Fluorescence lifetime imaging and electron microscopy: a correlative approach.

Fluorescence lifetime imaging microscopy (FLIM) allows the characterization of cellular metabolism by quantifying the rate of free and unbound nicotinamide adenine dinucleotide hydrogen (NADH). This study delineates the correlative imaging of cells with FLIM and electron microscopy (EM). Human fibroblasts were cultivated in a microscopy slide bearing a coordinate system and FLIM measurement was conducted. Following chemical fixation, embedding in Epon and cutting with an ultramicrotome, tomograms of selected cells were acquired with a scanning transmission electron microscope (STEM). Correlative imaging of antimycin A-treated fibroblasts shows a decrease in fluorescence lifetime as well as swollen mitochondria with large cavities in STEM tomography. To our knowledge, this is the first correlative FLIM and EM workflow. Combining the high sensitivity of FLIM with the high spatial resolution of EM could boost the research of pathophysiological processes involving cell metabolism, such as cancer, neurodegenerative disorders, and viral infection.

Fast-tunable femtosecond visible radiation via sum-frequency generation from a high power NIR NOPO.

We report on a high power ultra-broadband, quickly tunable non-collinear parametric oscillator with highly efficient intra-cavity sum-frequency generation. It simultaneously delivers femtosecond pulses in two synchronized output beams: up to 4.9 W tunable from 650 to 1050 nm in the near infrared and up to 1.9 W from 380 to 500 nm in the visible spectral range. The (to our knowledge) novel source is ideally suited for spectroscopy or multi-color imaging. First results of two-color functional microscopy are presented.

Simultaneous Phosphorescence and Fluorescence Lifetime Imaging by Multi-Dimensional TCSPC and Multi-Pulse Excitation.

TCSPC FLIM/PLIM is based on a multi-dimensional time-correlated single-photon counting process. The sample is scanned by a high-frequency-pulsed laser beam which is additionally modulated on/off synchronously with the pixels of the scan. FLIM is obtained by building up the distribution of the photons over the scanning coordinates and the times of the photons in the excitation pulse sequence, PLIM is obtained by building up the photon distribution over the scanning coordinates and the photon times in the modulation period. FLIM and PLIM data are thus obtained simultaneously within the same imaging process. Since the technique uses not only one but many excitation pulses for every phosphorescence signal period the sensitivity is much higher than for techniques that excite with a single pulse only. TCSPC FLIM/PLIM works both with one-photon and two-photon excitation, does not require a reduction of the laser pulse repetition rate by a pulse picker, and eliminates the need of high pulse energy for phosphorescence excitation.

Quenched coumarin derivatives as fluorescence lifetime phantoms for NADH and FAD.

Two-photon fluorescence lifetime imaging is a versatile laboratory technique in the field of biophotonics and its importance is also growing in the field of in vivo diagnostics for medical purposes. After years of experience in dermatology, endoscopic implementations of the technique are now posing new technical challenges. To develop, test, and compare instrumental solutions for this purpose suitable reference samples have been devised and tested. These reference samples can serve as reliable NADH- and FAD-mimicking optical phantoms for 2-photon fluorescence lifetime imaging, as they can be prepared relatively easily with reproducible and stable characteristics for this quite relevant diagnostic technique. The reference samples (mixtures of coumarin 1 and coumarin 6 in ethanol with suitable amounts of 4-hydroxy-TEMPO) have been tuned to exhibit spectral and temporal fluorescence characteristics very similar to those of NADH and FAD, the two molecules most frequently utilized to characterize cell metabolism.

Correlation of intracellular oxygen and cell metabolism by simultaneous PLIM of phosphorescent TLD1433 and FLIM of NAD(P)H.

During photodynamic therapy (PDT), disruption of cell respiration and metabolic changes could be one of the first events. Photophysical characteristics of the photosensitizer (PS) and its specific redox potential define consumption of molecular oxygen followed by generation of reactive oxygen species. The potential PS TLD1433 is based on transition metal Ru(II) and possess an oxygen-dependent luminescence. This enables the study of oxygen consumption by PS-phosphorescence lifetime imaging (PLIM) and simultaneously changes the cellular metabolic state by nicotinamide adenine dinucleotide (NAD(P)H)-fluorescence lifetime imaging (FLIM). Within this study, localization and cellular function of TLD1433 is investigated in bladder carcinoma cells using time-resolved and confocal laser scanning microscopy. Simultaneous FLIM/PLIM of NAD(P)H and TLD1433 during PDT correlated oxygen consumption, redox state and cellular energy metabolism. Our investigations aimed to provide a personalized protocol in theranostic PDT procedures and demonstrate the potential use of TLD1433 PDT also under hypoxic conditions, which are otherwise difficult to treat.

Correlative NAD(P)H-FLIM and oxygen sensing-PLIM for metabolic mapping.

