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PubMed1987

Local structure in a tryptic fragment of performic acid oxidized ribonuclease A corresponding to a proposed polypeptide chain-folding initiation site detected by tyrosine fluorescence lifetime and proton magnetic resonance measurements.

The effects of proline and X-Pro peptide bond conformations on the fluorescence properties of tyrosine in peptides corresponding to parts of a proposed chain-folding initiation site in bovine pancreatic ribonuclease A are examined by time-resolved and steady-state fluorescence spectroscopy. In peptides with Tyr-Pro sequences, the conformational constraints of proline on a preceding residue result in significant fluorescence quenching for both trans and cis peptide bond conformations. Small peptides containing Pro-Tyr sequences, on the other hand, do not exhibit fluorescence quenching compared to Ac-Tyr-NHMe. Studies of fluorescence decay in the tryptic fragment of performic acid oxidized ribonuclease corresponding to residues 105-124 (i.e., O-T-16) demonstrate the presence of at least two environments of the single tyrosine chromophore (in the sequence Asn113-Pro114-Tyr115). In these two (ensemble-averaged) environments, tyrosine has shorter and longer lifetimes, respectively, than in Ac-Tyr-NHMe. The fluorescence heterogeneity in O-T-16 does not correlate with X-Pro cis/trans conformational heterogeneity that can be detected by nuclear magnetic resonance (NMR) spectroscopy. Instead, the fluorescence heterogeneity in O-T-16 arises from the presence of multiple conformations with the same X-Pro peptide bond conformations which interconvert rapidly on the 1H NMR time scale (tau much less than 1 ms) but are distinguishable on the fluorescence lifetime time scale (tau greater than or equal to 1 ns). From comparisons with the tyrosine fluorescence decay of smaller synthetic peptides, it is concluded that the long-lifetime tyrosine fluorescence component of O-T-16 arises from interactions involving residues outside the Asn113-Pro114-Tyr115-Val116-Pro117 sequence, which either stabilize particular local conformations in the vicinity of Tyr115 or act directly to protect Tyr115 from efficient fluorescence quenching. The short-lifetime component of O-T-16 is also observed for the pentapeptide Ac-Asn-Pro-Tyr-Val-Pro-NHMe. The data provide evidence for a nonrandom polypeptide conformation of O-T-16 under conditions of solvent pH and temperature at which the complete disulfide-intact ribonuclease molecule is fully folded. Implications of this work for the interpretation of fluorescence-detected unfolding experiments are discussed.

Amino Acid Sequence↗

PubMed1996

Inhibitor-induced changes in the intrinsic fluorescence of human cyclooxygenase-2.

The steady state tryptophan fluorescence of apo-human cyclooxygenase-2 (hCox-2) is quenched approximately 40%-50% by the slow binding inhibitors diclofenac, indomethacin, ketoprofen, NS-398, and DuP-697. The effects of these inhibitors on tryptophan fluorescence are both time and concentration dependent. Addition of each inhibitor results in a rapid fluorescence decrease, followed by a slower time dependent quenching. The slow, time dependent loss of fluorescence follows first-order kinetics, the rate constants for the process increasing with inhibitor concentration in a saturation-type manner. The rapid fluorescence loss also increases with increasing inhibitor concentration in the same manner. These results are consistent with the initial formation of a rapid equilibrium complex of enzyme and inhibitor (EI), followed by the slower formation of a tightly bound enzyme-inhibitor complex (EI*). The fluorescence of the EI complex is not significantly different from that of the EI* complex. The kinetic parameters of each inhibitor derived for this process (Ki and kon) are close to those obtained by determination of the rate constants for the onset of enzyme inhibition, thereby linking the fluorescence changes with inhibitor binding. The reversible inhibitors ibuprofen and docosahexaenoic acid do not quench the protein fluorescence but do decrease both the rate of the slow fluorescence loss and the magnitude of the initial rapid fluorescence decrease caused by the slow binding inhibitors, consistent with their competitive behavior. ASA-acetylated apo-hCox-2 shows the same fluorescence-quenching behavior in the presence of most of the above inhibitors. However, acetylation apparently blocks the binding of diclofenac, whereas the affinity of ibuprofen is increased. The effects of the collisional quenching agents iodide and acrylamide on both the native and inhibited enzyme are small (< 20% quenching at 0.3 M), showing that inhibitor binding does not result in an increased solvent accessibility of protein tryptophans. The cause of the inhibitor-induced quenching of the intrinsic apo-hCox-2 fluorescence is likely energy transfer to the bound inhibitor. Calculations based on the inhibitor-tryptophan distances in ovine Cox-1 indicate that the distances are within the required range for significant quenching to occur.

