Fluorescence Lifetime Distribution Research Articles

Analysis of hydrolytic differences of free and “polyacrylic acid (PAAc)-conjugated trypsin and chymotrypsin” by using fluorescence lifetime distributions

Electroanalytic, photometric or fluorometric methods may provide information about the presence of proteolysis in a related sample, but they cannot accurately identify which protease belongs to the proteolytic activity. In other words, they cannot distinguish the proteases according to the differences in their activities. Previous studies on the chymotrypsin and trypsin have shown that the activities of proteases can be distinguished from each other by fluorescence lifetime distributions. In this study, it is aimed to show the sensitivity of the distributional model on the proteolytic activities of the two proteases. For this purpose, the proteolytic activities have been reduced by making covalent conjugation with polyacrylic acid (PAAc) and the effects of the low activities were examined on Bovine Serum Albumin (BSA) which excimer emission character was incorporated into its structure by modification with N-(1-pyrenyl)maleimide. The time-resolved spectrofluorometer was used to collect fluorescence decay data at λ(excimer) = 464 nm, which were analyzed by using Exponential Series Method (ESM) to obtain the changes of lifetime distributions. The results showed a significant decline in the activities. Despite the very low activities, the changes of fluorescence lifetime distributions exhibited remarkable specific differences that proved the sensitivity of ESM analysis.

Inferring quantity and qualities of superimposed reaction rates from single molecule survival time distributions

Actions of molecular species, for example binding of transcription factors to chromatin, may comprise several superimposed reaction pathways. The number and the rate constants of such superimposed reactions can in principle be resolved by inverse Laplace transformation of the corresponding distribution of reaction lifetimes. However, current approaches to solve this transformation are challenged by photobleaching-prone fluorescence measurements of lifetime distributions. Here, we present a genuine rate identification method (GRID), which infers the quantity, rates and amplitudes of dissociation processes from fluorescence lifetime distributions using a dense grid of possible decay rates. In contrast to common multi-exponential analysis of lifetime distributions, GRID is able to distinguish between broad and narrow clusters of decay rates. We validate GRID by simulations and apply it to CDX2-chromatin interactions measured by live cell single molecule fluorescence microscopy. GRID reveals well-separated narrow decay rate clusters of CDX2, in part overlooked by multi-exponential analysis. We discuss the amplitudes of the decay rate spectrum in terms of frequency of observed events and occupation probability of reaction states. We further demonstrate that a narrow decay rate cluster is compatible with a common model of TF sliding on DNA.

Fluorescence coupled with electrochemical approach at the bulk and the interface region of hydrogen-bonding self assemblies of urea derivatives with DDP dye in aqueous solution

Photophysical and electrochemical techniques were employed to hydrogen-bonding self assemblies forming solutes (Urea, Dimethylurea and Tetramethylurea) in the presence of 4-dicyanomethylene 2, 6-dimethyl-4H-pyran (DDP) dye. Addition of urea derivatives to DDP dye (Intramolecular Charge Transfer (ICT)) results in a fluorescence enhancement accompanied with a significant shift. Fluorescence lifetime behavior exhibits a tri-exponential decay with a large variation in the fluorescence lifetime and relative amplitude distribution. The coexistence of three different fluorescence lifetime components of DDP with urea derivatives signifies the existence of heterogeneous micro environment. The dye is surrounded by varying proportion of solute and water molecules are established from fluorescence lifetime studies. Urea derivatives govern the excited state characteristics of DDP dye resulting in the formation and promotion of different microenvironment which are clearly distinguishable. The existence of multi environment attributed to urea-water structural behaviour is authenticated by electrochemical impedance spectral studies (EIS). A large variation in the contour pattern, shape and intensity in 3D fluorescence contour spectra of dye with urea validate the existence of dye in a heterogeneous micro environment. The hydrophobicity of urea derivatives along with the hydrogen-bonding properties of urea-water and urea-urea influence the photophysical and electrochemical nature of dye is emphasized.

