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PubMed1975

Fluorescence intensity changes associated with contractile activation in frog muscle stained with Nile Blue A.

1. Extrinsic fluorescence intensity changes were studied in frog semitendinosus muscles stained with Nile Blue A in response to electrical stimulation. Muscles were stretched and put into hypertonic solutions to prevent movement. The muscles were illuminated at 90 degrees to their long axis with a narrow beam of light at a central wave-length of 6250 . Fluorescence emission was measured at 90 degrees to the exciting light using a filter which absorbed light of wave-lengths shorter than 6400 . 2. In response to a single stimulus the fluorescence intensity increases briefly. The fluorescence response is propagated at a constant velocity of about 1.5 m/sec. The average ratio of the maximum fluorescence intensity change to the resting fluorescence is 4.5 times 10-3 for supramaximal shocks. The fluorescence intensity change starts early in the falling phase of the action potential. 3. The fluorescence intensity change increases when nitrate replaces chloride and decreases when D2O replaces H2O. The rates of rise and fall of the fluorescence change was unaffected by nitrate replacement of chloride but are slowed where D2O replaces H2O. The rates of rise and fall of the fluorescence change increase with increasing temperature for all solutions used. The peak fluorescence intensity change, however, goes through a maximum at about 17 degrees C for aqueous chloride and nitrate solutions in the range of 10-25 degrees C. With D2O solutions, the peak fluorescence intensity increases monotonically in this range of temperatures. 4. The fluorescence intensity change in response to trains of action potentials are not additive. 5. Depolarization of muscles treated with tetrodotoxin using triangular-shaped fluid electrodes produces an increase in fluorescence at about the same threshold values required to elicit tension in preparations that are not fully stretched. The fluorescence intensity change precedes in time tension development. Near threshold depolarizations, the delay in onset of the fluorescence response can be 80 msec or longer. Byond threshold, delays become shorter and peak responses larger. During maintained depolarization, after the peak response, fluorescence declines to a plateau value. 6. The results suggest that the fluorescence intensity changes are associated with excitation-contraction coupling, possibly with changes in the transmembrane potential of the sarcoplasmic reticulum.

Action Potentials↗

PubMed1992

Pyrene-labeled cardiac troponin C. Effect of Ca2+ on monomer and excimer fluorescence in solution and in myofibrils.

The two cysteine residues (Cys-35 and Cys-84) of bovine cardiac troponin C (cTnC) were labeled with the pyrene-containing SH-reactive compounds, N-(1-pyrene) maleimide, and N-(1-pyrene)iodoacetamide in order to study conformational changes in the regulatory domain of cTnC associated with cation binding and cross-bridge attachment. The labeled cTnC exhibits the characteristic fluorescence spectrum of pyrene with two sharp monomer fluorescence peaks and one broad excimer fluorescence peak. The excimer fluorescence results from dimerization of adjacent pyrene groups. With metal binding (Mg2+ or Ca2+) to the high affinity sites of cTnC (sites III and IV), there is a small decrease in monomer fluorescence but no effect on excimer fluorescence. In contrast, Ca2+ binding to the low affinity regulatory (site II) site elicits an increase in monomer fluorescence and a reduction in excimer fluorescence. These results can be accounted for by assuming that the pyrene attached to Cys-84 is drawn into a hydrophobic pocket formed by the binding of Ca2+ to site II. When the labeled cTnC is incorporated into the troponin complex or substituted into cardiac myofibrils the monomer fluorescence is enhanced while the excimer fluorescence is reduced. This suggests that the association with other regulatory components in the thin filament might influence the proximity (or mobility) of the two pyrene groups in a way similar to that of Ca2+ binding. With the binding of Ca2+ to site II the excimer fluorescence is further reduced while the monomer fluorescence is not changed significantly. In myofibrils, cross-bridge detachment (5 mM MgATP, pCa 8.0) causes a reduction in monomer fluorescence but has no effect on excimer fluorescence. However, saturation of the cTnC with Ca2+ reduces excimer fluorescence but causes no further change in monomer fluorescence. Thus, the pyrene fluorescence spectra define the different conformations of cTnC associated with weak-binding, cycling, and rigor cross-bridges.

Animals↗

PubMed2002

Flavonoid distribution in tissues of Phillyrea latifolia L. leaves as estimated by microspectrofluorometry and multispectral fluorescence microimaging.