Cellular responses to oxygen tension have been studied extensively. Oxygen tension can be determined by considering the phosphorescence lifetime of a phosphorescence sensor. The simultaneous usage of FLIM of coenzymes as NAD(P)H and FAD(+) and PLIM of oxygen sensors could provide information about correlation of metabolic pathways and oxygen tension. We investigated correlative NAD(P)H-FLIM and oxygen sensing-PLIM for simultaneously analyzing cell metabolism and oxygen tension. Cell metabolism and pO2 were observed under different hypoxic conditions in squamous carcinoma cell cultures and in complex ex vivo systems. Increased hypoxia induced an increase of the phosphorescence lifetime of Ru(BPY)3 and in most cases a decrease in the lifetime of NAD(P)H which is in agreement to the expected decrease of the protein-bound NAD(P)H during hypoxia. Oxygen was modulated directly in the mitochondrial membrane. Blocking of complex III and accumulation of oxygen could be observed by both the decrease of the phosphorescence lifetime of Ru(BPY)3 and a reduction of the lifetime of NAD(P)H which was a clear indication of acute changes in the redox state of the cells. For the first time simultaneous FLIM/PLIM has been shown to be able to visualize intracellular oxygen tension together with a change from oxidative to glycolytic phenotype.

PDT with TOOKAD(®) studied in the chorioallantoic membrane of fertilized eggs.

BACKGROUND: The potential application of TOOKAD(®)-PDT for the treatment of blood vessels was investigated. TOOKAD(®) (WST09), a novel palladium-bacteriopheophorbide absorbs light in the near IR with a high quantum yield of intersystem crossing. Our study assessed the efficacy of this drug in inducing vascular damage with a view to its possible use in the treatment of age-related macular degeneration. METHODS: Vascular damage of TOOKAD(®)-PDT was studied in neovessels of the chorioallantoic membrane of fertilized eggs. Pharmacokinetic investigations were done by video microscopy and laser scanning microscopy. To induce damage vessels were irradiated with 763nm light from a diode laser. RESULTS: TOOKAD(®) was accumulated in the vessels in the first minutes following injection. TOOKAD(®) fluorescence was seen predominantly in the lumen and not in the vascular endothelial layer. Although fluorescence was very weak it could be attributed to TOOKAD(®) from the fluorescence spectrum in the circulation. Damage assessment was done 24h after application of 763nm light. No significant difference in the degree of damage was observed with different short drug-light intervals (1-10min), but damage increased with the light energy dose. Closure of smaller vessels and vanished capillaries could be achieved by irradiation with 5J/cm(2) and a TOOKAD(®) dose of 33µg/embryo, corresponding to a phototoxic efficacy of 0.0062. CONCLUSIONS: From the results discussed in this work, TOOKAD(®) could be a potential drug for the PDT of age-related macular degeneration in which the growth of new vessels in the choroids can lead to loss of vision.

Time-resolved microspectrofluorometry and fluorescence lifetime imaging of photosensitizers using picosecond pulsed diode lasers in laser scanning microscopes.

This work describes the time-resolved fluorescence characteristics of two different photosensitizers in single cells, in detail mTHPC and 5-ALA induced PPIX, which are currently clinically used in photodynamic therapy. The fluorescence lifetime of the drugs was determined in the cells from time-gated spectra as well as single photon counting, using a picosecond pulsed diode laser for fluorescence excitation. The diode laser, which emits pulses at 398 nm with 70 ps full width at half maximum duration, was coupled to a confocal laser scanning microscope. For time-resolved spectroscopy a setup consisting of a Czerny Turner spectrometer and a MCP-gated and -intensified CCD camera was used. Time-gated spectra within the cells were acquired by placing the laser beam in "spot scan" mode. In addition, a time-correlated single photon counting module was used to determine the fluorescence lifetime from single spots and to record lifetime images. The fluorescence lifetime of mTHPC decreased from 7.5 to 5.5 ns during incubation from 1 to 6 h. This decrease was probably attributed to enhanced formation of aggregates during incubation. Fluorescence lifetime imaging showed that longer lifetimes were correlated with accumulation in the cytoplasm in the neighborhood of the cell nucleus, whereas shorter lifetimes were found in the outer cytoplasm. For cells that were incubated with 5-ALA, a fluorescence lifetime of 7.4 ns was found for PPIX; a shorter lifetime at 3.6 ns was probably attributed to photoproducts and aggregates of PPIX. In contrast from fluorescence intensity images alone, different fluorescence species could not be distinguished. However, in the lifetime image a structured fluorescence distribution in the cytoplasm was correlated with the longer lifetime and probably coincides with mitochondria. In conclusion, picosecond diode lasers coupled to a laser scanning microscope equipped with appropriate detection units allows time-resolved spectroscopy and lifetime imaging with high spatial resolution and provides numerous possibilities in cellular and pharmaceutical research.

Spectrally resolved fluorescence lifetime imaging to investigate cell metabolism in malignant and nonmalignant oral mucosa cells.