Acetylation↗

PubMed1997

Fluorescence quenching by chlorophyll cations in photosystem II.

Although fluorescence is widely used to study photosynthetic systems, the mechanisms that affect the fluorescence in photosystem II (PSII) are not completely understood. The aim of this study is to define the low-temperature steady-state fluorescence quenching of redox-active centers that function on the electron donor side of PSII. The redox states of the electron donors and acceptors were systematically varied by using a combination of pretreatments and illumination to produce and trap, at low temperature, a specific charge-separated state. Electron paramagnetic resonance spectroscopy and fluorescence intensity measurements were carried out on the same samples to obtain a correlation between the redox state and the fluorescence. It was found that illumination of PSII at temperatures between 85 and 260 K induced a fluorescence quenching state in two phases. At 85 K, where the fast phase was most prominent, only one electron-transfer pathway is active on the donor side of PSII. This pathway involves electron donation to the primary electron donor in PSII, P680, from cytochrome b559 and a redox-active chlorophyll molecule, ChlZ. Oxidized ChlZ was found to be a potent quencher of chlorophyll fluorescence with 15% of oxidized ChlZ sufficient to quench 70% of the fluorescence intensity. This implies that neighboring PSII reaction centers are energetically connected, allowing oxidized ChlZ in a few centers to quench most of the fluorescence. The presence of a well-defined quencher in PSII may make it possible to study the connectivity between antenna systems in different sample preparations. The other redox-active components on the donor side of PSII studied were the O2-evolving complex, the redox-active tyrosines (YZ and YD), and cytochrome b559. No significant changes in fluorescence intensity could be attributed to changes in the redox state of these components. The fast phase of fluorescence quenching is attributed to the rapid photooxidation of ChlZ, and the slow phase is attributed to multiple turnovers providing for further oxidation of ChlZ and irreversible photoinhibition. Significant photoinhibition only occurred at Chl concentrations below 0.7 mg/mL and above 150 K. The reversible oxidation of ChlZ in intact systems may function as a photoprotection mechanism under high-light conditions and account for a portion of the nonphotochemical fluorescence quenching.

Cations↗

PubMed2006

Heterogeneity of fluorescence in psoriasis after application of 5-aminolaevulinic acid: an immunohistochemical study.

BACKGROUND: Psoriasis has been shown to highly accumulate protoporphyrin IX (PpIX), but a variable distribution within plaques after fluorescence diagnosis is seen. It is unknown what causes this heterogeneity of fluorescence in psoriatic skin, despite adequate keratolytic treatment. Variations in fluorescence might explain the variable and the mostly partial clinical response of psoriasis seen after photodynamic therapy (PDT). OBJECTIVES: This study examines morphological and immunohistochemical differences in inhomogeneous PpIX-induced fluorescence in stable plaque psoriasis. MATERIALS AND METHODS: Fourteen patients with stable plaque psoriasis were included in this study. In each patient one psoriatic plaque was incubated with 20% 5-aminolaevulinic acid (ALA) ointment for 3 h after keratolytic treatment. Fluorescence diagnosis with ALA-induced porphyrins (FDAP) was performed and subsequently high- and low-fluorescent psoriatic skin samples were biopsied. Biopsies were investigated with respect to histological hyperkeratosis (thickness of stratum corneum), proliferation (Ki-67 antigen), keratinization (K10, filaggrin) and inflammation (CD3). Digital images acquired with FDAP were analysed using image analysis software. RESULTS: Inhomogeneous fluorescence was seen in 12 of the 14 plaques. A significantly thicker stratum corneum was found in low-fluorescent psoriatic skin compared with highly fluorescent skin. Fluorescence intensity and thickness of the stratum corneum proved to be negatively correlated. The variable-fluorescent parts of the lesional psoriatic skin showed no differences in epidermal proliferation, keratinization or inflammation. CONCLUSIONS: Heterogeneous ALA-induced fluorescence in psoriasis plaques related to inhomogeneous distribution of PpIX in the epidermis may result from differences in penetration of ALA and/or light within a plaque caused by differences in stratum corneum thickness. The variable clinical response seen after PDT in psoriasis could be explained by this. These findings are consistent with the general assumption that optimal desquamation prior to FDAP or PDT is required for the most favourable results.