A new method for detecting the tissue-specific distribution of flavonoids has been developed by coupling microspectrofluorometry and multispectral fluorescence microimaging techniques. Fluorescence responses of cross sections taken from 1 year old Phillyrea latifolia leaves exposed to full (sun leaves) or 15% (shade leaves) solar radiation in a coastal area of Southern Tuscany were analyzed. Fluorescence spectra of different tissue layers, each normalized at its fluorescence maximum, that were stained or not stained with Naturstoff reagent A (in ethanol), under excitation with UV light (lambdaexc = 365 nm) or blue light (lambdaexc = 436 nm) were recorded. The shape of the fluorescence spectra of tissue layers from shade and sun leaves differed only under UV excitation. The fluorescence of stained cross sections from sun and shade leaves as well as from different layers of sun leaves received a markedly different contribution from the blue (470 nm) and the yellow-red (580 nm) wavebands. Such changes in tissue fluorescence signatures were related to light-induced changes of extractable caffeic acid derivatives and flavonoid glycosides, namely quercetin 3-O-rutinoside and luteolin 7-O-glucoside. Wall-bound phenolics, i.e. hydroxycinnamic acids (p-coumaric, ferulic and caffeic acid) and flavonoids (apigenin and luteolin derivatives), did not substantially differ between sun and shade leaves. A Gaussian deconvolution analysis of fluorescence spectra was subsequently performed to estimate the contribution of flavonoids (emitting at 600 nm, F600 [red fluorescence contribution = signal integrated over a Gaussian band centered at about 600 nm]) relative to the tissue fluorescence (Ftot [total fluorescence = signal integrated over the whole fluorescence spectrum]). The F600/ Ftot ratios sharply differed between analogous tissues of sun and shade leaves, as well as among tissue layers within each leaf type. A highly resolved picture of the tissue flavonoid distribution was finally provided through a fluorescence microimaging technique by acquiring fluorescence images at the blue (fluorescence at about 470 nm [F470]) and yellow-red (fluorescence at about 580 nm [F580]) wavelengths and correcting the F580 image for the contribution of nonflavonoids to the fluorescence at 580 nm. Monochrome images were elaborated by adequate computing functions to visualize the exclusive accumulation of flavonoids in different layers of P. latifolia leaves. Our data show that in shade leaves flavonoids almost exclusively occurred in the adaxial epidermal layer. In sun leaves flavonoids largely accumulated in the adaxial epidermal and subepidermal cells and followed a steep gradient passing from the adaxial epidermis to the inner spongy layers. Flavonoids also largely occurred in the abaxial epidermal cells and constituted the exclusive class of phenylpropanoids synthesized by the cells of glandular trichomes. The proposed method also allowed for the discrimination of the relative abundance of hydroxycinnamic derivatives and flavonoids in different layers of the P. latifolia leaves.

Flavonoids↗

PubMed2006

[Study of enhancement effects in X-ray fluorescence analysis for multi-layer samples].

The enhancement effects in X-ray fluorescence analysis for multi-layer samples were studied. A computer program was developed based on the theoretical equations for X-ray fluorescence intensity calculation and used to calculate the intensities of primary fluorescence and secondary fluorescence, the ratios of intensities of secondary fluorescence to primary fluorescence, and the portions of intensities of secondary fluorescence in total fluorescence for Fe Kalpha line in Zn/Fe and Fe/Zn film samples. It was found that the intensities of a characteristic line of an element in primary fluorescence and secondary fluorescence, the ratios of intensities of secondary fluorescence to primary fluorescence, and the portions of intensities of secondary fluorescence in total fluorescence were dependent not only on the thickness of the films but also on the position of the films in case of constant excitation conditions when multi-layer samples were tested by X-ray fluorescence analysis. When the thickness of Zn layer is the same as that of Fe layer, the calculated ratio of intensity of secondary fluorescence to total fluorescence of Fe Kalpha varies with the thickness, and reaches up to 9% for Fe/Zn sample while up to 35% for Zn/Fe sample.

English Abstract↗

PubMed1998

Simulations on the selectivity of 5-aminolaevulinic acid-induced fluorescence in vivo.

