection has become an indispensable tool in modern biosensing and imaging. Although a variety of excitation/detection optics designs and unmixing schemes have been proposed to achieve multiplexed detection, successful differentiation and quantification of multiple fluorophores at each imaging pixel is still challenging. Recently, fluorescence lifetime imaging microscopy (FLIM) in combination with the phasor plot analysis has shown many advantages over other multiplexed detection methods. Being an intrinsic property of a fluorescent molecule, fluorescence lifetime measured by FLIM is not biased by excitation power or probe abundance and can reveal information on the probe’s microenvironment (ions, pH, oxygen content, electrical signals, index of refractions, etc.). In addition, FLIM is one of the most robust ways of quantifying FRET for studying protein-protein interactions. Moreover, by combining lifetime information with a spectral reading at each pixel, spectral FLIM adds two more dimensions to the spatiotemporal information collected by a conventional confocal setup, resulting in a 6-dimenional (x, y, z, λ, τ, t) dataset. The major challenges to the spectral FLIM method lie in the data acquisition speed and the complexity in post processing and analysis. Here, we present a new time-resolved spectral detector with parallel 16-channel digital frequency domain FLIM (FastFLIM) readouts, for fast spectral FLIM data acquisition. The 16-channel FastFLIM can produce unbiased spectral FLIM data for phasor analysis that unambiguously discriminate and quantitate fluorescent species with unique spectral and lifetime features at each image pixel. Our spectral FastFLIM method offers new opportunities for monitoring multiple dynamic signaling events in live specimens, providing insights into complex biological systems.

SwissSPAD3 is the latest of a family of widefield time-gated SPAD imagers developed for fluorescence lifetime imaging (FLI) applications. Its distinctive features are (i) the ability to define shorter gates than its predecessors (width W < 1 ns), (ii) support for laser repetition rates up to at least 80 MHz and (iii) a dual-gate architecture providing an effective duty cycle of 100%. We present widefield macroscopic FLI measurements of short lifetime NIR dyes, analyzed using the phasor approach. The results are compared with those previously obtained with SwissSPAD2 and to theoretical predictions.

Molecular imaging tools that can image plant metabolism and effects of external agricultural treatments in the micro-environment of plant tissues are significant for further understanding plant biology and optimizing the formulation of new agricultural products. Mass spectrometry, a common tool used by plant biologists, is unable to resolve nano-crystalline active ingredients (AIs) on the leaf surface nor achieve 3D molecular imaging of living plants. To address that, multiphoton microscopy (MPM) and fluorescence lifetime imaging microscopy (FLIM) are combined to achieve sub-cellular, depth-resolved fluorescence lifetime of both AIs and intrinsic proteins/pigments (e.g., chlorophyll and/or cytosolic NADH) after the herbicide treatment application. Here we present a method using a custom-designed, high-speed MPM-FLIM system, “Instant FLIM”, to achieve real-time, unlabeled 3D functional molecular imaging of intrinsic proteins and pigments in optically thick and highly scattering plant samples with the application of external treatments. To validate the capability of MPM-FLIM to measure intrinsic proteins and pigments within plant tissues, we present the results of unlabeled bluegrass blades samples. To demonstrate simultaneous imaging of 3D molecular plant tissue and the agricultural AI nano-crystals deposition and formation, we evaluate the performance of the MPM-FLIM by applying commercial herbicide product to gamagrass blade sample. Additionally, to measure the herbicide-induced cellular-level functional responses within living plant tissues, 3D time-resolved molecular MPM-FLIM imaging of hemp dogbane leaf with herbicide is performed. Results demonstrate MPM-FLIM is capable of 3D simultaneous functional imaging of label-free living plant tissues and the quantitative measurements of the location and formation of AI nanocrystals within the plant tissues.

The ability to simultaneously detect and control neuronal activity grants the capacity to unravel the causal relationships behind brain processing. Nevertheless, scaling-up this technology to the whole encephalon of a vertebrate is a challenging task, and this impedes reaching a global comprehension of how the brain works. To this aim, we developed an optical system that enables fast noninvasive functional imaging of the whole brain of the zebrafish larva and, at the same time, allows us to optogenetically stimulate the activity of arbitrary sets of neurons in the volume. Our preliminary results show that with this optical system we can both consistently evoke activation of neurons in the stimulation site and identify distantly-located functionally-connected neurons placed downstream in the activated circuits. The expansibility of this concept paves the way for the brain-wide mapping of functional connectivity in the zebrafish larva.