Graphene setting the stage: tracking DNA hybridization with nanoscale resolution

In this study we use nanophotonic effects of graphene to study DNA hybridization: the z−4 nanoscale distance-dependence of the fluorescence lifetime for fluorophores located in the vicinity of graphene is for the first time used to track a DNA hybridization reaction with nanoscale resolution in real time. First, a nanostaircase with ≈2 nm steps from 0 to a total height of 48 nm is used as a nanoruler to confirm the distance dependence law. We find that the axial sensitivity is suited to determine the nanoscale surface roughness of these samples. The proof-of-concept DNA experiments in aqueous medium involve the hybridization of fluorescently labelled DNA beacons attached to CVD grown graphene with complementary (target) DNA added in solution. We track the conformational changes of the beacons statistically by determining the fluorescence lifetimes of the labelling dye and converting them into nanoscale distances from the graphene. In this way, we are able to monitor the vertical displacement of the label during DNA-beacon unfolding with an axial resolution reaching down to 1 nm. The measured distance increase during the DNA hybridization reaction of about 10 nm matches the length of the target DNA strand. Furthermore, the width of the fluorescence lifetime distributions could be used to estimate the molecular tilt angle of the hybridized ds-DNA configuration. The achieved nanoscale sensitivity opens innovation opportunities in material engineering, genetics, biochemistry and medicine.

Label-free assessment of pre-implantation embryo quality by the Fluorescence Lifetime Imaging Microscopy (FLIM)-phasor approach

Development of quantitative, safe and rapid techniques for assessing embryo quality provides significant advances in Assisted Reproductive Technologies (ART). Instead of assessing the embryo quality by the standard morphologic evaluation, we apply the phasor-FLIM (Fluorescence Lifetime Imaging Microscopy) method to capture endogenous fluorescent biomarkers of pre-implantation embryos as a non-morphological caliber for embryo quality. Here, we identify, under hypoxic and non-hypoxic conditions, the unique spectroscopic trajectories at different stages of mouse pre-implantation development, which is referred to as the developmental, or “D-trajectory”, that consists of fluorescence lifetime from different stages of mouse pre-implantation embryos. The D-trajectory correlates with intrinsic fluorescent species from a distinctive energy metabolism and oxidized lipids, as seen with Third Harmonic Generation (THG) that changes over time. In addition, we have defined a non-morphological Embryo Viability Index (EVI) to distinguish pre-implantation embryo quality using the Distance Analysis (DA), a machine learning algorithm to process the fluorescence lifetime distribution patterns. We show, under our experimental conditions, that the phasor-FLIM approach provides a much-needed non-invasive quantitative technology for identifying healthy embryos at the early compaction stage with 86% accuracy. The DA and phasor-FLIM method may provide the opportunity to improve implantation success rates for in vitro fertilization clinics.

Specific orientation of dipole moments in azocrown cetocyanine dyes determined by electrooptical absorption measurements (EOAM)

From electrooptical absorption measurements (EOAM) follows that the dipole moments of azacrown ketocyanine dyes 2,5-di{(E)-1-[4-(4,7,10,13-tetraoxa-1-azacyclopentadecyl)phenyl]methylidene}-1-cyclopentanone (Compound 1) and (E)-1-(2-hydroxy-4methoxyphenyl)-3-[4-(4,7,10,13-tetraoxa-1-azacyclopentadecyl)phenyl]-2-propen-1-one (Compound 2) in the equilibrium ground state μg and the change of dipole moments upon transition to the excited Franck–Condon state Δaμ are large. This alteration causes a significant long-wavelength shift of the absorption and emission spectra, as well as large fluorescence Stokes shift with increasing polarity of the solvent.From the DFT calculations follows that the terminal groups of the dyes are located outside the plane of the central part of the molecules by the torsion angles that allow good conjugation of the nitrogen atoms lone pairs with π-system of the molecule. The transition dipole moment of Compound 1 ma is perpendicular to the dipole moment μg in the equilibrium ground state and for Compound 2 ma is parallel to μg. This is due to the differences in the geometric structure of the dyes.The distribution of fluorescence lifetime of Compound 1 significantly depends on the polarity of the solvent. Thus, ketocyanine dyes with azacrown cycles can serve not only as ion indicators but also as fluorescent probes for studying the polarity of the environment.

Excitons and Biexciton Dynamics in Single CsPbBr3 Perovskite Quantum Dots.

Colloidal lead halide perovskite quantum dots, due to their optical versatility and facile solution processability, have been recently recognized as components of various optoelectronic devices. Detailed understanding of their exciton recombination dynamics at the single-particle level is necessary for utilizing their full potential. We conducted spectroscopic studies of the excitons and biexciton dynamics in single CsPbBr3 perovskite quantum dots. It was found that while the rates of radiative recombination remain essentially constant, the overall relaxation process is dominated by nonradiative recombination of single excitons and biexcitons. The radiative lifetime scaling is determined to be ∼1.0 for single exciton and ∼4.4 for biexcitons. A linear dependence of fluorescence lifetime vs intensity distribution agrees well with the prediction of the model of multiple recombination centers. The blinking mechanism of CsPbBr3 quantum dots is addressed by considering the trion states under higher excitation powers.