The knowledge of the exact time course of a photosensitizer in tumour and surrounding host tissue is fundamental for effective photodynamic therapy (PDT) and fluorescence-based diagnosis. In this study the time course of porphyrin fluorescence following topical application of 5-aminolaevulinic acid (ALA) using different formulations, concentrations and incubation times has been measured in amelanotic melanomas (A-Mel-3) (n = 54) grown in transparent dorsal skinfold chambers of Syrian golden hamsters and in human basal cell carcinomas (BCCs) (n = 40) in vivo. To simulate the accumulation of ALA-induced protoporphyrin IX (Pp IX), a three-compartment model has been developed and rate constants have been determined. The kinetics of both the A-Mel-3 tumours and the BCCs show a significantly higher fluorescence intensity in tumour as compared to normal surrounding host tissue. Maximal fluorescence intensity in A-Mel-3 tumours as a percentage of the reference standard used occurs 150 min post incubation (p.i.) using a 1, 3 or 10% (vol.) ALA solution buffered to pH 7.4 and 1 h incubation time. After a 4 h incubation time maximal fluorescence intensity in tumour is measured shortly p.i. A concentration of 10% ALA does not increase the fluorescence intensity as compared to 3% ALA following 4 h incubation, but either 3 or 10% ALA yields a significantly higher fluorescence after 4 h incubation time as compared to 1 h. The fluorescence intensity following an 8 h incubation reaches its maximum directly p.i. for all concentrations and then decreases exponentially. The fluorescence intensity in the surrounding host tissue shows no statistically significant difference regarding concentration or incubation time. At least during the first hour p.i., the fluorescence intensity measured in the surrounding tissue is lower as compared to that in the tumour in all groups. 24 h after topical application hardly any fluorescence is detectable in tumour or surrounding host tissue in all experimental groups. Incubating human BCCs with a 20% ALA cream (water-in-oil emulsion) or a 20% ALA gel (containing 40% dimethyl sulfoxide) for approximately 2 h yields a similar fluorescence intensity directly after incubation for either cream or gel. However, while yielding a maximum 120 min p.i. with cream, the fluorescence intensity increases for a longer time (about 2-3 h p.i.) and up to higher values using the gel formulation. In surrounding normal skin, cream as well as gel formulation yields a similar fluorescence intensity directly after incubation. Afterwards the fluorescence intensity decreases slowly using the cream whereas a further increase of the fluorescence intensity is measured in the normal skin with a maximum 240 min p.i. using the gel formulation. The results of the proposed three-compartment model indicate that the observed selectivity of accumulated porphyrins following topical application of ALA is mainly governed by an increased ALA penetration of the stratum corneum of the skin, an accelerated ALA uptake into the cell and a higher porphyrin formation in tumour as compared to normal skin tissue, but not by a reduced ferrocheletase activity.

Aminolevulinic Acid↗

PubMed2006

Fluorescence studies of anti-cancer drugs--analytical and biomedical applications.

The fluorescence properties of anticancer drugs (ACDs), including steady-state native fluorescence, time-resolved fluorescence, fluorescence polarization, excimer and exciplex emission, laser-induced fluorescence (LIF) with one- or two-photon excitation are reviewed, as well as the use of fluorogenic labels and fluorescent probes for the non-fluorescent ACDs. The interest of monitoring the fluorescence spectral changes to study the interactions of ACDs with biomolecules, such as DNA, proteins, vesicles, and the formation of complexes is discussed. The fluorescence methodologies used for ACDs studies, including fluorescence with two-photon excitation, liquid chromatography and capillary electrophoresis with fluorescence and laser-induced fluorescence (LIF) detection, and fluorescence microscopy, are also surveyed. Analytical and bioanalytical applications of fluorescence, indicating good selectivity and very low limits of detection at the nanomolar and picomolar level for most ACDs, are described. Biomedical and clinical applications of the fluorescence methods, mostly oriented towards the evaluation of the cytoxicity and anti-tumor potential of ACDs in single cells as well as in biological fluids, including blood, serum, plasma, cerebrospinal fluid, urine and feces, are also discussed in detail. This review is based on selected literature published in the last decade (1994-2003).

Animals↗

PubMed2005

[Three-dimensional excitation emission matrix fluorescence spectroscopic characterization of dissolved organic matter].

Three-dimensional excitation emission matrix fluorescence spectroscopy (3DEEM) was applied to characterize the fluorescence properties of dissolved organic matter (DOM) in lakes, rivers, streams, and ground waters. The results showed that the 3DEEM of DOM in aquatic environments mainly have four fluorescence peaks: peak A and C was referred to as fulvic-like fluorescence, and peak B and D was referred to as protein-like fluorescence. Results of river water DOM typically showed strong fulvic-like fluorescence. Polluted river waters often showed strong protein-like fluorescence. Four peaks were also found in the 3DEEM of lake DOM, which can origin from terrestrial runoff or from sources within the lakes. In Lake Baihua, strong protein-like fluorescence was found owing to the pollution by municipal wastewaters. Groundwater DOM has relatively lower DOC concentrations at 0.56-0.85 mg x L(-1) and is characterized by fulvic-like fluorescence only if it was polluted by municipal wastewaters, and then it has strong protein-like fluorescence. The authors demonstrate that for all DOM samples, fluorescence intensity at peak C and absorption at 254 nm both showed a strong correlation with DOC concentrations (r2 = 0.82 and 0.95, respectively). Also a strong linear correlation between UV-fulvic-like fluorescence and visible fulvic-like fluorescence was found (r2 = 0.96). The fulvic-like fluorescence at peak A and C varied in accordance to each other with the pH of the DOM samples, and the maximum fluorescence occurred at pH 10, while the maximum value of protein-like fluorescence (peak B) occurred at pH around 8.5.

English Abstract↗

PubMed1995

Laser-induced fluorescence microscopy of normal colon and dysplasia in colonic adenomas: implications for spectroscopic diagnosis.

