Mean Fluorescence Lifetime Research Articles

Investigation of Mitochondrial Metabolic Response to Doxorubicin in Prostate Cancer Cells: An NADH, FAD and Tryptophan FLIM Assay

Prostate cancer (PCa) is one of the leading cancers in men in the USA. Lack of experimental tools that predict therapy response is one of the limitations of current therapeutic regimens. Mitochondrial dysfunctions including defective oxidative phosphorylation (OXPHOS) in cancer inhibit apoptosis by modulating ROS production and cellular signaling. Thus, correction of mitochondrial dysfunction and induction of apoptosis are promising strategies in cancer treatment. We have used Fluorescence Lifetime Imaging Microscopy (FLIM) to quantify mitochondrial metabolic response in PCa cells by tracking auto-fluorescent NAD(P)H, FAD and tryptophan (Trp) lifetimes and their enzyme-bound fractions as markers, before and after treatment with anti-cancer drug doxorubicin. A 3-channel FLIM assay and quantitative analysis of these markers for cellular metabolism show in response to doxorubicin, NAD(P)H mean fluorescence lifetime (τm) and enzyme-bound (a2%) fraction increased, FAD enzyme-bound (a1%) fraction was decreased, NAD(P)H-a2%/FAD-a1% FLIM-based redox ratio and ROS increased, followed by induction of apoptosis. For the first time, a FRET assay in PCa cells shows Trp-quenching due to Trp-NAD(P)H interactions, correlating energy transfer efficiencies (E%) vs NAD(P)H-a2%/FAD-a1% as sensitive parameters in predicting drug response. Applying this FLIM assay as early predictor of drug response would meet one of the important goals in cancer treatment.

Fluorescence lifetimes of drusen in age‐related macular degeneration

PurposeFluorescence Lifetime Imaging Ophthalmoscopy (FLIO) allows non‐invasive in vivo measurement of fluorescence lifetimes of natural fluorophores of the retina upon excitation with a laser light. FLIO in different retinal diseases showed that the technique is useful to detect early metabolic changes and can differentiate between retinal deposits.The aim of this study was to characterize fluorescence lifetimes in retinal drusen due to age‐related macular degeneration (AMD).MethodsFLIO imaging was performed using a Fluorescence Lifetime Imaging Ophthalmoscope (Heidelberg Engineering, Germany). Autofluorescence was elicited with a 473 nm laser and decay times were measured in a short (498–560 nm) and in a long (560–720 nm) spectral channel. Fluorescence lifetime data was compared with autofluorescence intensity images, optical coherence tomography (OCT) and color fundus images. Soft drusen and reticular pseudodrusen were analysed and compared to an age matched control group.Results64 eyes from 64 patients with AMD and retinal drusen (age: mean ± SD 78 ± 8.5 years) were investigated and compared to 20 age matched healthy controls. Mean fluorescence lifetime in patients with AMD was significantly prolonged compared to the healthy control eyes (mean ± SEM; SSC 486 ± 18 ps vs 332 ± 11 ps, p > 0.0001; LSC: 493 ± 9 ps vs 382 ± 17 ps, p > 0.0001). Areas of drusen featured a broader range of fluorescence lifetime values. Long lifetimes were identified in areas of atrophy and intraretinal hyperreflective deposits. Areas of short lifetime corresponded to deposits within the photoreceptor outer segment band.ConclusionsMean retinal autofluorescence lifetimes in AMD were significantly prolonged compared to healthy control eyes. Intraretinal deposits caused prolonged lifetimes whereas deposits in the area of the outer photoreceptor segments lead to short fluorescence lifetimes.

In vivo label-free mapping of the effect of a photosystem II inhibiting herbicide in plants using chlorophyll fluorescence lifetime