Effect of Indole-3-Carbinol on Dimyristoylphosphatidylcholine Multilamellar Vesicles.

This study reports the interaction of indole-3-carbinol (I3C), which is a chemopreventive reagent, with an artificial model membrane {(dimyristoylphosphatidylcholine (DMPC) multilamellar vesicles (MLVs)}, using the intrinsic fluorescence properties of I3C, extrinsic fluorescence properties of Nile Red (NR), differential scanning calorimetry (DSC), dynamic light scattering (DLS), and confocal microscopy. The intrinsic fluorescence of I3C helps to provide information about its location, partitioning ability, and sensitivity toward the phase-transition temperature of liposomes, confirmed by cetylpyridinium chloride (CPC) quenching study, partition coefficient values {(4.60 ± 0.1) × 105 (solid gel phase) and (7.29 ± 0.1) × 105 M-1 (liquid crystalline phase)} and temperature-dependent emission behavior of I3C. I3C perturbs the DMPC MLVs above 15 mol %, as observed using the fluorescence properties of NR, DSC, and DLS data. This perturbation occurs as a consequence of interfacial hydration of the DMPC MLVs, which was clearly indicated by the fluorescence properties (emission intensity, fluorescence lifetime, and nonextensive distribution analysis) of NR.

Intramolecular Distance Distribution Reveals Mechanisms in Protein Folding and Dynamics

Flexibility and dynamics in polypeptides and proteins give rise to conformational and structural heterogeneity. Quantification of the level of heterogeneity is essential for providing deeper understanding of molecular mechanisms in protein folding. Fluorescence lifetime distribution generated by the maximum entropy method (MEM) provides an unbiased estimate of intramolecular distance distribution. This article discusses several examples where MEM-generated lifetime distribution brings out site-specific conformational and structural heterogeneity in proteins. Use of the information on intamolecular distance distribution in revealing molecular mechanisms of protein folding is also discussed.

In cell measurement of fluorescence lifetime imaging microscopy revealed C-terminal conformation changes of Ferroportin upon addition of Mn2+

Fluorescence microscopy, as a sensitive method to detect microenvironment of molecules, is widely used in protein conformation and dynamic studies in live cells. Fluorescence lifetime imaging microscopy (FLIM), which is independent of fluorophore concentrations, scattering and bleaching, is a suitable tool to analyze membrane proteins in a single cell. Ferroportin (FPN), a multi-ion exporter in vertebrates, was modulated by metal ions with unknown mechanism. Herein, we fused green fluorescence protein on C-terminal of FPN (FPN-eGFP) and applied fluorescence lifetime to monitor conformation changes of FPN in a live cell. The fluorescence lifetime distribution showed a shift to shorter lifetime upon Mn2+ treatment, suggesting a preference conformation of FPN in Mn2+ exposure. It is also observed that the lifetime (rather than intensity) measurement was not strongly influenced by laser power. The observed fluorescence lifetime changes of FPN-eGFP upon Mn2+ treatments indicated that extracellular metal ions can modulate FPN through conformation exchanges between several different states.

Enhanced spontaneous DNA twisting/bending fluctuations unveiled by fluorescence lifetime distributions promote mismatch recognition by the Rad4 nucleotide excision repair complex.

Rad4/XPC recognizes diverse DNA lesions including ultraviolet-photolesions and carcinogen-DNA adducts, initiating nucleotide excision repair. Studies have suggested that Rad4/XPC senses lesion-induced helix-destabilization to flip out nucleotides from damaged DNA sites. However, characterizing how DNA deformability and/or distortions impact recognition has been challenging. Here, using fluorescence lifetime measurements empowered by a maximum entropy algorithm, we mapped the conformational heterogeneities of artificially destabilized mismatched DNA substrates of varying Rad4-binding specificities. The conformational distributions, as probed by FRET between a cytosine-analog pair exquisitely sensitive to DNA twisting/bending, reveal a direct connection between intrinsic DNA deformability and Rad4 recognition. High-specificity CCC/CCC mismatch, free in solution, sampled a strikingly broad range of conformations from B-DNA-like to highly distorted conformations that resembled those observed with Rad4 bound; the extent of these distortions increased with bound Rad4 and with temperature. Conversely, the non-specific TAT/TAT mismatch had a homogeneous, B-DNA-like conformation. Molecular dynamics simulations also revealed a wide distribution of conformations for CCC/CCC, complementing experimental findings. We propose that intrinsic deformability promotes Rad4 damage recognition, perhaps by stalling a diffusing protein and/or facilitating ‘conformational capture’ of pre-distorted damaged sites. Surprisingly, even mismatched DNA specifically bound to Rad4 remains highly dynamic, a feature that may reflect the versatility of Rad4/XPC to recognize many structurally dissimilar lesions.