OBJECTIVES: To determine what structures fluoresce and to what extent in normal colon and colonic adenomas to fully exploit laser-induced fluorescence spectroscopy as a tool for the diagnosis of dysplasia at endoscopy. METHODS: Unstained frozen sections of normal colon and colonic adenomas were studied by fluorescence microscopy under 351-364-nm argon ion laser excitation. Tissue fluorescence was observed and compared to morphology in serial sections stained with hematoxylin and eosin (H&E), Movat pentachrome, mucicarmine, and oil red O. RESULTS: In normal colon, fluorescence correlated morphologically with connective tissue fibers (principally collagen) in all layers of the bowel wall and with cytoplasmic granules within eosinophils present between the crypts in the lamina propria of the mucosa. Fluorescence of absorptive cells in normal crypts was very faint, and Goblet cells did not fluoresce. However, marked fluorescence was observed in the cytoplasms of dysplastic epithelial cells in the crypts of colonic adenomas. Fewer fluorescent connective tissue fibers were present in the lamina propria of colonic adenomas resulting in decreased fluorescence intensity as compared to that of normal colon. Fluorescent eosinophil granules were present in larger numbers in adenomas as compared with normal colon. CONCLUSION: Laser-induced fluorescence in normal colon and colonic adenomas correlates with morphology. Previous reported differences in laser-induced fluorescence emission spectra of normal colon and colonic adenomas obtained in vitro and in vivo may be due to differences in the cytoplasmic fluorescence between the dysplastic epithelium in colonic adenomas and normal colonic epithelium. Laser-induced fluorescence spectroscopy may be useful in studying other forms of epithelial dysplasia such as that which occurs in ulcerative colitis.

Adenoma↗

PubMed1998

In vivo detection of metastatic ovarian cancer by means of 5-aminolevulinic acid-induced fluorescence in a rat model.

STUDY OBJECTIVES: To determine the feasibility of macroscopic visualization of small ovarian cancer metastases in vivo by fluorescence after intravenous administration of 5-aminolevulinic acid (ALA); to assess the time after drug injection when fluorescence of small metastases is maximum; and to correlate macroscopic in vivo fluorescence with both microscopic ex vivo fluorescence and histologic findings. DESIGN: Controlled animal study (Canadian Task Force classification I). SETTING: University-based facility. SUBJECTS: Twenty-four healthy, female Fischer rats. INTERVENTION: Diffuse peritoneal metastatic cancer was induced in Fischer 344 rats by intraperitoneal injection of 1 million syngeneic ovarian cancer cells (NuTu-19). Four weeks after induction ALA100 mg/kg was injected intravenously, and diagnostic laparotomy was performed 1, 3, 6, or 9 hours thereafter. MEASUREMENTS AND MAIN RESULTS: The peritoneal cavity was illuminated with the Wood's lamp (ultraviolet light). Fluorescence was determined by direct visualization and compared with a calibrated fluorescent disk. Tissues were collected, sectioned, and examined by fluorescence and conventional light microscopy. Within 1 to 3 hours after intravenous injection of ALA, in vivo fluorescence of tumor nodules (diameter 0.4-5.0 mm) was macroscopically visible. Tumor-free peritoneum did not show fluorescence and was significantly distinguishable from cancer nodules. Fluorescence from intestinal tissues was comparable with tumor nodules. Microscopic fluorescence analysis showed similar values for tumor nodules and peritoneum. Stained histologic specimens of peritoneal surface revealed a superficial layer of cancer cells responsible for fluorescence. The time course of the fluorescence curve in the intestine peaked twice, at 1 and 6 hours after ALA injection. Macroscopically fluorescing nodules were histology confirmed as malignant. CONCLUSIONS: Fluorescence detection of small cancer nodules after intravenous injection of ALA is feasible for nodules smaller than 0.5 mm on the peritoneum. One to 3 hours after drug injection is optimal for diagnosis of metastases.

Aminolevulinic Acid↗

PubMed2003

Microanatomical and biochemical origins of normal and precancerous cervical autofluorescence using laser-scanning fluorescence confocal microscopy.

Clinical studies have shown that in vivo fluorescence spectroscopy can improve the diagnosis of cervical precancer. Recent work suggests that epithelial fluorescence increases, whereas stromal fluorescence decreases, with precancer. However, the microanatomic and biochemical sources of fluorescence in living cervical tissue have not yet been established. This study aims to characterize the origins of living normal and precancerous cervical fluorescence at microscopic levels using laser-scanning fluorescence confocal microscopy. Ten pairs of colposcopically normal and abnormal biopsies were obtained; transverse, 200 microm thick, short-term tissue cultures were prepared and imaged when viable with UV (351-364 nm) and 488 nm excitation before and after addition of the vital dye, Mitotracker Orange. In normal epithelium basal epithelial cells showed cytoplasmic fluorescence; parabasal, intermediate and superficial cells showed fluorescence only at the periphery of the cell. In low-grade precancers cytoplasmic fluorescence was visible in the bottom one-third of the epithelium; in high-grade precancers cytoplasmic fluorescence was visible throughout the lower two-thirds of the epithelium. Cytoplasmic fluorescence was colocalized with the MitoTracker probe and is attributed to mitochondrial reduced form of nicotinamide adenine dinucleotide at UV excitation and mitochondrial flavin adenine dinucleotide at 488 nm excitation. Stromal fluorescence originated from matrix fibers; with the development of precancer the density and fluorescence intensity of matrix fibers decrease. Autofluorescence properties of precancerous cervix reflect an increased number of metabolically active mitochondria in epithelial cells and a reduced stromal fluorescence, which can be an indicator for altered communication between precancerous epithelium and stroma. These changes can explain differences in in vivo fluorescence spectra of normal and precancerous cervical tissue.

