Time-gated Fluorescence Spectroscopy Research Articles

Fluorescence Imaging of Breast Tumors and Gastrointestinal Cancer.

Optical imaging offers a high potential for noninvasive detection and therapy of cancer in humans. Recent advances in instrumentation for diffuse optical imaging have led to new capabilities for the detection of cancer in highly scattering tissue such as the female breast. In particular, fluorescence imaging was made applicable as a sensitive technique to image molecular probes in vivo. We review recent developments in the detection of breast cancer and fluorescence-guided surgery of the breast by contrast agents available for application on humans. Detection of cancer has been investigated with the unspecific contrast agents "indocyanine green" and "omocianine" so far. Hereby, indocyanine green was found to offer high potential for the differentiation of malignant and benign lesions by exploiting vessel permeability for macromolecules as a cancer-specific feature. Tumor-specific molecular targeting and activatable probes have been investigated in clinical trials for fluorescence-guided tumor margin detection. In this application, high spatial resolution can be achieved, since tumor regions are visualized mainly at the tissue surface. As another example of superficial tumor tissue, imaging of lesions in the gastrointestinal tract is discussed. Promising results have been obtained on high-risk patients with Barrett´s esophagus and with ulcerative colitis by administering 5-aminolevulinic acid which induces accumulation of protoporphyrin IX serving as a tumor-specific fluorescent marker. Time-gated fluorescence imaging and spectroscopy are effective ways to suppress underlying background from tissue autofluorescence. Furthermore, recently developed tumor-specific molecular probes have been demonstrated to be superior to white-light endoscopy offering new ways for early detection of malignancies in the gastrointestinal tract.

Performance Evaluation of a Time-Gated Fluorescence Spectroscopy Measurement System for the Classification and Recycling of Plastics.

Plastics have many unrivaled properties and are thus indispensable materials with an ever-increasing production. Since the plastics recycling rates are still small, the plastic waste that ends up on landfills, in the environment including oceans, and incineration plants also steadily grows. Large-scale automated sorting of waste plastics may help solve the problem. However, most state-of-the-art approaches are limited to a (very) small number of plastic types that can be simultaneously sorted and have severe difficulties with black plastics. The labeling of plastics with fluorescent tracers ("markers") during their production has the potential to enable the fast and highly reliable classification and automated sorting of many different plastic types simultaneously. Unfortunately, the unique marker fluorescence ("optical fingerprint") might be obscured by the autofluorescence (AF) often emitted by plastics. Recently, we have shown that time-gated fluorescence spectroscopy (TGFS) can be successfully applied to eliminate the influences from AF. In this study, we present the prototype of a TGFS measurement system for the classification and sorting of plastics and evaluate its performance in a typical industrial situation. With 150 000 investigated plastics flakes from 10 different plastic types that were labeled with binary combinations of six fluorescent markers, an overall plastics recovery rate (mean true positive rate ) of 99.76%, and a plastics purity (positive predictive value ) of 99.88% were achieved. Additional simulations that were carried out based upon the experiments allow a prediction of the performance as a function of the signal-to-noise (S/N) ratio such that various system parameters can be adapted to the particular situation.

Optimized Time-Gated Fluorescence Spectroscopy for the Classification and Recycling of Fluorescently Labeled Plastics.

For the production of high-quality parts from recycled plastics, a very high purity of the plastic waste to be recycled is mandatory. The incorporation of fluorescent tracers ("markers") into plastics during the manufacturing process helps overcome typical problems of non-tracer based optical classification methods. Despite the unique emission spectra of fluorescent markers, the classification becomes difficult when the host plastics exhibit (strong) autofluorescence that spectrally overlaps the marker fluorescence. Increasing the marker concentration is not an option from an economic perspective and might also adversely affect the properties of the plastics. A measurement approach that suppresses the autofluorescence in the acquired signal is time-gated fluorescence spectroscopy (TGFS). Unfortunately, TGFS is associated with a lower signal-to-noise (S/N) ratio, which results in larger classification errors. In order to optimize the S/N ratio we investigate and validate the best TGFS parameters-derived from a model for the fluorescence signal-for plastics labeled with four specifically designed fluorescent markers. In this study we also demonstrate the implementation of TGFS on a measurement and classification prototype system and determine its performance. Mean values for a sensitivity of [Formula: see text] = 99.93% and precision [Formula: see text] = 99.80% were achieved, proving that a highly reliable classification of plastics can be achieved in practice.