Administration, Cutaneous↗

PubMed2006

Characteristics of 5-aminolaevulinic acid-induced protoporphyrin IX fluorescence in human skin in vivo.

BACKGROUND/PURPOSE: Topical photodynamic therapy (PDT) is increasingly being used to treat skin cancers. Knowledge of the detailed characteristics of 5-aminolaevulinic acid (ALA)-induced protoporphyrin IX (PpIX) fluorescence in diseased and normal skin is incomplete. Understanding the characteristics of PpIX fluorescence in normal skin may facilitate optimization of PDT regimes while minimizing side effects in the surrounding normal skin. We investigated the characteristics of ALA-induced PpIX fluorescence in normal skin. METHODS: ALA was applied to the arm, back and leg skin of 21 healthy volunteers for 1-6 h, and PpIX fluorescence was measured for up to 24 h after ALA application using a fluorescent spectrometer. The effect of tape stripping on fluorescence was also examined. RESULTS: Considerable inter-subject variation was observed in the time to reach peak PpIX fluorescence. Intra-subject variation in the time to peak fluorescence was dependent on ALA application time. Six hours after ALA application, no significant difference was observed in the degree of fluorescence achieved irrespective of ALA application times ranging between 1 and 6 h. PpIX fluorescence was reduced on the leg and increased by tape stripping. CONCLUSIONS: Marked inter- and intra-subject variation in ALA-induced PpIX fluorescence occurs in normal human skin. ALA application time, body site and the state of the stratum corneum are all determinants of PpIX fluorescence within subjects and these factors need to be taken into account in optimization of PDT regimes.

Adult↗

PubMed2005

Carboxyfluorescein diacetate succinimidyl ester fluorescent dye for cell labeling.

Our objective was to study the properties of the carboxyfluorescein diacetate succinimidyl ester (CFDA-SE) and the methodology of cell labeling using CFDA-SE fluorescent dye. First, we analyzed the kinetics of CFDA-SE fluorescent dye intensity over time. Second, we determined the optimal concentration of CFDA-SE fluorescent dye for cell labeling. Third, we tested the toxicity of CFDA-SE fluorescent dye on labeled cells. Finally, we determined the optimal staining time of CFDA-SE fluorescent dye for cell labeling. The results show that the optimal concentration of CFDA-SE fluorescent dye for cell labeling varies according to different cell types. CFDA-SE fluorescent dye is non-toxic to cells as the cell death rate caused by CFDA-SE labeling is below 5%. The optimal cell labeling time was determined to be 8 min of incubation with CFDA-SE fluorescent dye. We concluded that the advantages of using CFDA-SE fluorescent dye for cell labeling are as follows: (1) the binding of CFDA-SE fluorescent dye to cells is stable; (2) CFDA-SE fluorescent dye is not toxic and does not modify the viability of labeled cells; and (3) CFDA-SE fluorescent dye is a suitable fluorochrome for cell labeling.

Animals↗

PubMed2006

Quantify single nucleotide polymorphism (SNP) ratio in pooled DNA based on normalized fluorescence real-time PCR.

BACKGROUND: Conventional real-time PCR to quantify the allele ratio in pooled DNA mainly depends on PCR amplification efficiency determination and Ct value, which is defined as the PCR cycle number at which the fluorescence emission exceeds the fixed threshold. Because of the nature of exponential calculation, slight errors are multiplied and the variations of the results seem too large. We have developed a new PCR data point analysis strategy for allele ratio quantification based on normalized fluorescence ratio. RESULTS: In our method, initial reaction background fluorescence was determined based upon fitting of raw fluorescence data to four-parametric sigmoid function. After that, each fluorescence data point was first subtracted by respective background fluorescence and then each subtracted fluorescence data point was divided by the specific background fluorescence to get normalized fluorescence. By relating the normalized fluorescence ratio to the premixed known allele ratio of two alleles in standard samples, standard linear regression equation was generated, from which unknown specimens allele ratios were extrapolated using the measured normalized fluorescence ratio. In this article, we have compared the results of the proposed method with those of baseline subtracted fluorescence ratio method and conventional Ct method. CONCLUSION: Results demonstrated that the proposed method could improve the reliability, precision, and repeatability for quantifying allele ratios. At the same time, it has the potential of fully automatic allelic ratio quantification.