Cervix Uteri↗

PubMed1994

Relationships between glycation and oxidation related fluorescences in rat collagen during aging. An in vivo and in vitro study.

BACKGROUND: Glycation and oxidation are spontaneous chemical modifications of body proteins. Usually these reactions have been studied separately by assessing their fluorescent final products. Glycation of protein and its related fluorescence increases during aging, whereas the level of the fluorescence related to protein adducts from lipoperoxidation side products is unknown. Moreover, no data on the fluorescence, at different wavelengths, connected to the two reactions in the same sample are available. Nevertheless recent in vitro studies support the possibility of an interaction between the two spontaneous reactions. EXPERIMENTAL DESIGN: In this study, we evaluated the modification of proteins due to glycation and to lipoperoxidation side products, by measuring their specific fluorescence levels in the collagen of 65 healthy Wistar rats during the aging process. The relationships among the fluorescence at different wavelengths were also reported. The fluorescence pattern of insoluble collagen was characterized by a tridimensional study after the incubation of insoluble collagen with probable precursors of protein glycation (ribose) and oxidation (malondialdehyde and hydroxynonenal); the maximum peaks of fluorescence were recognized and compared. RESULTS: An increase of all fluorescence intensities was observed in rat collagen during aging: the glycation-related ones (y370/440 = 28.3 e0.08x, r = 0.808, p < 0.01; y335/385 = 66.7 e0.06x, r = 0.798, p < 0.01) and the hydroxynonenal adduct-related (y356/460 = 44.3 e0.06x, r = 0.810, p < 0.01) were exponential, whereas that derived from MDA-adduct was almost linear (y390/460 = 17.7 + 4.1x, r = 0.661, p < 0.01). A different accumulation rate might explain this result. Significant correlation coefficients were found within the age-adjusted fluorescence intensities of both reactions, suggesting a close relationship between glycation and oxidation, besides a mutual influence due to the broad spectrum area. The in vitro study confirmed a good specificity of collagen fluorescence after incubation with a reducing sugar (ribose 0.5 M for 6 hours) for protein glycation, and after incubation with malondialdehyde (0.1 mM for 3 hours) for lipoperoxidation adducts; surprisingly enough hydroxynonenal (0.5 mM for 3 hours) significantly increased the fluorescence related to pentosidine-like products (335 nm excitation/385 nm emission) suggesting that this compound might be the precursor of products with a fluorescence similar to pentosidine or of pentosidine itself. CONCLUSIONS: The in vivo results of this study confirm that nonenzymatic reactions, glycation and oxidation, significantly modify collagen fluorescence during aging and can play a role in tissue damage related to age. The close relationships among fluorescences may be due to a reciprocal interconnection rather than to a parallel increase of both reactions during aging; this hypothesis is supported by the in vitro findings of this study.

Aging↗

PubMed1995

Fluorescence spectroscopy.

Although providing extensive detail, the time-resolved experiments described here can be quite complex and the instrumentation is not always readily available. However, a number of national fluorescence user facilities funded by the National Institutes of Health or the National Science Foundation are currently in operation and are dedicated to the use of fluorescence spectroscopy in the biomedical and biophysical sciences. These centers include the Laboratory for Fluorescence Dynamics at the University of Illinois and the Center for Fluorescence Spectroscopy at the University of Maryland. Even in the absence of the sophisticated equipment necessary for carrying out the time-resolved experiments, a great deal of information can be obtained from steady-state fluorescence profiles if one is careful to monitor all of the available fluorescence observables, namely intensity, wavelength or color, and polarization. Steady-state measurements of ANS binding are also quite informative. The combination of kinetic as well as equilibrium approaches, as with folding studies using any technique, will provide further insight into the pathways and stable and transient intermediates in the folding and unfolding reactions. Fluorescence spectroscopy offers a very sensitive window into the structural and dynamic characteristics of macromolecules. Recent advances in data acquisition and analysis combined with available structure information from NMR and crystallographic studies have led to increasingly greater insight into the structural and dynamic determinants of fluorescence decay parameters in the native states of proteins. As our understanding of the fluorescence properties of native proteins has grown, fluorescence spectroscopists have begun to investigate what fluorescence can tell us about the denatured states of proteins as well as the folding/unfolding transitions and pathways. A great deal of progress has been made in the characterization and interpretation of the response of the various fluorescence parameters to protein folding and denaturation. There remain, however, a number of unanswered questions, particularly concerning the structural and dynamic determinants of the fluorescence properties of the denatured states of proteins. Future studies will undoubtedly be aimed toward this goal, and progress in this area will certainly result from systematic comparisons of fluorescence studies with a number of other biophysical and biochemical approaches.

