Two-photon Fluorescence Lifetime Microscopy Research Articles

Label-free single-cell live imaging reveals fast metabolic switch in T lymphocytes.

T-cell activation induces a metabolic switch generating energy for proliferation, survival, and functions. We used noninvasive label-free two-photon fluorescence lifetime microscopy (2P-FLIM) to map the spatial and temporal dynamics of the metabolic NAD(P)H co-enzyme during T lymphocyte activation. This provides a readout of the OXPHOS and glycolysis rates at a single-cell level. Analyzes were performed in the CD4+ leukemic T cell line Jurkat, and in human CD4+ primary T cells. Cells were activated on glass surfaces coated with activating antibodies mimicking immune synapse formation. Comparing the fraction of bound NAD(P)H between resting and activated T cells, we show that T-cell activation induces a rapid switch toward glycolysis. This occurs after 10 min and remains stable for one hour. Three-dimensional analyzes revealed that the intracellular distribution of fraction of bound NAD(P)H increases at the immune synapse in activated cells. Finally, we show that fraction of bound NAD(P)H tends to negatively correlate with spreading of activated T cells, suggesting a link between actin remodeling and metabolic changes. This study highlights that 2P-FLIM measurement of fraction of bound NAD(P)H is well suited to follow a fast metabolic switch in three dimensions, in single T lymphocytes with subcellular resolution.

Improving longitudinal resolution of Airy beams two-photon volume imaging with fluorescence lifetime imaging

The non-diffracting Airy beams have been reported to increase acquisition speed by projecting the axially significantly elongated information to a two-dimensional (2D) image. Due to the elongated focal length, an Airy volumetric image can cover information as much as stack images with multiple Gaussian images at different imaging depths, which significantly increases the volumetric imaging rate. However, the projection of three-dimensional (3D) structures on a 2D plane is usually indistinguishable, especially those that overlap along the axis. Therefore, we proposed an axially resolved volume imaging technique based on a two-photon fluorescence lifetime microscopy imaging (FLIM) by using Airy beams.

NAD(P)H autofluorescence lifetime imaging enables single cell analyses of cellular metabolism of osteoblasts in vitro and in vivo via two-photon microscopy.

Two-photon fluorescence lifetime microscopy (2P-FLIM) is a non-invasive optical technique that can obtain cellular metabolism information based on the intrinsic autofluorescence lifetimes of free and enzyme-bound NAD(P)H, which reflect the metabolic state of single cells within the native microenvironment of the living tissue. NAD(P)H 2P-FLIM was initially performed in bone marrow stromal cell (BMSC) cultures established from Col (I) 2.3GFP or OSX-mCherry mouse models, in which osteoblastic lineage cells were labelled with green or red fluorescence protein, respectively. Measurement of the mean NAD(P)H lifetime, τM, demonstrated that osteoblasts in osteogenic media had a progressively increased τM compared to cells in regular media, suggesting that osteoblasts undergoing mineralization had higher NAD+/NAD(P)H ratio and may utilize more oxidative phosphorylation (OxPhos). In vivo NAD(P)H 2P-FLIM was conducted in conjunction with two-photon phosphorescence lifetime microscopy (2P-PLIM) to evaluate cellular metabolism of GFP+ osteoblasts as well as bone tissue oxygen at different locations of the native cranial bone in Col (I) 2.3GFP mice. Our data showed that osteocytes dwelling within lacunae had higher τM than osteoblasts at the bone edge of suture and marrow space. Measurement of pO2 showed poor correlation of pO2 and τM in native bone. However, when NAD(P)H 2P-FLIM was used to examine osteoblast cellular metabolism at the leading edge of the cranial defects during repair in Col (I) 2.3GFP mouse model, a significantly lower τM was recorded, which was associated with lower pO2 at an early stage of healing, indicating an impact of hypoxia on energy metabolism during bone tissue repair. Taken together, our current study demonstrates the feasibility of using non-invasive optical NAD(P)H 2P-FLIM technique to examine cellular energy metabolism at single cell resolution in living animals. Our data further support that both glycolysis and OxPhos are being used in the osteoblasts, with more mature osteoblasts exhibiting higher ratio of NAD+/NAD(P)H, indicating a potential change of energy mode during differentiation. Further experiments utilizing animals with genetic modification of cellular metabolism could enhance our understanding of energy metabolism in various cell types in living bone microenvironment.