BackgroundIn order to better understand and improve the mode of action of agrochemicals, it is useful to be able to visualize their uptake and distribution in vivo, non-invasively and, ideally, in the field. Here we explore the potential of plant autofluorescence (specifically chlorophyll fluorescence) to provide a readout of herbicide action across the scales utilising multiphoton-excited fluorescence lifetime imaging, wide-field single-photon excited fluorescence lifetime imaging and single point fluorescence lifetime measurements via a fibre-optic probe.ResultsOur studies indicate that changes in chlorophyll fluorescence lifetime can be utilised as an indirect readout of a photosystem II inhibiting herbicide activity in living plant leaves at three different scales: cellular (~μm), single point (~1 mm2) and macroscopic (~8 × 6 mm2 of a leaf). Multiphoton excited fluorescence lifetime imaging of Triticum aestivum leaves indicated that there is an increase in the spatially averaged chlorophyll fluorescence lifetime of leaves treated with Flagon EC—a photosystem II inhibiting herbicide. The untreated leaf exhibited an average lifetime of 560 ± 30 ps while the leaf imaged 2 h post treatment exhibited an increased lifetime of 2000 ± 440 ps in different fields of view. The results from in vivo wide-field single-photon excited fluorescence lifetime imaging excited at 440 nm indicated an increase in chlorophyll fluorescence lifetime from 521 ps in an untreated leaf to 1000 ps, just 3 min after treating the same leaf with Flagon EC, and to 2150 ps after 27 min. In vivo single point fluorescence lifetime measurements demonstrated a similar increase in chlorophyll fluorescence lifetime. Untreated leaf presented a fluorescence lifetime of 435 ps in the 440 nm excited chlorophyll channel, CH4 (620–710 nm). In the first 5 min after treatment, mean fluorescence lifetime is observed to have increased to 1 ns and then to 1.3 ns after 60 min. For all these in vivo plant autofluorescence lifetime measurements, the plants were not dark-adapted.ConclusionsWe demonstrate that the local impact of a photosystem II herbicide on living plant leaves can be conveniently mapped in space and time via changes in autofluorescence lifetime, which we attribute to changes in chlorophyll fluorescence. Using portable fibre-optic probe instrumentation originally designed for label-free biomedical applications, this capability could be deployed outside the laboratory for monitoring the distribution of herbicides in growing plants.

The fluorescence intensities ratio is not a reliable parameter for evaluation of protein unfolding transitions.

Monitoring the fluorescence of proteins, particularly the fluorescence of intrinsic tryptophan residues, is a popular method often used in the analysis of unfolding transitions (induced by temperature, chemical denaturant, and pH) in proteins. The tryptophan fluorescence provides several suitable parameters, such as steady-state fluorescence intensity, apparent quantum yield, mean fluorescence lifetime, position of emission maximum that are often utilized for the observation of the conformational/unfolding transitions of proteins. In addition, the fluorescence intensities ratio at different wavelengths (usually at 330 nm and 350 nm) is becoming an increasingly popular parameter for the evaluation of thermal transitions. We show that, under certain conditions, the use of this parameter for the analysis of unfolding transitions leads to the incorrect determination of thermodynamic parameters characterizing unfolding transitions in proteins (e.g., melting temperature) and, hence, can compromise the hit identification during high-throughput drug screening campaigns.

Monitoring macular pigment changes in macular holes using fluorescence lifetime imaging ophthalmoscopy.

To investigate the impact of macular pigment (MP) on fundus autofluorescence (FAF) lifetimes invivo by characterizing full-thickness idiopathic macular holes (MH) and macular pseudo-holes (MPH). A total of 37 patients with MH and 52 with MPH were included. Using the fluorescence lifetime imaging ophthalmoscope (FLIO), based on a Heidelberg Engineering Spectralis system, a 30° retinal field was investigated. FAF decays were detected in a short (498-560nm; ch1) and long (560-720nm; ch2) wavelength channel. τm , the mean fluorescence lifetime, was calculated from a three-exponential approximation of the FAF decays. Macular coherence tomography scans were recorded, and macular pigment's optical density (MPOD) was measured (one-wavelength reflectometry). Two MH subgroups were analysed according to the presence or absence of an operculum above the MH. A total of 17 healthy fellow eyes were included. A longitudinal FAF decay examination was conducted in nine patients, which were followed up after surgery and showed a closed MH. In MH without opercula, significant τm differences (p<0.001) were found between the hole area (MHa) and surrounding areas (MHb) (ch1: MHa 238±64ps, MHb 181±78ps; ch2: MHa 275±49ps, MHb 223±48ps), as well as between MHa and healthy eyes or closed MH. Shorter τm , adjacent to the hole, can be assigned to areas with equivalently higher MPOD. Opercula containing MP also show short τm . In MPH, the intactness of the Hele fibre layer is associated with shortest τm . Shortest τm originates from MP-containing retinal layers, especially from the Henle fibre layer. Fluorescence lifetime imaging ophthalmoscope (FLIO) provides information on the MP distribution, the pathogenesis and topology of MH. Macular pigment (MP) fluorescence may provide a biomarker for monitoring pathological changes in retinal diseases.