Depth-Dependent Membrane Ordering by Hemagglutinin Fusion Peptide Promotes Fusion.

Membrane fusion, one of the most fundamental processes in life, occurs when two separate lipid membranes merge into a single continuous bilayer. Membrane fusion is essential for the entry of lipid-sheathed viruses such as influenza and HIV. Influenza virus is internalized via receptor-mediated endocytosis and then fuses with the endosomal membrane at low pH. Hemagglutinin, a glycoprotein found on the surface of influenza virus, is responsible for the fusion of the viral sheath with the endosomal membrane. The ∼20 amino acid long N-terminus of hemagglutinin, known as the fusion peptide, plays a crucial role in the viral fusion process. Although there exists vast literature on the importance and role of the fusion peptide in promoting membrane fusion, there is no consensus on the mechanism by which it promotes fusion. A recent report suggested that the fusion peptide occupies and orders space in the outer leaflets of contacting bilayers so as to promote acyl chain protrusion into interbilayer space and promote fusion "stalk" formation. We report here the effect of the wild type, G1S, G1V, and W14A mutants of hemagglutinin fusion peptide on depth-dependent ordering of model membranes along the bilayer normal. We utilized fluorescence anisotropy, lifetime measurements, and lifetime distribution analyses of different anthroyloxy stearic acid probes (n-AS) in order to examine the effect of fusion peptides at various depths along the bilayer normal. Wild type peptide uniquely ordered a region ∼12 Å from the bilayer midpoint, W14A and G1S mutants mainly ordered the bilayer interface, while G1V had little ordering influence. On the basis of recent analysis of the effects of these peptides on fusion, ordering of the mid-upper region of the bilayer appears to promote fusion pore formation, while ordering of the bilayer interface inhibits it.

Nucleosome Opening Kinetics and the Influence of Histone Modifications Studied by Single Molecule FRET

Nucleosomes compact the genome and regulate access by wrapping and unwrapping DNA. Here we characterized NaCl-induced opening of mononucleosomes by single-molecule FRET, using donor/acceptor labels on the DNA and various histones. By species-selective fluorescence lifetime and photon distribution analysis we identified new nucleosome opening intermediates and developed a kinetic model [1]. Opening proceeds through a weakening of the H2A-H2B dimer/(H3-H4)2 tetramer interface on a 0.1 ms time scale, then by a slower two-step release of the dimers coupled to DNA unwrapping, extending from several ms to minutes. Nucleosome opening and detachment of histone dimers proceed asymmetrically depending on the DNA sequence.Mutations at the H2A/H3 interface (H2A R81A, R88A, R81A/R88A, R81E/R88E) facilitate the initial opening, confirming the importance of the dimer:tetramer interface for nucleosome stability. This is also supported by molecular dynamics (MD) simulations that show enhanced DNA fluctuations for the mutants. Partially opened states such as described here might be a convenient nucleation point for DNA-recognizing proteins.For characterizing the role of histone tails in nucleosome interaction and stability, we performed MD simulations in explicit and implicit solvent on the H4 tail interacting with the acidic patch of a neighboring nucleosome. The simulations show that H4K16 acetylation decreases the interactions between H4 tail and the acidic patch of the neighboring nucleosome, a process that is central to the regulation of chromatin compaction [2,3].

Quantitative deconvolution of autocorrelations and cross correlations from two-dimensional lifetime decay maps in fluorescence lifetime correlation spectroscopy

Fluorescence correlation spectroscopy (FCS) is a widely used method for measuring molecular diffusion and chemical kinetics. However, when a mixture of fluorescent species is taken into account, the conventional FCS method has limitations in extracting autocorrelations for different species and cross correlations between different species. Recently developed fluorescence lifetime correlation spectroscopy (FLCS) based on time-tagged time-resolved (TTTR) photon recording, which can record the global and micro arrival time for each individual photon, has been used to discriminate different species according to fluorescence lifetime. Here, based on two-dimensional lifetime decay maps constructed from TTTR photon stream, we have developed a quantitative lifetime-deconvolution FCS model (LDFCS) to extract precise chemical rates for chemical conversions in multi-species systems. The key point of LDFCS model is separation of different species according to the global distribution of fluorescence lifetime and then deconvolution of autocorrelations and cross-correlations from the two-dimensional lifetime decay maps constructed by the micro arrival times of photon pairs at each delay time.