Fluorescence-guided diagnosis of tumor tissue is in many cases insufficient, because false positive results interfere with the outcome. Improvement through observation of cell metabolism might offer the solution, but needs a detailed understanding of the origin of autofluorescence. With respect to this, spectrally resolved multiphoton fluorescence lifetime imaging was investigated to analyze cell metabolism in metabolic phenotypes of malignant and nonmalignant oral mucosa cells. The time-resolved fluorescence characteristics of NADH were measured in cells of different origins. The fluorescence lifetime of bound and free NADH was calculated from biexponential fitting of the fluorescence intensity decay within different spectral regions. The mean lifetime was increased from nonmalignant oral mucosa cells to different squamous carcinoma cells, where the most aggressive cells showed the longest lifetime. In correlation with reports in the literature, the total amount of NADH seemed to be less for the carcinoma cells and the ratio of free/bound NADH was decreased from nonmalignant to squamous carcinoma cells. Moreover for squamous carcinoma cells a high concentration of bound NADH was found in cytoplasmic organelles (mainly mitochondria). This all together indicates that oxidative phosphorylation and a high redox potential play an important role in the energy metabolism of these cells.

Fluorescence investigation of the detachment of aluminum phthalocyanine molecules from aluminum phthalocyanine nanoparticles in monocytes/macrophages and skin cells and their localization in monocytes/macrophages.

BACKGROUND: Nanoparticles made from aluminum phthalocyanine (AlPc) are non-fluorescent in the nanoparticle form. Once AlPc molecules become detached from the particle, fluorescence occurs. Preliminary work showed the benefit of using aluminum phthalocyanine nanoparticles (nAlPc) for the rating of the rejection risk of skin autografts in mice by measuring fluorescence intensities of detached AlPc. Skin autografts showing a high fluorescence intensity were finally rejected suggesting an inflammatory process. In contrast, autografts with normal autofluorescence were accepted. This work was focused on the mechanism of this finding. The aim is detecting inflammatory processes and the potential use of nAlPc for PDT as a new treatment modality. METHODS: The effect of the lipopolysaccharide-stimulated monocyte/macrophage murine cell line J774A.1 on the monomerization of internalized nAlPc was tested. Further, we investigated the influence of J774A.1 cells and the normal skin cell lines L-929 or HaCaT on the dissolution of nAlPc by laser scanning microscopy and flow cytometry. Localization of AlPc molecules after uptake and dissolution of nanoparticles by the cells was surveyed. RESULTS: In co-culture models composed of J774A.1 and HaCaT/L-929 cells, the AlPc fluorescence intensity in J774A.1 cells is 1.38/1.89 fold higher, respectively. According to localization measurements in J774A.1 cells it can be assumed that nAlPc is taken up via endocytosis and remains in endosomes and/or lysosomes dissolving there. Detached molecules of AlPc cause rapture of the endosomal and/or lysosomal membrane after irradiation to become quite uniformly distributed in the cytoplasm. CONCLUSIONS: Evidence for monocytes/macrophages being the origin of the measured AlPc fluorescence in rejected skin autografts was confirmed.

Role of mitochondria in cell death induced by Photofrin-PDT and ursodeoxycholic acid by means of SLIM.

The present study was undertaken to find new ways to improve efficacy of photodynamic therapy (PDT). We investigated the combinatory effect of the photosensitizer Photofrin and ursodeoxycholic acid (UDCA). UDCA is a relatively non-toxic bile acid which is used inter alia as a treatment for cholestatic disorders and was reported to enhance PDT efficiency of two other photosensitizers. Since besides necrosis and autophagic processes apoptosis has been found to be a prominent form of cell death in response to PDT for many cells in culture, several appropriate tests, such as cytochrome c release, caspase activation and DNA fragmentation were performed. Furthermore spectral resolved fluorescence lifetime imaging (SLIM) was used to analyse the cellular composition of Photofrin and the status of the enzymes of the respiratory chain. Our experiments with two human hepatoblastoma cell lines revealed that the combination of Photofrin with UDCA significantly enhanced efficacy of PDT for both cell lines even though the underlying molecular mechanism for the mode of action of Photofrin seems to be different to some extent. In HepG2 cells cell death was clearly the consequence of mitochondrial disturbance as shown by cytochrome c release and DNA fragmentation, whereas in Huh7 cells these features were not observed. Other mechanisms seem to be more important in this case. One reason for the enhanced PDT effect when UDCA is also applied could be that UDCA destabilizes the mitochondrial membrane. This could be concluded from the fluorescence lifetime of the respiratory chain enzymes which turned out to be longer in the presence of UDCA in HepG2 cells, suggesting a perturbation of the mitochondrial membrane. The threshold at which PDT damages the mitochondrial membrane was therefore lower and correlated with the enhanced cytochrome c release observed post PDT. Thus enforced photodamage leads to a higher loss of cell viability.