Effect of pharmacologically induced retinal degeneration on retinal autofluorescence lifetimes in mice

Fluorescence lifetime imaging ophthalmoscopy (FLIO) was used to investigate retinal autofluorescence lifetimes in mouse models of pharmacologically induced retinal degeneration over time. Sodium iodate (NaIO3, 35 mg/kg intravenously) was used to induce retinal pigment epithelium (RPE) degeneration with subsequent loss of photoreceptors (PR) whereas N-methyl-N-nitrosourea (MNU, 45 mg/kg intraperitoneally) was employed for degeneration of the photoreceptor cell layer alone. All mice were measured at day 3, 7, 14, and 28 after the respective injection of NaIO3, MNU or NaCl (control). Fluorescence lifetime imaging was performed using a fluorescence lifetime imaging ophthalmoscope (Heidelberg Engineering, Heidelberg, Germany). Fluorescence was excited at 473 nm and fluorescence lifetimes were measured in a short and a long spectral channel (498–560 nm and 560–720 nm). Corresponding optical coherence tomography (OCT) images were consecutively acquired and histology was performed at the end of the experiments. Segmentation of OCT images and histology verified the cell type-specific degeneration process over time. Retinal autofluorescence lifetimes increased from day 3 to day 28 in mice after NaIO3 treatment. Finally, at day 28, fluorescence lifetimes were prolonged by 8% in the short and 61% in the long spectral channel compared to control animals (p = 0.21 and p = 0.004, respectively). In mice after MNU treatment, the mean retinal autofluorescence lifetimes were already decreased at day 3 and retinal lifetimes were finally shortened by 27% in the short and 51% in the long spectral channel at day 28 (p = 0.0028). In conclusion, degeneration of the RPE with subsequent photoreceptor degeneration by NaIO3 lead to longer mean fluorescence lifetimes of the retina compared to control mice, whereas during specific degeneration of the photoreceptor layer induced by MNU shorter lifetimes were measured. Therefore, short retinal fluorescence lifetimes may originate from the RPE and may be modified by the overlaying retinal layers.

Using fluorescence lifetime microscopy to study the subcellular localization of anthocyanins.

Anthocyanins are flavonoid pigments that accumulate in most seed plants. They are synthesized in the cytoplasm but accumulate inside the vacuoles. Anthocyanins are pigmented at the lower vacuolar pH, but in the cytoplasm they can be visualized based on their fluorescence properties. Thus, anthocyanins provide an ideal system for the development of new methods to investigate cytoplasmic pools and association with other molecular components. We have analyzed the fluorescence decay of anthocyanins by fluorescence lifetime imaging microscopy (FLIM), in both invitro and invivo conditions, using wild-type and mutant Arabidopsis thaliana seedlings. Within plant cells, the amplitude-weighted mean fluorescence lifetime (τm ) correlated with distinct subcellular localizations of anthocyanins. The vacuolar pool of anthocyanins exhibited shorter τm than the cytoplasmic pool. Consistently, lowering the pH of anthocyanins in solution shortened their fluorescence decay. We propose that FLIM is a useful tool for understanding the trafficking of anthocyanins and, potentially, for estimating vacuolar pH inside intact plant cells.

Fundus autofluorescence lifetimes are increased in non-proliferative diabetic retinopathy.

To discriminate non-proliferative diabetic retinopathy (NPDR) patients from healthy controls by fluorescence lifetime imaging ophthalmoscopy (FLIO). A prototype FLIO (Heidelberg-Engineering, Heidelberg, Germany) was used to examine the retina of 33 patients and 28 controls. As increased fluorescence of the diabetic lens is known, the lenses of 34 patients and 24 controls were investigated as well. Time-resolved decay was detected in two spectral channels (ch1: 498-560nm, ch2: 560-720nm) and approximated by a series of three exponential functions yielding in lifetimes (τ1 , τ2 , τ3 ), amplitudes (α1 , α2 , α3 ) and their amplitude-weighted means (τm ). Significant differences between patients and controls were found for all fundus lifetime components (τm , τ1 -τ3 ) as for the amplitude α3 in both spectral channels. Channel 1 showed the largest differences: the average of mean fluorescence lifetime τm in the macula was 259±137ps in the patients versus 147±69ps in the controls. A logistic regression model allowed discrimination between study and control group with a sensitivity of 90.09% and a specificity of 71.4% (area under the curve: 0.865). Significantly shorter τm in the patients group than in the control group was detected in channel 2 in the crystalline lens (1587±326ps versus 1854±384ps, p=0.006). Fundus Fluorescence lifetimes are significantly increased in NPDR while lens lifetimes are shorter in the patient group. Lifetime changes might be indicative for the accumulation of advanced glycation end products (AGEs) which enables detection of the disease with high sensitivity and specificity possibly bearing diagnostic merit.