Time-gated fluorescence spectroscopy improves endoscopic detection of low-grade dysplasia in ulcerative colitis

Dysplasia in ulcerative colitis is frequently missed with 4-quadrant biopsies. An experimental setup recording delayed fluorescence spectra simultaneously with white light endoscopy was recently developed. We compared detection of invisible flat intraepithelial neoplasia with protoporphyrin IX fluorescence and standard 4-quadrant biopsies. Prospective, crossover design without randomization of the order of procedures. Gastroenterology Department, Humboldt University, Charité, Berlin, Germany. Forty-two patients with extensive ulcerative colitis of more than 10 years' duration were included. Colonoscopy with 4-quadrant biopsies and targeted biopsies of macroscopic lesions and time-gated fluorescence-guided colonoscopy were performed 2 weeks apart by 2 blinded endoscopists. Three independent pathologists examined the biopsy specimens. The primary outcome criterion was detection rate of invisible flat intraepithelial neoplasia. Invisible flat intraepithelial neoplasia was detected in 3 (7%) patients by white light 4-quadrant biopsies and in 10 (24%) patients by fluorescence-guided endoscopy (P = .02). The sensitivity and specificity for differentiating patients with and without dysplasia were 100% and 81%, respectively. Dysplastic and nondysplastic mucosa could be discriminated with a sensitivity and specificity of 73% and 81%, respectively. The trial was not randomized. The detection rate of intraepithelial neoplasia in patients with ulcerative colitis can be improved by fluorescence-guided colonoscopy.

Investigating Desorption of Native Pyrene from Sediment on Minute- to Month-Timescales by Time-Gated Fluorescence Spectroscopy

We investigated desorption of native pyrene from field-aged sediments using time-gated, laser-induced fluorescence (LIF) spectroscopy. LIF is superior to conventional analytical methods for the measurement of quickly changing dissolved pyrene because it allows observations at minute-scale resolution, has a low detection limit (approximately 1 ng/L), and minimizes sample loss and disturbance since it requires no system subsampling and chemical analysis. The efficacy of LIF was demonstrated in studies of pyrene desorption from Boston Harbor sediment segregated into different size-fractions (38-75, 75-106, and 180-250 microm diameter) and used in varying solid-to-water ratios (20, 70, and 280 mg(solid)/L). The effects of particle size and solid loading on desorption were consistent with diffusion physics. For suspension conditions between 20 and 280 mg(solids)/L, we observed desorption continuing toward an apparent plateau level over the course of weeks to months. This implies that the characteristic desorption time of pyrene from fine sediments and, by inference, other sediment-bound hydrophobic organic compounds (HOCs) of similar hydrophobicity, exceeds the typical characteristic times for pore water flushing and resuspension events. Consequently, the assumption of local sorption equilibrium in modeling efforts would be inappropriate.

Endoscopic detection of dysplasia in inflammatory bowel disease using time-gated laser-induced fluorescence spectroscopy

Endoscopic detection of dysplasia in inflammatory bowel disease using time-gated laser-induced fluorescence spectroscopy

Endoscopic detection of dysplasia in inflammatory bowel disease using time-gated laser-induced fluorescence spectroscopy

Endoscopic detection of dysplasia in inflammatory bowel disease using time-gated laser-induced fluorescence spectroscopy

Energy transfer spectroscopy for measuring mitochondrial metabolism in living cells.