Revealing the dynamics of a mitochondrial microenvironment during apoptosis under two-photon fluorescence lifetime microscopy using a cyclic iridium(iii) complex

A mitochondrial viscosity-sensitive two-photon fluorescent probe (Mito-Ap) to reflect apoptosis was rationally developed. The apoptotic process was accurately detected by two-photon fluorescence lifetime imaging in real-time.

Simultaneous NAD(P)H and FAD fluorescence lifetime microscopy of long UVA\u2013induced metabolic stress in reconstructed human skin

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 the UVA1-induced metabolic stress response in reconstructed human skin with multicolor two-photon fluorescence lifetime microscopy (FLIM). Simultaneous imaging of 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. 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 oxidative phosphorylation or oxidative stress possibly correlated to ROS generation. NAD(P)H and FAD biomarkers have unique temporal dynamic and sensitivity to skin cell types and UVA1 dose. While the FAD biomarker is UVA1 dose-dependent in keratinocytes, the NAD(P)H biomarker shows no dose dependence in keratinocytes, but is directly affected after exposure in fibroblasts, thus reflecting different skin cells sensitivities to oxidative stress. Finally, we show that a sunscreen including a UVA1 filter prevents UVA1 metabolic stress response from occurring.

NIR-II Excitation and NIR-I Emission Based Two-photon Fluorescence Lifetime Microscopic Imaging Using Aggregation-induced Emission Dots

Near-infrared(NIR) lights are powerful tools to conduct deep-tissue imaging since NIR-I wavelengths hold less photon absorption and NIR-II wavelengths serve low photon scattering in the biological tissues compared with visible lights. Two-photon fluorescence lifetime microscopy(2PFLM) can utilize NIR-II excitation and NIR-I emission at the same time with the assistance of a well-designed fluorescent agent. Aggregation induced emission(AIE) dyes are famous for unique optical properties and could serve a large two-photon absorption(2PA) cross-section as aggregated dots. Herein, we report two-photon fluorescence lifetime microscopic imaging with NIR-II excitation and NIR-I emission using a novel deep-red AIE dye. The AIE-gens held a 2PA cross-section as large as 1.61×104 GM at 1040 nm. Prepared AIE dots had a two-photon fluorescence peak at 790 nm and a stable lifetime of 2.2 ns under the excitation of 1040 nm femtosecond laser. The brain vessels of a living mouse were vividly reconstructed with the two-photon fluorescence lifetime information obtained by our home-made 2PFLM system. Abundant vessels as small as 3.17 µm were still observed with a nice signal-background ratio at the depth of 750 µm. Our work will inspire more insight into the improvement of the working wavelength of fluorescent agents and traditional 2PFLM.

Assessing the severity of psoriasis through multivariate analysis of optical images from non-lesional skin

Patients with psoriasis represent a heterogeneous population with individualized disease expression. Psoriasis can be monitored through gold standard histopathology of biopsy specimens that are painful and permanently scar. A common associated measure is the use of non-invasive assessment of the Psoriasis Area and Severity Index (PASI) or similarly derived clinical assessment based scores. However, heterogeneous manifestations of the disease lead to specific PASI scores being poorly reproducible and not easily associated with clinical severity, complicating the efforts to monitor the disease. To address this issue, we developed a methodology for non-invasive automated assessment of the severity of psoriasis using optical imaging. Our analysis shows that two-photon fluorescence lifetime imaging permits the identification of biomarkers present in both lesional and non-lesional skin that correlate with psoriasis severity. This ability to measure changes in lesional and healthy-appearing skin provides a new pathway for independent monitoring of both the localized and systemic effects of the disease. Non-invasive optical imaging was conducted on lesions and non-lesional (pseudo-control) skin of 33 subjects diagnosed with psoriasis, lesional skin of 7 subjects diagnosed with eczema, and healthy skin of 18 control subjects. Statistical feature extraction was combined with principal component analysis to analyze pairs of two-photon fluorescence lifetime images of stratum basale and stratum granulosum layers of skin. We found that psoriasis is associated with biochemical and structural changes in non-lesional skin that can be assessed using clinically available two-photon fluorescence lifetime microscopy systems.

Frequency-doubled FDML-MOPA laser in the visible.