Simultaneous uptake of a Förster transfer dye pair by diatoms: Application in determination of staining density

The simultaneous uptake of PDMPO and Rhodamine B as two fluorescent dyes forming a Förster transfer pair by the diatom Cyclotella meneghiniana is demonstrated by in vivo-fluorochromation. The incorporation density in the cell walls was high enough for achieving resonant energy transfer between the two dyes as detected by fluorescence and excitation spectroscopy. The mean fluorescence lifetime of the donor is shortened in the presence of the acceptor by a factor of 0.75. By determining the mean lifetime from the fluorescence decay fitted by three eponentials, the efficiency of the energy transfer and the acceptor concentration is calculated assuming a homogenous distribution. For an initial concentration of both dyes of 5μM in the culture medium which is at the saturation limit of incorporation, an acceptor incorporation density of 0.6mM is obtained. In addition to such quantitative determinations, efficient emitting systems based on resonant energy transfer between two laser dyes may be useful in photonic applications of the hybrid biomineral. By achieving stimulated emission, the presence of optical modes in diatom frustules, which may act as photonic resonators due to the refractive index contrast to the environment in combination with the more or less regular pore pattern, may be characterized further.

Longitudinal in vivo tracking of adverse effects following topical steroid treatment.

Topical steroids are known for their anti-inflammatory properties and are commonly prescribed to treat many adverse skin conditions such as eczema and psoriasis. While these treatments are known to be effective, adverse effects including skin atrophy are common. In this study, the progression of these effects is investigated in an in vivo mouse model using multimodal optical microscopy. Utilizing a system capable of performing two-photon excitation fluorescence microscopy (TPEF) of reduced nicotinamide adenine dinucleotide (NADH) to visualize the epidermal cell layers and second harmonic generation (SHG) microscopy to identify collagen in the dermis, these processes can be studied at the cellular level. Fluorescence lifetime imaging microscopy (FLIM) is also utilized to image intracellular NADH levels to obtain molecular information regarding metabolic activity following steroid treatment. In this study, fluticasone propionate (FP)-treated, mometasone furoate (MF)-treated and untreated animals were imaged longitudinally using a custom-built multimodal optical microscope. Prolonged steroid treatment over the course of 21 days is shown to result in a significant increase in mean fluorescence lifetime of NADH, suggesting a faster rate of maturation of epidermal keratinocytes. Alterations to collagen organization and the structural microenvironment are also observed. These results give insight into the structural and biochemical processes of skin atrophy associated with prolonged steroid treatment.

Sperm metabolism is altered during storage by female insects: evidence from two-photon autofluorescence lifetime measurements in bedbugs.

We explore the possibility of characterizing sperm cells without the need to stain them using spectral and fluorescence lifetime analyses after multi-photon excitation in an insect model. The autofluorescence emission spectrum of sperm of the common bedbug, Cimex lectularius, was consistent with the presence of flavins and NAD(P)H. The mean fluorescence lifetimes showed smaller variation in sperm extracted from the male (tau m, τm = 1.54–1.84 ns) than in that extracted from the female sperm storage organ (tau m, τm = 1.26–2.00 ns). The fluorescence lifetime histograms revealed four peaks. These peaks (0.18, 0.92, 2.50 and 3.80 ns) suggest the presence of NAD(P)H and flavins and show that sperm metabolism can be characterized using fluorescence lifetime imaging. The difference in fluorescence lifetime variation between the sexes is consistent with the notion that female animals alter the metabolism of sperm cells during storage. It is not consistent, however, with the idea that sperm metabolism represents a sexually selected character that provides females with information about the male genotype.

Impact of Macular Pigment on Fundus Autofluorescence Lifetimes.