We applied a multiphoton tomograph for multimodal multiphoton imaging of extracted human teeth based on two-photon excited fluorescence, phosphorescence, and second harmonic generation. Fine details of the dentin and enamel could be revealed at and below the surface. Spectral and time decay analysis indicated the presence of a signal in the red spectral region, which could originate from the caries infestation. Fluorescence lifetime imaging showed clearly mono- and bi-exponential decay characteristics for parts of the enamel and dentin. Furthermore, weak phosphorescence with a decay time of approximately 90 µs could be recorded from the dentin of the root section. The fact that whole teeth could be directly used for multiphoton tomography without any histological treatment or preparation and, nevertheless, provided a high information content confirms the huge potential for the study of physiological and pathological conditions in dentistry and for femtosecond laser-guided treatment by multiphoton ablation.

Recent studies have shown that common anticancer treatments can induce cell senescence rather than death, a critical phenotype governing tumor recurrence. This calls for the urgent development of safe, precise, and quick tools to unveil critical Therapy-Induced Senescence (TIS). Merging different coherent Raman and multiphoton techniques, we present label-free multimodal nonlinear optical (NLO) microscopy as a powerful tool to spot early TIS. We home-built a microscope including different NLO modalities: Stimulated Raman Scattering (SRS), forward and epi-detected Coherent Anti-Stokes Raman Scattering (CARS and E-CARS), and Two-Photon Excited Fluorescence (TPEF). The infrared laser source outputs synchronized narrowband 780 nm pump pulses and 950-1050 nm tunable Stokes pulses, so to match the CH-stretching region of the Raman spectrum. Thanks to the co-registration of these diverse techniques applied on label-free TIS cells and controls, we exposed quantitative hallmarks of early TIS, confirmed by comparing different optical signals monitored over 72 hours of treatment. TPEF from metabolic coenzymes combined with E-CARS from cardiolipin and cytochrome C indicated an early shrinking of mitochondria. CARS and SRS revealed lipid vesicles overproduction and accumulation. Nuclei enlarged irregularly, visualized via subtraction of SRS signals of proteins and lipids, and CARS from deoxyribose. We consider our results will strongly influence anticancer pre-clinical studies and translated clinical applications, helping to identify quickly, non-invasively, and quantitatively TIS in human tumors.

Coherent excitonic coupling in a dimer of a GFP-analogue, Venus-A206, was discovered previously by using circular dichroism and a combination of photon antibunching and FCS. To further elucidate this effect, here we use a new approach based on the measurements of the two-photon polarization ratio (Omega) as a function of excitation wavelength. In a Venus tandem dimer we observe significant changes of Omega across the spectrum, compared to a negative control with only one chromophore. Our model suggests that only a fraction of dimers are coupled excitonically. Their constituent dipoles make an angle of ~40 degree in an oblique arrangement.

Two decades ago, the very first prototype of a multiphoton tomograph was built and delivered as a high-resolution skin imaging device to the cosmetic company Beiersdorf AG on November 2, 2001. The tomograph was based on a tunable 80 MHz NIR femtosecond laser system and able to detect two-photon excited autofluorescence intensity and fluorescence lifetime based on time-correlated single photon counting (TCSPC) as well as second harmonic generation (SHG) with 300 nm lateral resolution and 250 ps temporal resolution. Two-photon fluorescence lifetime imaging (FLIM) was performed based in a biexponential decay and false-color coding. The first clinical in vivo study on melanocytic lesions with the DermaInspect started at the Department of Dermatology of the University Jena on September 5, 2003. The study was performed on 53 patients and resulted in optical biopsies based on 2108 optical sections. The tomograph worked under clinical conditions without any problem. Finally, the CE0118 certificate for the multiphoton tomograph DermaInspect as medical device was granted by the European Certified Body Thuringian State Authority of Metrology and Verification (LMET) on December 22, 2004. The DermaInspect became the world’s first femtosecond laser medical device for clinical imaging. The CE0118 certificate for the 2nd generation of multiphoton tomographs, the portable medical device with a tunable titanium:sapphire laser, an optical arm and a 360° measurement head mounted on a mechanical arm, the “MPTflex”, was granted on Nov 8, 2010. The third generation, the multimodal multiphoton tomograph MPTcompact, is based on a chiller-free ultracompact femtosecond fiber laser and miniaturized detectors to measure autofluorescence, SHG, FLIM, and confocal reflectance. The first two MPTcompact prototypes were delivered to hospitals in Heidelberg and Gera in 2018 to realize a multicenter clinical study on the detection of malignant melanoma. JenLab’s multiphoton tomographs have been used to realize the word’s first clinical in vivo two-photon imaging of patients, including (i) the first two-photon fluorescence lifetime imaging, (ii) the first microendoscopy, the first (iii) diseased skin imaging, (iv) brain imaging, and (v) the first CARS studies.