Circular Dichroism↗

PubMed1997

Fluorescent dots in fluorescein angiography and fluorescein leukocyte angiography using a scanning laser ophthalmoscope in humans.

PURPOSE: The purpose of the study is to disclose the nature of fluorescent dots and segments traditionally observed with fluorescein angiography (FA) using a scanning laser ophthalmoscope (SLO 101; Rodenstock, München, Germany). The authors developed a new method, called fluorescein leukocyte angiography (FLA), to display directly the movement of leukocytes in human retinal vessels. METHODS: Fluorescein angiography was performed on two normal volunteers using a scanning laser ophthalmoscope and fluorescent dots and segments were observed. Fluorescein leukocyte angiography, using an injection of fluorescent buffy coat layer from which the fluorescent plasma and nonfluorescent erythrocytes have been removed externally, was performed on seven normal volunteers. Injection fluid smears were examined through a fluorescent microscope. Peripheral blood smears taken during midphase of FA and FLA also were examined. In addition, 15 early-phase FAs of central serous chorioretinitis (CSC) were studied retrospectively. RESULTS: In the FAs of normal volunteers, fluorescent dots were detected only in perimacular capillaries at early phase. Eight of the 15 CSC FAs examined showed both fluorescent dots and segments. In the FLAs, fluorescent dots were detected in whole retinal vessels for more than 30 minutes. Fluorescent segments were observed in FA but not in FLA. Injected fluid smears from one FLA showed fluorescent leukocytes and small platelets. However, in peripheral blood smears of the FLA, leukocytes and platelets were more visible and exhibited higher contrast than those of an FA due to background plasma fluorescence. The mean velocity of 21 flowing leukocytes in perifoveal capillaries was 1.37 +/- 0.35 mm/second in 2 FAs and that of 89 flowing leukocytes was 1.41 +/- 0.29 mm/second in 7 FLAs. CONCLUSIONS: The authors' observations suggest that fluorescent dots in scanning laser ophthalmoscope imaging are fluorescein-stained leukocytes, whereas fluorescent segments are the hyperfluorescent plasma that is located between rouleaux formations of erythrocytes. The velocity of the fluorescent dots could be measured in the perimacular capillaries by either FA or FLA; however, only FLA can display the flow of fluorescent leukocytes in large vessels.

Adult↗

PubMed2006

Monitoring dynamic systems with multiparameter fluorescence imaging.

A new general strategy based on the use of multiparameter fluorescence detection (MFD) to register and quantitatively analyse fluorescence images is introduced. Multiparameter fluorescence imaging (MFDi) uses pulsed excitation, time-correlated single-photon counting and a special pixel clock to simultaneously monitor the changes in the eight-dimensional fluorescence information (fundamental anisotropy, fluorescence lifetime, fluorescence intensity, time, excitation spectrum, fluorescence spectrum, fluorescence quantum yield, distance between fluorophores) in real time. The three spatial coordinates are also stored. The most statistically efficient techniques known from single-molecule spectroscopy are used to estimate fluorescence parameters of interest for all pixels, not just for the regions of interest. Their statistical significance is judged from a stack of two-dimensional histograms. In this way, specific pixels can be selected for subsequent pixel-based subensemble analysis in order to improve the statistical accuracy of the parameters estimated. MFDi avoids the need for sequential measurements, because the registered data allow one to perform many analysis techniques, such as fluorescence-intensity distribution analysis (FIDA) and fluorescence correlation spectroscopy (FCS), in an off-line mode. The limitations of FCS for counting molecules and monitoring dynamics are discussed. To demonstrate the ability of our technique, we analysed two systems: (i) interactions of the fluorescent dye Rhodamine 110 inside and outside of a glutathione sepharose bead, and (ii) microtubule dynamics in live yeast cells of Schizosaccharomyces pombe using a fusion protein of Green Fluorescent Protein (GFP) with Minichromosome Altered Loss Protein 3 (Mal3), which is involved in the dynamic cycle of polymerising and depolymerising microtubules.

Algorithms↗

PubMed1984

Interaction of bimane-labeled fluorescent tubulin with the isolated mitotic apparatus.