To characterize the macular region and to investigate the influence of the macular pigment (MP) on fundus autofluorescence (FAF) lifetimes in vivo. Forty-eight healthy subjects with a mean age of 24.1 ± 3.6 years (range, 20-37 years) were included. A 30° retinal field was investigated using the fluorescence lifetime imaging ophthalmoscope (FLIO), based on a Heidelberg Engineering Spectralis system, detecting FAF decays in a short (498-560 nm; ch1)- and a long (560-720 nm; ch2)-wavelength channel. The mean fluorescence lifetime τm was calculated from a 3-exponential approximation of the FAF decays. Macular pigment optical density (MPOD) was measured by one-wavelength reflectometry, and macular optical coherence tomogram (OCT) scans were recorded. Correlations between τm and MPOD were analyzed. The τm showed shortest values at the macular region with a mean of 82 ps (ch1) and 126 ps (ch2). We found a strong correlation of τm to the MPOD (ch1: r = -0.760; ch2: r = -0.663; P < 0.001), as well as a topologic agreement of shortest τm with highest MPOD. Macular pigment, which is known to have very short fluorescence decays, considerably contributes to the macular autofluorescence (AF). This study gives indirect evidence for a strong impact of MP on macular τm, although no direct measurement of MP autofluorescence lifetimes in vivo is possible at this point. Potentially, imaging the FAF lifetimes could lead to a novel methodology for the detection of macular pigment properties and pathology-induced changes in the living human retina.

SU‐C‐303‐02: Correlating Metabolic Response to Radiation Therapy with HIF‐1alpha Expression

Purpose:To understand radiation induced alterations in cellular metabolism which could be used to assess treatment or normal tissue response to aid in patient‐specific adaptive radiotherapy. This work aims to compare the metabolic response of two head and neck cell lines, one malignant (UM‐SCC‐22B) and one benign (Normal Oral Keratinocyte), to ionizing radiation. Responses are compared to alterations in HIF‐1alpha expression. These dynamics can potentially serve as biomarkers in assessing treatment response allowing for patient‐specific adaptive radiotherapy.Methods:Measurements of metabolism and HIF‐1alpha expression were taken before and X minutes after a 10 Gy dose of radiation delivered via an orthovoltage x‐ray source. In vitro changes in metabolic activity were measured via fluorescence lifetime imaging (FLIM) to assess the mean lifetime of NADH autofluorescence following a dose of 10 Gy. HIF‐1alpha expression was measured via immunohistochemical staining of in vitro treated cells and expression was quantified using the FIJI software package.Results:FLIM demonstrated a decrease in the mean fluorescence lifetime of NADH by 100 ps following 10 Gy indicating a shift towards glycolytic pathways for malignant cells; whereas this benign cell line showed little change in metabolic signature. Immunohistochemical analysis showed significant changes in HIF‐1alpha expression in response to 10 Gy of radiation that correlate to metabolic profiles.Conclusion:Radiation induces significant changes in metabolic activity and HIF‐1alpha expression. These alterations occur on time scales approximating the duration of common radiation treatments (approximately tens of minutes). Further understanding these dynamics has important implications with regard to improvement of therapy and biomarkers of treatment response.

Fluorescence lifetime imaging in retinal artery occlusion.

Fluorescence lifetime imaging ophthalmoscopy is a technique to measure decay times of endogenous retinal fluorophores. The purpose of this study was to investigate fluorescence lifetimes in eyes with central and branch retinal artery occlusion. Twenty-four patients with central or branch retinal artery occlusion were included in this study. The contralateral unaffected fellow eye was used as control. Measurements were performed using a fluorescence lifetime imaging ophthalmoscope based on a HRA Spectralis system. Fluorescence excitation wavelength was 473 nm, and mean lifetimes were measured in a short (498-560 nm) and in a long (560-720 nm) spectral channel. Fluorescence lifetimes in the area of retinal artery occlusion were measured and compared to corresponding areas in contralateral unaffected eyes. Additionally, findings were correlated to optical coherence tomography measurements. Retinal lifetime images of 24 patients with retinal artery occlusion were analyzed. Mean retinal fluorescence lifetimes were prolonged by 50% in the short and 20% in the long spectral channel in ischemic retinal areas up to 3 days after retinal artery occlusion compared to the contralateral unaffected eyes. In the postacute disease stage there was no difference between the lifetimes of affected areas and unaffected fellow eyes. Retinal artery occlusion leads to significantly longer fluorescence lifetimes of the retina in the acute phase and may serve as a useful indicator for acute ischemic retinal damage.