Microscopic energy transfer spectroscopy was established using mixed solutions of reduced nicotinamide adenine dinucleotide (NADH) and the mitochondrial marker rhodamine 123 (R123). This method was applied to probe mitochondrial malfunction of cultivated endothelial cells from calf aorta incubated with various inhibitors of specific enzyme complexes of the respiratory chain. Autofluorescence of the coenzyme NADH as well as energy transfer efficacy from excited NADH molecules (energy donor) to R123 (energy acceptor) were measured by time-gated fluorescence spectroscopy. Because intermolecular distances in the nanometer range are required for radiationless energy transfer, this method is suitable to probe selectively mitochondrial NADH. Autofluorescence of endothelial cells usually exhibited a weak increase after specific inhibition of enzyme complexes of the respiratory chain. In contrast, pronounced and statistically significant changes of energy transfer efficacy were observed after inhibition of the same enzyme complexes. Detection of NADH and R123 in different nanosecond time gates following the exciting laser pulses enhances the selectivity and improves quantification of fluorescence measurements. Therefore, time-gated energy transfer spectroscopy is suggested to be an appropriate tool for probing mitochondrial malfunction.

Time-gated microscopic energy transfer measurements for probing mitochondrial metabolism

Spectroscopic and microscopic methods for probing mitochondrial malfunction were established using cultivated endothelial cells from the calf aorta and various inhibitors of the respiratory chain, which is located at the inner mitochondrial membrane. Time-gated fluorescence spectroscopy was used to measure autofluorescence of the coenzyme NADH as well as “energy transfer efficacy” from excited NADH molecules (energy donor) to the mitochondrial marker rhodamine-123 (energy acceptor). Autofluorescence usually exhibited a weak increase after specific inhibition of enzyme complexes of the respiratory chain. In contrast, a pronounced increase in energy transfer efficacy was observed after inhibition of the same enzyme complexes. The detection of donor (NADH) and acceptor (R123) fluorescence in different nanosecond time gates following the exciting laser pulses enhances selectivity and improves quantification of energy transfer measurements. Therefore, timegated energy transfer spectroscopy is suggested to be an appropriate tool for probing mitochondrial malfunction.

Microspectrofluorometry, fluorescence imaging and confocal microscopy of an endogenous pigment of the marine ciliate Fabrea salina

Fabrea salina, an apparently colorless marine ciliate in the order Heterotrichida, shows positive phototaxis and photophobic step-down reactions. Fluorescence microscopy and imaging of living cells reveal only a scarcely detectable emission, but dead cells are characterized by a strong red fluorescence, results indicating the presence of an endogenous fluorescent pigment. Single cell microspectrofluorometry, fluorescence imaging and laser confocal microscopy studies, together with time-gated fluorescence spectroscopy of cell suspensions, were performed in order to investigate the nature and localization of this pigment. The fluorescence emission spectrum at different excitation wavelenghts (from 337 to 425 nm) exhibits two peaks: the main one between 590 and about 615 nm, depending on the cell treatment, and the other one at about 655 nm. The time decay of fluorescence shows three distinct components, with lifetimes ranging from a few hundred picoseconds to some nanoseconds and relative contributions varying with the chemical treatment. Time-gated spectroscopy allows us also to observe differences in the espectral features of the distinct emitting species. Laser confocal microscopy reveals that the pigment is localized just below the cell membrane. The spectral and temporal fluorescence features of the endogenous pigment present in F. salina are very similar to the ones of blepharismin, the pigment of Blepharisma japonicum, thus suggesting that the F. salina pigment might also be a hypericin-like pigment.

Time-gated microscopic imaging and spectroscopy in medical diagnosis and photobiology [also Erratum 33(11)3828(Nov1994)

An experimental setup was developed for time-gated (nanosecond) fluorescence spectroscopy and imaging of microscopic samples. This makes it possible to depict individual components of complex fluorophores and to measure specific metabolites on the basis of their decay times. The field of applications includes the selective detection of intrinsic fluorophores and of photosensitizers in single cells, skin, and teeth, as well as of photosynthetic pigments in plants under various stress conditions.