Here, we present a new handheld multiphoton endomicroscopic system designed for tumor diagnosis in the head and neck region. It consists of an approximate 25-cm-long rigid endomicroscopic probe with two variants (0° and 45° bended tip), connected to a handheld scan-head. The system can achieve a field of view ⪆600 µm for Coherent Anti-stokes Raman Scattering (CARS) and other nonlinear imaging modalities by a non-de-scanned detection and using a de-scanned confocal imaging channel to detect light from tissue labeled with Indocyanine Green (ICG). Furthermore, high-power femtosecond laser pulses can be transmitted through the system for precise tissue ablation which was considered in the optical design of the probe.

We report on a simultaneous 2-photon and 3-photon signal acquisition method for label-free multiphoton microscopy. With dual excitation wavelengths of 1580 nm and 790 nm and a set of filter designs, multiple contrasts including second harmonic generation, third harmonic generation, and two-photon-excitation-fluorescence images are detected and separated. The spectrum of the nonlinear signals is measured and verified by a spectrometer. Depth-resolved multimodal images are demonstrated on a silicon photonic chip and leaf samples. The simultaneous 2- and 3-photon microscopy is shown to have great potential for label-free in-vivo imaging.

Articular cartilage in the tibiofemoral joint contains unique tissue microstructures that serve specific functions, including reduction of friction and distributing the dynamic and static cyclic loading at the ends of diarthrodial joints. A proficient understanding of these microstructures can lead to significant clinical advances in diagnosing orthopedic diseases such as osteoarthritis and improving cartilage repairs. The surface of tibiofemoral condyles can be roughly separated into loadbearing and meniscus-covered areas. Due to the difference in mechanical loading between the two regions, we hypothesize that their microstructures differ. To test this hypothesis, we used cartilage punches harvested from the tibial condyle of porcine knee joints as an example tissue and a custom nonlinear optical microscope for performing a dye-free imaging study. The custom nonlinear optical microscope could simultaneously acquire Two-Photon excitation Auto-Fluorescence (TPAF) and Second Harmonic Generation (SHG) images. Through the TPAF channel, elastin fibers are visible along with chondrocytes. The SHG channel was utilized for observing the vast collagen network and its evident orientation throughout the tibial condyle. Images were analyzed by ImageJ to reveal alignment angles of the collagen network and elastin fibers. The load-bearing region exhibits a denser uniform collagen network with minimum elastin fibers. In contrast, the meniscuscovered areas have a distinctive collagen orientation with a greater magnitude of co-localized elastin fibers. The biological differences are likely derived from their different biomechanical environments in the tibiofemoral joint.

We present spectrally-resolved multi-photon induced fluorescence (TPIF) and second harmonic generation (SHG) imaging of porcine arterial wall tissue sections, both native tissues and damaged tissues subject to a proprietary therapy based on photo-activated crosslinking of collagen and other structural proteins in the arterial wall, a proposed treatment for peripheral artery disease termed non-invasive vascular scaffolding (NVS). We employ a spectrally-resolved multi-photon imaging system, based on a closed loop piezoelectric stage, a transmission grating and an EMCCD. From the spectrally-resolved multiphoton emission, we form ratiometric images of select spectral bands associated primarily with collagen (SHG) and elastin (TPIF). The ratiometric images aid in identifying representative regions for comparing the native and treated tissue sections, as well as reducing sensitivity to variations in intensity caused by scattering or attenuation of the excitation beam. Our aim is to use these ratios as a metric of the tissue structure and composition, indicating the relative contribution of collagen and elastin to the observed nonlinear signals. We note the photochemical modification results in a recovery of SHG intensity similar to the native artery, with the hypothesis that cross-linking of the compressed collagen fibrils in the arterial wall during the light activation step results in the formation of the NVS.