Use of time-resolved fluorescence spectroscopy to evaluate diagnostic value of collagen degradation products.

The concentration of collagen degradation products (CDPs) may reflect the process of left ventricular remodeling (LVR). The aim of this study was to evaluate the potential diagnostic usefulness of time-resolved fluorescence spectroscopy (TRFS) in assessment of CDPs. The preliminary experiment was designed to establish if CDPs’ characteristics might be visible by mean fluorescence lifetime (FLT) in determined conditions. The in vitro model of CDPs was prepared by conducting the hydrolysis of type III collagen. The FLT of samples was measured by the time-resolved spectrometer Life Spec II with the subnanosecond pulsed 360-nm EPLED diode. The FLTs were obtained by deconvolution analysis of the data using a multiexponential model of fluorescence decay. In order to determine the limit of traceability of CDPs, a comparison of different collagen/plasma ratio in samples was performed. The results of our study showed that the increase of added plasma to hydrolyzed collagen extended the mean FLT. Thus, the diagnosis of LVR based on measurements using TRFS is possible. However, it is important to point out the experiment was preliminary and further investigation in this field of research is crucial.

Non-linear imaging and characterization of atherosclerotic arterial tissue using combined SHG and FLIM microscopy.

Atherosclerosis is one of the leading causes of death in the Western World and its characterization is extremely interesting from the diagnostic point of view. Here, we employed combined SHG-FLIM microscopy to characterize arterial tissue with atherosclerosis. The shorter mean fluorescence lifetime measured within plaque depositions (1260 ± 80 ps) with respect to normal arterial wall (1480 ±100ps) allowed discriminating collagen from lipids. SHG measurements and image analysis demonstrated that the normal arterial wall has a more anisotropic Aspect Ratio (0.37 ± 0.02) with respect to plaque depositions (0.61 ± 0.02) and that the correlation length can be used for discriminating collagen fibre bundles (2.0± 0.6µm) from cholesterol depositions (4.1 ± 0.6µm). The presented method has the potential to find place in a clinical setting as well as to be applied in vivo in the near future. Graphic composition of SHG and FLIM images representing normal arterial wall and plaque depositions.

Effect of Ca²⁺ on the steady-state and time-resolved emission properties of the genetically encoded fluorescent sensor CatchER.

We previously designed a calcium sensor CatchER (a GFP-based Calcium sensor for detecting high concentrations in the high calcium concentration environment such as ER) with a capability for monitoring calcium ion responses in various types of cells. Calcium binding to CatchER induces the ratiometric changes in the absorption spectra, as well as an increase in fluorescence emission at 510 nm upon excitation at both 395 and 488 nm. Here, we have applied the combination of the steady-state and time-resolved optical methods and Hydrogen/Deuterium isotope exchange to understand the origin of such calcium-induced optical property changes of CatchER. We first demonstrated that calcium binding results in a 44% mean fluorescence lifetime increase of the indirectly excited anionic chromophore. Thus, CatchER is the first protein-based calcium indicator with the single fluorescent moiety to show the direct correlation between the lifetime and calcium binding. Calcium exhibits a strong inhibition on the excited-state proton transfer nonadiabatic geminate recombination in protic (vs deuteric) medium. Analysis of CatchER crystal structures and the MD simulations reveal the proton transfer mechanism in which the disrupted proton migration path in CatchER is rescued by calcium binding. Our finding provides important insights for a strategy to design calcium sensors and suggests that CatchER could be a useful probe for FLIM imaging of calcium in situ.