Alleles↗

PubMed2004

Time-resolved confocal fluorescence imaging and spectrocopy system with single molecule sensitivity and sub-micrometer resolution.

We present novel technical features and results from a two channel confocal fluorescence lifetime microscope, which allows to efficiently investigate fluorescence dynamics down to the single molecule level. The MicroTime 200 time-resolved fluorescence microscope offers a multicolor excitation where different picosecond diode lasers are used. For imaging and positioning purposes we utilize a compact Piezo scanner which allows, due to a novel scanning algorithm and synchronisation technique, a superior movement and positioning accuracy. The data acquisition is completely based on time-correlated single photon counting, where every photon is detected and stored individually with its specific timing information (Time-Tagged Time-Resolved mode). This multiparameter data acquisition scheme offers the opportunity to analyse the parameter dependencies in a multitude of different ways. Standard intensity analysis can be used to reconstruct 2D-images or the temporal evolution (time trace) of the fluorescence of a single spot. The information from the two distinct detector channels additionally allows to investigate the polarisation of the emitted light or its spectral composition, for example for analysis of Fluorescence Resonance Energy Transfer (FRET). The timing information down to a picosecond scale offers the possibility not only to reconstruct fluorescence decay constants of each pixel for the purpose of Fluorescence Lifetime Imaging (FLIM) but also to analyze the fluorescence fluctuation correlation function of any single spot of interest. The flexible multichannel detector scheme enables in this case also a cross-correlation between spectrally separated parts of the emission light, or even identical parts of the fluorescence to eliminate detector artifacts. The photon arrival coincidence analysis can also be expanded in the sub-ns range to study fluorescence antibunching in the fluorescence emission of single molecules. The ability of combining these different pieces of temporal information allows the construction of extremely powerful analysis methods and assays. We demonstrate a variety of these capabilities with results obtained from fluorescently labeled latex beads, biological samples, and single molecules excited in the blue or red wavelength region.

Equipment Design↗

PubMed1998

Excimer laser spectroscopy: influence of tissue ablation on vessel wall fluorescence.

Limited steerability and injury to the normal vessel wall are major drawbacks of laser coronary angioplasty. To overcome these limitations a new generation of laser systems has been developed which allows not only to eliminate the atherosclerotic plaque but to guide the laser beam by analyzing the laser induced tissue fluorescence (= spectroscopy) for the treatment of the atherosclerotic vessel. An excimer laser (MAX 10 LP, 308 nm, Technolas, Munich, Germany) was used with an emitting (phi 1070 microns) and a detecting (phi 130 microns) optical fiber to induce tissue fluorescence which was analyzed quantitatively by a computerized system. Specimens from the descending (thoracic) aorta were obtained from 24 patients (mean age 68.1 years, range 44-92). Tissue fluorescence was induced with ablating (26-30 mJ/mm2) and nonablating (3 mJ/cm2) laser activations. The emitted fluorescence (range 380-575 nm) was normalized to a wavelength of 380 nm; as a measure of tissue fluorescence the intensity ratio at 500 nm divided by 400 nm was calculated in normal (n = 78), mildly atherosclerotic (n = 40), and severely atherosclerotic (n = 48) tissue samples. Repeated laser activations were carried out and tissue fluorescence was checked until the fluorescence spectrum was normalized. All tissue samples were analyzed histologically by a semiquantitative score. Normal tissue samples showed the highest intensity ratios (5.9 +/- 3.4), whereas mildly (2.9 +/- 1.3) and severely atherosclerotic (2.1 +/- 1.0) samples elicited a significantly reduced fluorescence. Repeated tissue ablations were associated with a normalization of fluorescence intensity ratios in the mildly (7.0) as well as in the severely diseased (4.9) vessels. A curvilinear relationship between intensity ratio and the semiquantitative score was observed (r = 0.66) as well as between intensity ratio and intimal wall thickness (r = 0.62). No gender related differences were found but there was an inverse relationship between fluorescence intensity ratio and age (r = 0.56) as well as between intimal thickness and age (r = 0.41). Excimer laser spectroscopy allows reliable detection of atherosclerotic vessel alterations. Fluorescence intensity ratio is inversely proportional to the intimal wall thickness and the severity of the histologic alterations. There is an age dependency of fluorescence intensity ratio which can be explained by an increase in intimal wall thickness. Successful tissue ablation can be obtained by laser angioplasty and allows determination of the optimal point where complete tissue ablation is achieved by laser activation. Thus, excimer laser spectroscopy is an effective method for selective tissue ablation by laser angioplasty.