Wavelength-swept lasers enable high-speed measurements in absorption spectroscopy, Raman spectroscopy, nonlinear Raman hyperspectral microscopy, rapid confocal microscopy, short impulse generation, and most importantly for high-speed optical coherence tomography, with speeds up to video-rate volumetric imaging. Recently, we introduced a pulsed wavelength-swept laser based on the Fourier domain mode-locked (FDML) laser principle combined with a master-oscillator power amplifier (MOPA) architecture. The high peak powers reached with this laser enabled rapid two-photon microscopy and two-photon fluorescence lifetime microscopy and high-speed light detection and ranging measurements. Here, we present the extension of this laser into the visible wavelength range by frequency doubling the output from 1064 nm to 532 nm via second harmonic generation in a deuterated potassium dihydrogen phosphate crystal. The result is a wavelength-swept laser source around 532 nm that outputs a pulse train of distinct, almost monochromatic wavelengths at an 88 MHz pulse repetition rate and 342 kHz sweep repetition rate. This swept-source laser in the visible can open up new research applications in spectroscopy, metrology, sensing, and high-speed imaging.

Quantitative Spatial Analysis of Metabolic Heterogeneity Across in vivo and in vitro Tumor Models.

Metabolic preferences of tumor cells vary within a single tumor, contributing to tumor heterogeneity, drug resistance, and patient relapse. However, the relationship between tumor treatment response and metabolically distinct tumor cell populations is not well-understood. Here, a quantitative approach was developed to characterize spatial patterns of metabolic heterogeneity in tumor cell populations within in vivo xenografts and 3D in vitro cultures (i.e., organoids) of head and neck cancer. Label-free images of cell metabolism were acquired using two-photon fluorescence lifetime microscopy of the metabolic co-enzymes NAD(P)H and FAD. Previous studies have shown that NAD(P)H mean fluorescence lifetimes can identify metabolically distinct cells with varying drug response. Thus, density-based clustering of the NAD(P)H mean fluorescence lifetime was used to identify metabolic sub-populations of cells, then assessed in control, cetuximab-, cisplatin-, and combination-treated xenografts 13 days post-treatment and organoids 24 h post-treatment. Proximity analysis of these metabolically distinct cells was designed to quantify differences in spatial patterns between treatment groups and between xenografts and organoids. Multivariate spatial autocorrelation and principal components analyses of all autofluorescence intensity and lifetime variables were developed to further improve separation between cell sub-populations. Spatial principal components analysis and Z-score calculations of autofluorescence and spatial distribution variables also visualized differences between models. This analysis captures spatial distributions of tumor cell sub-populations influenced by treatment conditions and model-specific environments. Overall, this novel spatial analysis could provide new insights into tumor growth, treatment resistance, and more effective drug treatments across a range of microscopic imaging modalities (e.g., immunofluorescence, imaging mass spectrometry).

Cerebral metabolism in a mouse model of Alzheimer's disease characterized by two-photon fluorescence lifetime microscopy of intrinsic NADH.

.Disruptions and alterations to cerebral energy metabolism play a vital role in the onset and progression of many neurodegenerative disorders and cerebral pathologies. In order to precisely understand the complex alterations underlying Alzheimer’s disease (AD) progression, in vivo imaging at the microscopic level is required in preclinical animal models. Utilizing two-photon fluorescence lifetime imaging microscopy and the phasor analysis method, we have observed AD-related variations of endogenous fluorescence of reduced nicotinamide adenine dinucleotide (NADH) in vivo. We collected NADH FLIM images from the cerebral cortices of both APPswe:PS1dE9 mice to model amyloid plaque accumulation and corresponding age-matched wildtype controls. Distinct variations in NADH fluorescence lifetime between wildtype and AD mice, as well as variations related to proximity from amyloid plaques, are obvervable via the phasor method. The combination of NADH FLIM and phasor analysis allows for a minimally invasive, high-resolution technique to characterize the adverse effects of amyloid accumulation on mitochondrial energy metabolism and could guide our understanding of preclinical AD pathology.

A novel lysosome-to-mitochondria signaling pathway disrupted by amyloid-β oligomers.