Chronic kidney disease (CKD) is one of the most prevalent kidney disorders with no distinct symptoms and gradual loss of kidney function over time. The underlying mechanism in the pathogenesis of CKD with various causes such as high blood pressure, diabetes, high cholesterol, and kidney infection is not well understood. Following up on the pathophysiological alterations, longitudinal in vivo cellular-level imaging during CKD progression can provide unique insights and valuable information for diagnosing and managing CKD. In this study, with two-photon kidney intravital imaging, we achieved a repetitive longitudinal visualization of the kidney in an adenine diet-induced CKD mouse model over 30 days. Interestingly, based on a second-harmonics generation signal, we clearly visualized and quantified the formation of 2,8-dihydroxyadenine (DHA) crystals over time in a label-free manner. The imaging results were well correlated with CKD progression monitored by a standard blood test of blood urea nitrogen (BUN) and cystatin C levels. This result suggests the potential of the label-free second-harmonics generation crystal imaging as a novel optical technique for in vivo CKD progression monitoring.

Chronic inflammation is one of the most prominent features of Alzheimer's disease (AD). Little is known about how brain hemodynamics and oxygenation are affected by AD-related inflammation. Here, we use two-photon phosphorescence lifetime imaging with an oxygen-sensitive dye “Oxyphor 2P” to measure the partial pressure of oxygen (pO2) before and during endotoxin-induced neuroinflammation in cortical vessels of a mouse model of AD. Capillary red blood cell flux (RBC flux) was measured through two-photon phosphorescence intensity microscopy. To induce chronic inflammation, we injected lipopolysaccharide (LPS) intraperitoneally, daily for two weeks, in female APPswe: PS1dE9 mice and age-matched wild-type (WT) controls. Intravascular pO2 and RBC flux were measured in the somatosensory cortex before the LPS injection, on week 1 (day 7), and week 2 (day 15) during the LPS injection. Our results demonstrate that LPS-induced systemic inflammation leads to significant decreases in cortical intravascular pO2 while showing a negligible effect on capillary RBC flux. Moreover, AD mice are more susceptible to inflammation with more pronounced cortical pO2 reduction in comparison to WT mice. Our findings suggest that inflammation plays a key role in AD-related disruptions of cerebral tissue metabolism of oxygen.

Multiphoton microscopy is an established technique for deep in vivo neuroimaging. However, the volume of the adult brain in most model organisms (e.g. mouse) is beyond the reach of high resolution optical imaging techniques, limiting the study of neurobiological questions in adulthood. Danionella, a genus closely related to zebrafish, which have smaller brains and lack ossification above the brain in adulthood have been introduced for in vivo studying of brain circuits in adult stages. Here, we present a quantitative comparison of optical accessibility within the Danionella dracula brain with two- and three-photon microscopy.

We demonstrate that the entire brain of this animal is optically accessible in adulthood by imaging fluorescently-labeled vasculature throughout the deepest part of the brain. While both two- and three-photon microscopy can penetrate through the entire brain, images obtained by three-photon microscopy maintain higher contrast and optical sectioning in deeper regions. To determine the source of low signal to background ratio in the deep images we characterized the distribution of blood vessels to find the staining density in the brain and compared our findings to theoretical expectations of signal to background ratio as a function of depth in multiphoton microscopy. We found that low quality of images in not just due to background generation. Optical aberrations due to tissue inhomogeneity in the cone of light likely play a role in low image quality of the deeper regions. We also demonstrate longitudinal imaging enabled by the non-invasive nature of multiphoton microscopy.