Adult↗

PubMed2000

Fluorescence-guided resection of glioblastoma multiforme by using 5-aminolevulinic acid-induced porphyrins: a prospective study in 52 consecutive patients.

OBJECT: It has been established that 5-aminolevulinic acid (5-ALA) induces the accumulation of fluorescent porphyrins in glioblastoma multiforme (GBM), a phenomenon potentially exploitable to guide tumor resection. In this study the authors analyze the influence of fluorescence-guided resection on postoperative magnetic resonance (MR) imaging and survival in a series of patients who underwent surgery in the authors' department. METHODS: Fifty-two consecutive patients with GBM received oral doses of 5-ALA (20 mg/kg body weight) 3 hours before induction of anesthesia. Intraoperatively, tumor fluorescence was visualized using a modified operating microscope. Fluorescing tissue was removed whenever it was considered safely possible. Residual enhancement on early postoperative MR imaging was quantified and related to each patient's characteristics to determine which factors influenced resection. Survival was analyzed using the Kaplan-Meier method and multivariate analysis was performed in which the Karnofsky Performance Scale (KPS) score, residual fluorescence, patient age, and residual enhancement on MR images were considered. Intraoperatively, two fluorescence qualities were perceived: solid fluorescence generally reflected coalescent tumor, whereas vague fluorescence mostly corresponded to infiltrative tumor. Complete resection of contrast-enhancing tumor was accomplished in 33 patients (63%). Residual intraoperative tissue fluorescence left unresected for safety reasons predicted residual enhancement on MR images in 18 of the 19 remaining patients. Age, residual solid fluorescence, and absence of contrast enhancement in MR imaging were independent explanatory factors for survival, whereas the KPS score was significant only in univariate analysis. No perioperative deaths and one case of permanent morbidity were encountered. CONCLUSIONS: The observations in this study indicate the usefulness of 5-ALA-induced tumor fluorescence for guiding tumor resection. The completeness of resection, as determined intraoperatively from residual tissue fluorescence, was related to postoperative MR imaging findings and to survival in patients suffering from GBM.

Adult↗

PubMed2001

[Fluorescence diagnostics of premalignant and malignant changes in patients with Barrett's esophagus: doubts and hopes].

Increasing incidence of esophageal adenocarcinoma that mostly arises from Barrett's esophagus (BE) has been observed. Therefore sensitive methods for early detection of dysplasias and adenocarcinomas in BE are more and more necessary. Fluorescence endoscopy is an alternative or complementary to standard four-quadrant biopsies technique. The basics of diagnostics application of tissue autofluorescence and exogenous dyes fluorescence such as protoporphyrin IX, induced by 5-aminolevulinic acid are summarized. There are two methods of fluorescence diagnostics of dysplasia in BE: point fluorescence spectroscopy and real-time fluorescence imaging. Fluorescence spectroscopy is sensitive and specific but can be targeted only toward lesions visible in white-light endoscopy. Fluorescence imaging allows to examine large areas for occult foci of dysplasia but selectivity is somewhat limited by the background fluorescence of Barrett's mucosa. Laser induced fluorescent endoscopy (LIFE) images of esophageal dysplasia are presented. Up-to date results of fluorescence spectroscopy and fluorescence imaging of BE are reviewed. These results indicate fluorescence diagnostics promising and clinically useful for detection of early adenocarcinoma and dysplasia in BE.