The mechanisms of mitochondrial dysfunction in Alzheimer's disease are incompletely understood. Using two-photon fluorescence lifetime microscopy of the coenzymes, NADH and NADPH, and tracking brain oxygen metabolism with multi-parametric photoacoustic microscopy, we show that activation of lysosomal mechanistic target of rapamycin complex 1 (mTORC1) by insulin or amino acids stimulates mitochondrial activity and regulates mitochondrial DNA synthesis in neurons. Amyloid-β oligomers, which are precursors of amyloid plaques in Alzheimer's disease brain and stimulate mTORC1 protein kinase activity at the plasma membrane but not at lysosomes, block this Nutrient-induced Mitochondrial Activity (NiMA) by a mechanism dependent on tau, which forms neurofibrillary tangles in Alzheimer's disease brain. NiMA was also disrupted in fibroblasts derived from two patients with tuberous sclerosis complex, a genetic disorder that causes dysregulation of lysosomal mTORC1. Thus, lysosomal mTORC1 couples nutrient availability to mitochondrial activity and links mitochondrial dysfunction to Alzheimer's disease by a mechanism dependent on the soluble building blocks of the poorly soluble plaques and tangles.

Abstract 2191: Quantitative cell-level spatial analysis of tumor metabolism

Abstract Sub-populations of tumor cells exhibit distinct responses to treatment. This cell-level heterogeneity can lead to growth of resistant populations, and tumor recurrence. Many cancer therapies alter tumor cell metabolism, and the metabolic microenvironment can further contribute to tumor heterogeneity and drug response. However, the relationship between the spatial diversity of tumor cell metabolism and treatment response requires further characterization. Two-photon fluorescence lifetime microscopy (FLIM) monitors cellular metabolism by measuring NAD(P)H and FAD autofluorescence, and enables single-cell resolution of metabolic heterogeneity within 3D samples. Previous studies have shown that FLIM predicts treatment response of 2D and 3D cultures in vitro, and in vivo tumors. Additionally, these studies demonstrated responsive cells have lower post-treatment NAD(P)H mean lifetime (NAD(P)H τm) than non-responsive cells. Thus, these published FLIM data were used to develop an algorithm to quantify the spatial distribution of cellular metabolism in response to treatment. Density-based clustering analysis defined sub-populations in organoids based on NAD(P)H τm. Accuracy of cluster analysis classification was first evaluated on 2D co-culture images of two cell lines plated at varying proportions. Following this validation, metabolic sub-populations and their spatial distributions were characterized in control and drug-treated 3D tumor organoids of human squamous cell carcinoma (FaDu). Descriptive spatial metrics were developed using graph-based network analysis integrated with standard image analysis to establish local (sub-population level) and global (organoid level) spatial relationships. High classification accuracy (94.7%) of cell lines was achieved in 2D breast carcinoma co-cultures. In all FaDu 3D organoids, metabolic sub-populations were largely segregated into clusters of identical metabolic phenotype, with ≥78% of cell neighbors belonging to the same sub-population. Clustering density was greater in populations with low NAD(P)H τm (responsive), exhibiting shorter intercellular distances than high NAD(P)H τm (non-responsive) populations (p <0.05). Additionally, responsive and non-responsive cell sub-populations displayed similar organization relative to both adjacent sub-populations and organoid centers. Geographically weighted principal component analysis demonstrates variability of each metabolic and spatial parameter as a function of cell location. This analysis is under refinement for use with in vivo FaDu xenograft images. This algorithm to quantify spatial distributions of metabolic sub-populations within tumors could enhance our understanding of tumor progression and therapeutic resistance. Citation Format: Tiffany M. Heaster, Bennett A. Landman, Melissa C. Skala. Quantitative cell-level spatial analysis of tumor metabolism [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2191.

Nutrient-induced Mitochondrial Activation (NiMA): A Novel Lysosome-to-Mitochondria Signaling Pathway Disrupted by Amyloid-- Oligomers

The mechanisms of mitochondrial dysfunction in Alzheimer’s Disease (AD) are incompletely understood. Using two-photon fluorescence lifetime microscopy of NAD(P)H and tracking brain oxygen metabolism with multi-parametric photoacoustic microscopy, we show that activation of lysosomal mechanistic target of rapamycin complex 1 (mTORC1) by insulin or amino acids stimulates mitochondrial activity and regulates mitochondrial DNA synthesis in neurons. Amyloid-β oligomers, which are precursors of amyloid plaques in AD brain and stimulate mTORC1 protein kinase activity at the plasma membrane, but not at lysosomes, block this nutrient-induced mitochondrial activity (NiMA) by a mechanism dependent on tau, which forms neurofibrillary tangles in AD brain. NiMA was also disrupted in fibroblasts derived from a patient with tuberous sclerosis complex, a genetic disorder that causes dysregulation of lysosomal mTORC1. Thus, lysosomal mTORC1 couples nutrient availability to mitochondrial activity, and links mitochondrial dysfunction to AD by a mechanism dependent on soluble building blocks of plaques and tangles.