Fluorescent derivatives of cellular proteins that retain their native characteristics have become useful probes to investigate the dynamics of specific cytoskeletal proteins. In the experiments reported here, a previously characterized fluorescent derivative of tubulin, bimane-tubulin [Wadsworth and Sloboda, 1982a], was used to investigate microtubule assembly in vitro. The results demonstrate that bimane-tubulin was competent to assemble onto a variety of organizing centers in vitro, including microtubule organizing centers (MTOCs) present in homogenates of sea urchin eggs, isolated mitotic apparatuses (MAs), and lysed mitotic cells. When homogenates of fertilized sea urchin eggs containing MTOCs were incubated with bimane-tubulin at 37 degrees C, discrete areas of linear fluorescence were observed. Only diffuse fluorescence was observed when calcium or colchicine was added to the homogenate or if the temperature was maintained at 0 degrees C. Negative-stain electron microscopy of the fluorescent arrays revealed morphologically normal microtubules radiating from electron dense regions. When mitotic spindles, isolated in glycerol containing buffers and therefore cold stable, were incubated with bimane-tubulin, linear fluorescence was observed emanating from the spindle poles but not from the region occupied by the kinetochores. MAs incubated with bimane-labeled bovine serum albumin or bimane-labeled microtubule-associated proteins showed only diffuse fluorescence. However, when mitotic cells which were hypotonically lysed in the absence of detergents or microtubule stabilizing solvents, were perfused with bimane-tubulin intense fluorescence was observed in the asters and throughout the spindle. Two experiments suggested that the fluorescence observed in the results outlined above was due to the assembly of normal microtubules from the fluorescent subunits. First, the observed fluorescence was sensitive to cold temperature, which is known to disassemble microtubules. Second, when the isolated, fluorescent MAs were examined by thin section electron microscopy, microtubules of normal diameter were seen. No aggregated material appeared associated with the walls of the microtubules, which might have been expected if the fluorescent protein was nonspecifically adsorbed to the microtubules. The results of these experiments demonstrate that isolated, stabilized MAs support the growth of new microtubules from the spindle poles while labile spindles, present in lysed cells, incorporate fluorescent tubulin throughout the spindle and asters.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

PubMed2003

Chlorophyll a fluorescence rise induced by high light illumination of dark-adapted plant tissue studied by means of a model of photosystem II and considering photosystem II heterogeneity.

Chlorophyll a fluorescence rise (FLR) measured in vivo in dark-adapted plant tissue immediately after the onset of high light continuous illumination shows complex O-K-J-I-P transient. The steps typically appear at about 400 micros (K), 2 ms (J), 30 ms (I), and 200 - 500 ms (P) and a transient decrease of fluorescence to local minima (dips D) can be observed after the K, J, and I steps. As the FLR reflects a function of photosystem II (PSII) and to more understand the FLR, a PSII reactions model was formulated comprising equilibrium of excited states among all light harvesting and reaction centre pigments and P680, reversible radical pair formation and the donor and acceptor side functions. Such a formulated model is the most detailed and complex model of PSII reactions used so far for simulations of the FLR. By varying of selected model parameters (rate constants and initial conditions) several conclusions can be made as for the origin of and changes in shape of the theoretical FLR and compare them with in-literature-reported results. For homogeneous population of PSII and using standard in-literature-reported values of the model parameters, the simulated FLR is characterized by reaching the minimal fluorescence F(0) at about 3 ns after the illumination is switched on lasting to about 1 micros, followed by fluorescence rise to a plateau located at about 2 ms and subsequent fluorescence rise to a global maximum that is reached at about 60 ms. Varying of the values of rate constants of fast processes that can compete for utilization of the excited states with fluorescence emission does not change qualitatively the shape of the FLR. However, primary photochemistry of PSII (the charge separation, recombination and stabilization), non-radiative loss of excited states in light harvesting antennae and excited states quenching by oxidized plastoquisnone (PQ) molecules from the PQ pool seem to be the main factors controlling the maximum quantum yield of PSII photochemistry as expressed by the F(V)/F(M) ratio. The appearance of the plateau at about 2 ms in the FLR is affected by several factors: the height of the plateau in the FLR increases when the fluorescence quenching by oxidized P680(+) is not considered in the simulations or when the electron transfer from Q(A)(-) to Q(B)((-)) is slowed down whereas the height of the plateau decreases and its position is shifted to shorter times when OEC is initially in higher S state. The plateau at about 2 ms is changed into the local fluorescence maximum followed by a dip when the fluorescence quenching by oxidized PQ molecules or the charge recombination between P680(+) and Q(A)(-) is not considered in the simulations or when all OEC is initially in the S(0) state or when the S -state transitions of OEC are slowed down. Slowing down of the S -state transitions of OEC as well as of the electron transfer from Q(A)(-) to Q(B)((-)) also causes a decrease of maximal fluorescence level. In the case of full inhibition of the S -state transitions of OEC as well as in the case of full inhibition of the electron donation to P680(+) by Y(Z), the local fluorescence maximum becomes the global fluorescence maximum. Assuming homogeneous PSII population, theoretical FLR curve that only far resembles experimentally measured O-J-I-P transient at room temperature can be simulated when slowly reducing PQ pool is considered. Assuming heterogeneous PSII population (i.e. the alpha/beta and the Q(B) -reducing/Q(B)-non-reducing heterogeneity and heterogeneity in size of the PQ pool and rate of its reduction) enables to simulate the FLR with two steps between minimal and maximal fluorescence whose relative heights are in agreement with the experiments but not their time positions. A cause of this discrepancy is discussed as well as different approaches to the definition of fluorescence signal during the FLR.