Multiphoton fluorescence lifetime imaging microscopy (FLIM) is used to collect label-free metabolic information from biological samples via autofluorescence lifetime imaging of reduced nicotinamide adenine dinucleotide and nicotinamide adenine dinucleotide phosphate (NAD(P)H). However, FLIM has traditionally been limited by slow acquisition due to the limited bandwidth of analog electronics that perform photon counting and time-tagging. This slow acquisition has restricted the applicability of multiphoton FLIM of NAD(P)H by impeding the ability to accurately study biological problems that require characterization of fast dynamics. Faster image acquisition can be achieved by directly digitizing the amplified output of a hybrid photodetector and computationally determining photon counts via the Single- and multi-photon PEak Event Detection (SPEED) algorithm. This method, bypassing the limited-bandwidth analog electronics used for photon counting and time-tagging of photons in traditional FLIM, enables fast photon counting capabilities which are well suited for fast, high-dynamic range biological processes such as metabolic changes during apoptosis. Here, we utilize this technology to examine fast dynamics of apoptosis in 2D culture of normal and cancerous human breast cell lines, rat mammary tumor tissue-derived organoids, and in vivo rat mammary tumors. Results indicate that apoptosis-related metabolic dynamics are biological model-dependent and based on local pharmacokinetics, with tumor derived organoids in Matrigel showing a significantly slower response than in vivo or in vitro 2D cell models. Future work should carefully consider these implications when determining which tumor model to use for experimentation and should improve tumor models to better represent in vivo tumor apoptosis dynamics.

Using the structured illumination, single pixel detection imaging technique SPatIal Frequency modulation Imaging (SPIFI), we demonstrate a cascaded Wavelength Domain and Spatial Domain (WD-SD-SPIFI) system enabling real-time, in-line, second order dispersion compensation optimization for multiphoton imaging. Enhanced resolution is demonstrated by imaging a sub-diffractive 140 nm fluorescent nanodiamond with Two Photon Excitation Fluorescence (2PEF) to measure the Point Spread Function (PSF). With a 1034 nm pulsed laser through a Numerical Aperture (NA) of 0.5, a PSF Full Width at Half Max (FWHM) of 780 nm was measured with minimal post processing analysis that only requires Fast Fourier Transforms (FFTs).

We aim to use the resolving power of near-infrared (NIR) fluorescence lifetime microscopy (FLIM) to provide information on the fluorescence decay behavior of NIR FRET donor probes, both in solution and in vitro, and assess their impact on in vivo macroscopic FLI FRET (MFLI FRET) tumor imaging. Utilizing HER2 mAbs, i.e., trastuzumab (TZM), labeled with AlexaFluor 700 (AF700), and HER2 positive cancer cell lines (AU565 and SKOV-3), we have documented significant impacts of IRF extraction methods and probe labeling schemes on FLIM analysis. Additionally, we have noted marked variation in the intracellular distribution of the HER2-TZM binding complexes, as well as in average endosomal lifetime measurements between cell lines. Herein, we discuss optimal methods for IRF extraction and generating NIR probes, as well as results from the newly optimized NIR FLIM FRET assay demonstrating variations in the average intracellular lifetime of TZM-AF700. Because fluorescence lifetime is impacted by environmental factors, such as pH, refraction, viscosity, and proximity to other molecules, these variations imply differences in the way TZM interacts with the endosomal microenvironment of these cell lines. We hypothesize that different HER2 positive cancer types exhibit variations in endosomal trafficking of the HER2-drug complex that play a key role in primary/acquired resistance to TZM.

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.

Cholesterol is connected to multiple health problems. Although the biochemical pathway is well-understood, many disease-related mechanisms, such as subcellular regulation of cholesterol synthesis, are still unclear. It presents a grand challenge for conventional characterization methods, which have difficulties providing non-invasive, high-speed, and high-resolution measurements of metabolites in living cells. Stimulated Raman scattering (SRS) can selectively visualize metabolic biomolecules based on vibrational spectroscopic features. Here, by SRS imaging of subcellular sterol, we found an unconventional regulatory mechanism based on the distribution of sterols on the plasma membrane and lipid droplets. The combination of vibrational imaging and genetic modulation promises great potential in finding new regulatory mechanisms, which would open opportunities for diagnosing and prognosis of cholesterol-related diseases.