WE‐E‐BRE‐12: Tumor Microenvironment Dynamics Following Radiation

Purpose:This work aims to understand the radiation‐induced interplay between tumor oxygenation and metabolic activity. These dynamics can potentially serve as biomarkers in assessing treatment response allowing for patient‐specific adaptive radiotherapy.Methods:Using patient‐derived xenografts of head and neck cancer we assessed tumor oxygenation via fiber‐optic probe monitored hemoglobin saturation and Blood Oxygen Level Dependent (BOLD) MRI. Measurements were taken before and after a 10 Gy dose of radiation. Changes in metabolic activity were measured via Fluorescence Lifetime IMaging (FLIM) with the appropriate controls following a 10 Gy dose of radiation. FLIM can non‐invasively monitor changes in fluorescence in response to the microenvironment including being able to detect free and bound states of the intrinsically fluorescent metabolite NADH (Nicotinamide Adenine Dinucleotide). With this information FLIM can accurately quantify the metabolic state of cells that have been radiated. To model the observed changes, a two‐compartment, source‐sink simulation relating hemoglobin saturation and metabolic activity was performed using MATLAB.Results:Hemoglobin saturation as measured by interstitial probe and BOLD‐MRI decreased by 30% within 15 minutes following radiation. FLIM demonstrated a decrease in the mean fluorescence lifetime of NADH by 100 ps following 10 Gy indicating a shift towards glycolytic pathways. Simulation of radiation‐induced alterations in tumor oxygenation demonstrated that these changes can be the result of changes in either vasculature or metabolic activity.Conclusion:Radiation induces significant changes in hemoglobin saturation and metabolic activity. These alterations occur on time scales approximately the duration of common radiation treatments. Further understanding these dynamics has important implications with regard to improvement of therapy and biomarkers of treatment response.

CHARACTERIZING FLUORESCENCE LIFETIME OF NAD(P)H IN HUMAN LEUKEMIC MYELOID CELLS AND MONONUCLEAR CELLS

The aim of this ex vivo study was to explore the potential of using the fluorescence lifetime of intracellular reduced nicotinamide adenine dinucleotide (phosphate) (NAD(P)H) as a label-free indicator to characterize the differences between human leukemic myeloid cells and normal mononuclear cells (MNC). The steady-state and time-resolved autofluorescence of two human leukemic myeloid cell lines (K562, HL60) and MNC were measured by a spectrofluorimeter. According to excitation–emission matrix (EEM) analysis, the optimal emission of NAD(P)H in these cells suspensions occurred at 445 nm. Furthermore, the fluorescence lifetimes of NAD(P)H in leukemic myeloid cells and MNC were determined by fitting the time-resolved autofluorescence data. The mean fluorescence lifetimes of NAD(P)H in K562, HL60, and MNC cells were 5.57 ± 1.19, 4.45 ± 0.71, and 7.31 ± 0.60 ns, respectively. There was a significant difference in the mean lifetime of NAD(P)H between leukemic myeloid cells and MNC (p &lt; 0.05). The difference was essentially caused by the change in relative concentration of free and protein-bound NAD(P)H. This study suggests that the mean fluorescence lifetime of NAD(P)H might be a potential label-free indicator for differentiating leukemic myeloid cells from MNC.

Multiphoton laser tomography and fluorescence lifetime imaging of melanoma: morphologic features and quantitative data for sensitive and specific non-invasive diagnostics.

Multiphoton laser tomography (MPT) combined with fluorescence lifetime imaging (FLIM) is a non-invasive imaging technique, based on the study of fluorescence decay times of naturally occurring fluorescent molecules, enabling a non-invasive investigation of the skin with subcellular resolution. The aim of this retrospective observational ex vivo study, was to characterize melanoma both from a morphologic and a quantitative point of view, attaining an improvement in the diagnostic accuracy with respect to dermoscopy. In the training phase, thirty parameters, comprising both cytological descriptors and architectural aspects, were identified. The training set included 6 melanomas with a mean Breslow thickness±S.D. of 0.89±0.48 mm. In the test phase, these parameters were blindly evaluated on a test data set consisting of 25 melanomas, 50 nevi and 50 basal cell carcinomas. Melanomas in the test phase comprised 8 in situ lesions and had a mean thickness±S.D. of 0.77±1.2 mm. Moreover, quantitative FLIM data were calculated for special areas of interest. Melanoma was characterized by the presence of atypical short lifetime cells and architectural disorder, in contrast to nevi presenting typical cells and a regular histoarchitecture. Sensitivity and specificity values for melanoma diagnosis were 100% and 98%, respectively, whereas dermoscopy achieved the same sensitivity, but a lower specificity (82%). Mean fluorescence lifetime values of melanocytic cells did not vary between melanomas and nevi, but significantly differed from those referring to basal cell carcinoma enabling a differential diagnosis based on quantitative data. Data from prospective preoperative trials are needed to confirm if MPT/FLIM could increase diagnostic specificity and thus reduce unnecessary surgical excisions.