Time-gated fluorescence spectroscopy of porphyrin derivatives and aluminium phthalocyanine incorporated in vivo in a murine ascitic tumour model

The effect of systemic administration on drug uptake at cellular level was evaluated using time-gated fluorescence spectroscopy performed on a murine ascitic tumour model. Mice bearing L1210 leukaemia were injected intraperitoneally or intravenously with 25 mg per kg body weight hematoporphyrin derivative (HpD), 12.5 mg per kg body weight photofrin II (PII), 25 or 5 mg per kg body weight disulphonated aluminium phthalocyanine (AlS 2Pc). Every 2 h and for up to 22 or 30 h, mice were sacrificed, leukaemic cells extracted from the peritoneum, washed, and resuspended in buffer for fluorescence measurements. HpD and PII emission spectra were almost identical 12 h after intraperitoneal injection with main peaks at 630 nm and no appreciable changes afterwards. In the first 12 h, the PII fluorescence spectrum was constant, while in the case of HpD a shoulder at 615 nm was detectable. Similar fluorescence behaviour was observed after intravenous administration of porphyrin derivatives. These results seem to confirm that the tumour localizing fraction is the part actually retained by the cells. The AlS 2Pc spectrum peaked at 685 nm and did not change in any of our experiments. AlS 2Pc is incorporated more rapidly with respect to porphyrins, as was clearly observed in the case of intravenous administration, where the AlS 2Pc fluorescence was readily detectable after 2 h, whereas the PII emission became apparent only after 4–6 h.

Time-gated fluorescence spectroscopy of porphyrin derivatives incorporated into cells

Time-gated fluorescence spectroscopy was performed on the tumour-localizing fraction (TLF) of haematoporphyrin derivative (HPD) incorporated into cells. Three different cell lines were incubated with 20 and 5 μg ml −1 of TLF for various time periods; they were then washed and resuspended in buffer. Fluorescence decay measurements and time-integrated and time-gated spectra were then obtained from the cell suspensions. Similar experiments were repeated using HPD containing 60% of the active material. The experimental results show a modification of the emission spectra for both drugs depending on the incubation time; this modification is more significant for the TLF. In particular, the emission peak observed in aqueous solution at 615 nm is shifted to 630 nm as a consequence of incorporation into cells, and the gated spectra indicate that the fluorescence emission is mainly related to monomers and unfolded polymeric chains. The ratio between the intensities of the two peaks depends on the relative amount of the TLF; the peak at 615 nm is more pronounced for HPD. The results obtained seem to indicate that both the composition of the drug and the metabolic properties of the biological environment strongly influence the uptake process and the fluorescence behaviour of the incorporated sensitizer.

TIME‐GATED FLUORESCENCE SPECTROSCOPY OF THE TUMOR LOCALIZING FRACTION OF HpD IN THE PRESENCE OF CATIONIC SURFACTANT

Time-gated fluorescence spectroscopy was performed on the tumor localizing fraction (TLF) of HpD in buffer at different concentrations of cationic surfactant. This technique obtains emission spectra with programmable delay relative to the excitation pulse. According to the measured fluorescence decay-time constants (approximately 0.7, approximately 3 and approximately 15 ns) three gates were considered, delayed by 0, 5 and 18 ns, respectively, to evaluate the contribution of the emitting molecular species to the spectra. Simultaneous to these measurements, fluorescence decay waveforms and time-integrated spectra were also detected. In buffer and in detergent micelles the fluorescence spectra are given by the superposition of the emission of the different molecular species present in the solution, and no appreciable interaction among the chromophores is observed. On the contrary, in the pre-micellar range of the surfactant, evidence for the existence of an energy transfer mechanism was found. This effect seems to be related to the configurational state of the TLF polymeric chains and depends on the relative TLF/surfactant concentration.