Quantitative sampling of conformational heterogeneity of a DNA hairpin using molecular dynamics simulations and ultrafast fluorescence spectroscopy.

Molecular dynamics (MD) simulations and time resolved fluorescence (TRF) spectroscopy were combined to quantitatively describe the conformational landscape of the DNA primary binding sequence (PBS) of the HIV-1 genome, a short hairpin targeted by retroviral nucleocapsid proteins implicated in the viral reverse transcription. Three 2-aminopurine (2AP) labeled PBS constructs were studied. For each variant, the complete distribution of fluorescence lifetimes covering 5 orders of magnitude in timescale was measured and the populations of conformers experimentally observed to undergo static quenching were quantified. A binary quantification permitted the comparison of populations from experimental lifetime amplitudes to populations of aromatically stacked 2AP conformers obtained from simulation. Both populations agreed well, supporting the general assumption that quenching of 2AP fluorescence results from pi-stacking interactions with neighboring nucleobases and demonstrating the success of the proposed methodology for the combined analysis of TRF and MD data. Cluster analysis of the latter further identified predominant conformations that were consistent with the fluorescence decay times and amplitudes, providing a structure-based rationalization for the wide range of fluorescence lifetimes. Finally, the simulations provided evidence of local structural perturbations induced by 2AP. The approach presented is a general tool to investigate fine structural heterogeneity in nucleic acid and nucleoprotein assemblies.

The modifier effects of chymotrypsin and trypsin enzymes on fluorescence lifetime distribution of “N-(1-pyrenyl)maleimide–bovine serum albumin” complex

Chymotrypsin and trypsin are the well known proteolytic enzymes, both of which are synthesized in the pancreas as their precursors — the inactive forms; chymotrypsinogen and trypsinogen — and then are released into the duodenum to cut proteins into smaller peptides. In this paper, the effects of activities of chymotrypsin and trypsin enzymes on fluorescence lifetime distributions of the substrat bovine serum albumin (BSA) modified with N-(1-pyrenyl)maleimide (PM) were examined. In the labeling study of BSA with PM, it is aimed to attach PM to the single free thiol (Cys34) and to all the free amine groups in accessible positions in order to produce excimers of pyrene planes of the possible highest amount to form the lifetime distributions in the widest range, that may show specifically distinguishing changes resulting from the activities of the proteases. The time resolved spectrofluorometer was used to monitor fluorescence decays, which were analyzed by using the exponential series method (ESM) to obtain the changes of lifetime distributions. After the exposure of the synthesized substrat PM-BSA to the enzymes, the fluorescence lifetime distributions exhibited different structures which were attributed to the different activities of the proteases.

Ca2+ and Mg2+ modulate conformational dynamics and stability of downstream regulatory element antagonist modulator

Downstream Regulatory Element Antagonist Modulator (DREAM) belongs to the family of neuronal calcium sensors (NCS) that transduce the intracellular changes in Ca(2+) concentration into a variety of responses including gene expression, regulation of Kv channel activity, and calcium homeostasis. Despite the significant sequence and structural similarities with other NCS members, DREAM shows several features unique among NCS such as formation of a tetramer in the apo-state, and interactions with various intracellular biomacromolecules including DNA, presenilin, Kv channels, and calmodulin. Here we use spectroscopic techniques in combination with molecular dynamics simulation to study conformational changes induced by Ca(2+) /Mg(2+) association to DREAM. Our data indicate a minor impact of Ca(2+) association on the overall structure of the N- and C-terminal domains, although Ca(2+) binding decreases the conformational heterogeneity as evident from the decrease in the fluorescence lifetime distribution in the Ca(2+) bound forms of the protein. Time-resolved fluorescence data indicate that Ca(2+) binding triggers a conformational transition that is characterized by more efficient quenching of Trp residue. The unfolding of DREAM occurs through an partially unfolded intermediate that is stabilized by Ca(2+) association to EF-hand 3 and EF-hand 4. The native state is stabilized with respect to the partially unfolded state only in the presence of both Ca(2+) and Mg(2+) suggesting that, under physiological conditions, Ca(2+) free DREAM exhibits a high conformational flexibility that may facilitate its physiological functions.