Two-Photon Fluorescence Lifetime Imaging for Metabolic Profiling of Cochlear Dysfunction

More than 120,000 individuals treated with lifesaving antibiotics develop hearing or balance disorders annually. Although research has shown that of the two cochlear cell types, sensory and supporting cells, sensory cells are readily damaged due to age-related hearing loss, acoustic trauma and ototoxins, the reason for this remains unknown. Furthermore, cochlear sensory hair cells can be divided into two types, inner and outer hair cells (IHCs, OHCs). OHCs in the high-frequency region of the cochlea exhibit the greatest sensitivity to the above conditions. To determine if variations in sensory and supporting mitochondrial metabolism account for these differences, two-photon fluorescence lifetime microscopy (FLIM) was used to measure changes in the metabolic reporter molecule NADH in sensory and supporting cells from explanted murine cochleae. Mitochondrial uncouplers, inhibitors and an ototoxic antibiotic, gentamicin (GM), were used to assess high- and low-frequency IHC, OHC and supporting cell mitochondrial metabolism. Chemically induced changes in metabolic state resulted in a reorganization of specific NADH lifetimes into altered subcellular fluorescence lifetime pools. Variations in NADH intensity and average NADH lifetime were greatest in high-frequency OHCs. Pretreatment with GM significantly increased NADH intensity in high-frequency sensory cells but not supporting cells. Treatment with GM significantly increased the average NADH fluorescence lifetime within IHCs but not OHCs. GM also caused a significant increase of NADH concentration in OHCs, not IHCs. These results demonstrate: differences between sensory and supporting cell metabolism; GM alters mitochondrial metabolism; and IHCs and OHCs display differing metabolic effects when exposed to GM. Such fundamental differences between sensory and supporting cell mitochondrial metabolism indicate differing metabolic changes during antibiotic exposure. Understanding these antibiotic-induced metabolic changes may explain the ototoxic effects of these drugs which is crucial for preventing and treating numerous auditory deficits and diseases.

In vivo imaging of cerebral energy metabolism with two-photon fluorescence lifetime microscopy of NADH.

Minimally invasive, specific measurement of cellular energy metabolism is crucial for understanding cerebral pathophysiology. Here, we present high-resolution, in vivo observations of autofluorescence lifetime as a biomarker of cerebral energy metabolism in exposed rat cortices. We describe a customized two-photon imaging system with time correlated single photon counting detection and specialized software for modeling multiple-component fits of fluorescence decay and monitoring their transient behaviors. In vivo cerebral NADH fluorescence suggests the presence of four distinct components, which respond differently to brief periods of anoxia and likely indicate different enzymatic formulations. Individual components show potential as indicators of specific molecular pathways involved in oxidative metabolism.

Folate receptor targeting silica nanoparticle probe for two-photon fluorescence bioimaging

Narrow dispersity organically modified silica nanoparticles (SiNPs), diameter ~30 nm, entrapping a hydrophobic two-photon absorbing fluorenyl dye, were synthesized by hydrolysis of triethoxyvinylsilane and (3-aminopropyl)triethoxysilane in the nonpolar core of Aerosol-OT micelles. The surface of the SiNPs were functionalized with folic acid, to specifically deliver the probe to folate receptor (FR) over-expressing Hela cells, making these folate two-photon dye-doped SiNPs potential candidates as probes for two-photon fluorescence microscopy (2PFM) bioimaging. In vitro studies using FR over-expressing Hela cells and low FR expressing MG63 cells demonstrated specific cellular uptake of the functionalized nanoparticles. One-photon fluorescence microscopy (1PFM) imaging, 2PFM imaging, and two-photon fluorescence lifetime microscopy (2P-FLIM) imaging of Hela cells incubated with folate-modified two-photon dye-doped SiNPs were demonstrated.