Chlorophyll↗

PubMed1997

Dissociation of Ca2+ from sarcoplasmic reticulum Ca2+-ATPase and changes in fluorescence of optically selected Trp residues. Effects of KCl and NaCl and implications for substeps in Ca2+ dissociation.

Sequential dissociation of the two Ca2+ ions bound to non-phosphorylated sarcoplasmic reticulum Ca2+-ATPase was triggered by addition, in a stopped-flow experiment, of quin2, which acted both as a high-affinity chelator and as a Ca2+-sensitive fluorescent probe. The kinetics of Ca2+ dissociation were deduced from the observed changes in quin2 fluorescence in the visible region (with lambdaex = 313 nm), while fluorescence detection in the UV region (with lambdaex = 290 nm) made it possible to monitor the tryptophan fluorescence changes accompanying this dissociation under the same ionic conditions. In the absence of KCl or NaCl, at pH 6 or 7, the observed changes in quin2 fluorescence were monoexponential, with rate constants very close to those of the changes in ATPase tryptophan fluorescence, which also appeared monophasic. In the presence of 100 mM KCl, quin2 fluorescence changes, although still monoexponential, were faster than in the absence of the monovalent ions but distinctly slower than the changes in tryptophan fluorescence, which were accelerated to a larger extent. In addition, the apparent kinetics of the Trp fluorescence changes depended on the excitation wavelength. Using an excitation wavelength of 296 nm, the Trp fluorescence drop was still faster than with an excitation wavelength of 290 nm, and in the presence of NaCl it even displayed a clear undershoot. We conclude that in the presence of KCl or NaCl and with an excitation wavelength of 290 nm, the rapid drop in tryptophan fluorescence mainly monitors the dissociation of the first of the two Ca2+ ions to be released from Ca2+-ATPase, while excitation at 296 nm optically selects a subpopulation of Trp residues whose fluorescence level is lower in the ATPase species with one Ca2+ ion bound than in the Ca2+-deprived ATPase species. The latter conditions result in an initial drop in Trp fluorescence whose apparent rate constant (in single-exponential analysis) is faster than the true rate of dissociation of the first Ca2+ ion and in a subsequent slower rise related to dissociation of the second Ca2+ ion. The difference between results obtained in the absence and in the presence of K+ or Na+ is due to an antagonizing effect of these cations on proton-induced conformational rearrangement of Ca2+-free ATPase, a conformational rearrangement which changes the ATPase Trp fluorescence level and significantly affects the cooperativity of Ca2+ binding at equilibrium.

Aminoquinolines↗

PubMed1996

Kinetics and localisation of PpIX fluorescence after topical and systemic ALA application, observed in skin and skin tumours of UVB-treated mice.

In this study the kinetics and localisation of protoporphyrin IX (PpIX) fluorescence in skin and skin tumours were examined after topically (20% for 4h) or systemically (200 mg/kg,i.p.) administered 5-aminolaevulinic acid (ALA). As a model we used hairless mice with skin lesions (actinic keratoses and squamous cell carcinoma), which were induced by daily UVB irradiation. The epidermis of the skin surrounding the tumours (T) was altered (AS); owing to the UVB irradiation, the epidermis was thicker and less elastic. Therefore, non-UVB-irradiated mice were used to assess fluorescence of normal skin (NS). Light from a halogen lamp was used to excite at 500 +/- 20 nm and fluorescence was detected through a filter that passes light of 670 +/- 50 nm. Maximal fluorescence following i.p. ALA was observed 2 h post injection (p.i.) and was three times less than after topically applied ALA. Furthermore, after i.p. ALA a lower T selectively (T/NS) could be obtained than after topically applied ALA. Maximal fluorescence following topically applied ALA was achieved 6 h after the end of the 4 h application time. At that interval fluorescence of T was twice as high as directly after the application period. Furthermore, T selectivity (T/NS) after topical ALA at the interval of maximal fluorescence was higher than at the interval directly after application. With fluorescence cryomicroscopy localisation of fluorescence in the skin at the interval of maximal fluorescence was determined after both administration routes. For both cases fluorescence was mainly located in T, epidermis and hair follicles. Fluorescence in subcutis could only be observed at 2 h post i.p. ALA and a 6 h post topical ALA. No fluorescence could be observed in muscle. We conclude that, in this model and with these ALA doses, a higher fluorescence intensity and selectivity (T/NS) was achieved after topically applied ALA than after systemically administered ALA. These results make topically applied ALA more favourable for ALA-PDT of superficial skin tumours in this model. In general these results imply that by optimising the time after ALA application the efficacy and selectivity of topical ALA-PDT for skin tumours may be improved.

Administration, Topical↗