Steady state and time resolved fluorescence studies of azadioxatriangulenium (ADOTA) fluorophore in silica and PVA thin films

A cationic azadioxatriangulenium dye was entrapped in silica thin films obtained by the sol–gel process and in poly (vinyl) alcohol (PVA) thin films. Azadioxatriangulenium is a red emitting fluorophore with a long fluorescence lifetime of ∼20 ns. The fluorescent properties of azadioxatriangulenium in silica thin films and PVA films were studied by means of steady–state and time resolved fluorescence techniques. We have found that the azadioxatriangulenium entrapped in silica thin film has a wider fluorescence lifetime distribution (Lorentzian distribution), lower fluorescence efficiencies, shorter lifetimes compared to Azadioxatriangulenium in a PVA film. The local environment of azadioxatriangulenium molecules in the silica thin film is rich with water and ethanol, which creates the possibility of forming excited state aggregates due to high concentration of dye within a small confined area. In contrast to the PVA matrices, the porous silica films allow restricted rotations of Azadioxatriangulenium molecules, which result in faster and complex fluorescence anisotropy decays suggesting energy migration among dye molecules.

Single-molecule spectroscopy and femtosecond transient absorption studies on the excitation energy transfer process in ApcE(1-240) dimers.

ApcE(1-240) dimers with one intrinsic phycocyanobilin (PCB) chromophore in each monomer that is truncated from the core-membrane linker (ApcE) of phycobilisomes (PBS) in Nostoc sp. PCC 7120 show a sharp and significantly red-shifted absorption. Two explanations either conformation-dependent Förster resonance energy transfer (FRET) or the strong exciton coupling limit have been proposed for red-shifted absorption. This is a classic example of the special pair in the photosynthetic light harvesting proteins, but the mechanism of this interaction is still a matter of intense debate. We report the studies using single-molecule and transient absorption spectra on the interaction in the special pair of ApcE dimers. Our results demonstrate the presence of conformation-dependent FRET between the two PCB chromophores in ApcE dimers. The broad distributions of fluorescence intensities, lifetimes and polarization difference from single-molecule measurements reveal the heterogeneity of local protein-pigment environments in ApcE dimers, where the same molecular structures but different protein environments are the main reason for the two PCB chromophores with different spectral properties. The excitation energy transfer rate between the donor and the acceptor about (110 ps)(-1) is determined from transient absorption measurements. The red-shifted absorption in ApcE dimers could result from more extending conformation, which shows another type of absorption redshift that does not depend on strong exciton coupling. The results here stress the importance of conformation-controlled spectral properties of the chemically identical chromophores, which could be a general feature to control energy/electron transfer, widely existing in the light harvesting complexes.

Metabolic Profiling of the Skin to Monitor the Onset and Progression of Squamous Cell Carcinoma through Time- and Wavelength-Resolved Fluorescence Lifetime Imaging

Every year millions of people in the United States are affected by skin cancer, and like other cancers, the key to an effective treatment is early detection and monitoring of disease progression. To this end, we have used intensity-, time-, and wavelength-resolved multiphoton microscopy to measure changes in cellular metabolism. By analyzing the fluorescence emission of metabolic coenzymes such as the reduced form of Nicotinamide Adenine Dinucleotide (NADH) and the oxidized form of flavin cofactors, slight changes in the mitochondrial microenvironment and cellular metabolism can be quantified. We used dual-channel fluorescence lifetime imaging (FLIM) to quantify metabolic changes both within and between high- and low-HER2 expressing squamous cell carcinoma cell lines (SCC-74B and SCC-74A, respectively). Chemically-induced changes in the metabolic state led to measureable changes in lifetime distributions and fluorescence intensities within each cell line. In addition, SCC-74A and −74B could clearly be distinguished on the basis of the fluorescence intensity dynamic range (difference between uncoupled and inhibited metabolic states) and lifetime distributions. The metabolic imaging technique has also been adapted to living skin in vivo. In particular, fluorescence signals originating from metabolically active and in-active sources are identified and separated based on a combination of the two-photon-excited fluorescence excitation/emission spectra and second harmonic generation (SHG).This study was conducted at the Creighton University Integrative Bio-Imaging Facility (CU-IBIF) and was supported by NIH DC 02053, NSF-EPSCoR EPS-0701892 (CFD 47.076), NIH P20 RR16469 (NCRR) and 5P20GM103427 (NIGMS) as well as the Ferlic Summer Research Program at Creighton University.