Adenocarcinoma↗

PubMed2003

Fluorescence confocal microscopy and image analysis of bladder cancer using 5-aminolevulinic acid.

5-aminolevulinic acid mediated changes in tissue specific fluorescence were studied in bladder cancer. Bladders of normal patients and also patients diagnosed with cancer were instilled with 5-aminolevulinic acid and the resultant protoporphyrin IX mediated fluorescence intensity was imaged and quantified with confocal laser microscopy and fluorescence image analysis. Urothelial tumour cells were observed to fluoresce more intensely than normal urothelial cells. Submucosa and muscle tissues exhibited minimal fluorescence compared to urothelial cells of malignant origin and also normal urothelial cells. Degree of fluorescence intensity was in the order of malignant urothelium > normal urothelium > normal submucosa > normal muscles. Fluorescence intensity was also found to increase with duration of ALA instillation. Grade 3 malignant cells produced more fluorescence compared to grade 2 and grade 1. Similarly, T1 transitional cell carcinoma (TCC) showed increased fluorescence intensity than that of Ta TCC. Also, tumour blood vessels fluoresced more intensely compared to blood vessels found in normal bladder tissue. Tissue specific ALA mediated PpIX micro fluorescence can be used as a diagnostic technique for early detection of neoplasms and confocal laser microscopy and fluorescence image analysis are advantageous diagnostic tools for the photodynamic diagnosis of bladder neoplasms in vivo.

Administration, Intravesical↗

PubMedDate not supplied

Thiol-disulfide status and acridine orange fluorescence of mammalian sperm nuclei.

The relationship between thiol-disulfide status and acridine orange fluorescence of testicular, epididymal, and ejaculated spermatozoa in several mammalian species was investigated. Spermatozoa were fixed with acetic alcohol, stained with acridine orange, and examined with a fluorescence microscope. The majority of the nuclei of testicular spermatozoa of the hamster, mouse, and rabbit exhibited red acridine orange fluorescence. The proportion of sperm nuclei with red acridine orange fluorescence decreased as the spermatozoa descended the epididymis. Red acridine orange fluorescence was replaced by green acridine orange fluorescence. The site in the epididymis where 100% of the nuclei exhibited green fluorescence was the distal caput in the mouse, the corpus in the rabbit, and the proximal cauda in the hamster. In semen samples from men with proven fertility, normal semen parameters, or both, about 60% to 90% of the nuclei exhibited green acridine orange fluorescence. The proportion of sperm nuclei exhibiting green acridine orange fluorescence was higher in the spermatozoa pellet (containing highly motile spermatozoa) obtained by centrifugation through a Percoll gradient. From experiments using disulfide-reducing, thiol-oxidizing and thiol-detecting agents, we concluded that sperm nuclei fluoresce red when they are treated with acid while their DNA-associated protamines are poor in disulfides. Under such conditions, DNA is vulnerable to denaturation. Acridine orange binds to denatured (single-stranded) DNA as aggregates and emits red fluorescence. In contrast, when sperm nuclei are treated with acid while their DNA-associated protamines are rich in disulfides, DNA is resistant to denaturation. Acridine orange binds to native (double-stranded) DNA as a monomer and emits green fluorescence.(ABSTRACT TRUNCATED AT 250 WORDS)

Acridine Orange↗

PubMed1984

Immobilization-dependent fluorescence of colchicine.

Colchicine fluoresces when bound to tubulin but not in water, dioxane, or benzene. The basis of the fluorescence has now been investigated. Colchicine fluoresces in higher alcohols and shows a blue shift as a function of chain length. Glycerol produces a higher fluorescence efficiency and a further blue shift. Plots of 1/fluorescence versus T/eta yield straight lines for both alcohols and glycerol/water mixtures. Fluorescence in glycerol/dimethyl sulfoxide mixtures, in which the dielectric constant remains unchanged, varies as a function of solvent viscosity. Even highly nonpolar solvents such as dioxane require a threshold viscosity for fluorescence to occur. When solvent polarity was decreased at constant viscosity, there was also an enhancement of colchicine fluorescence, but this effect appeared to be smaller than that obtained with increasing viscosity. Immobilization by covalent attachment of desacetylcolchicine to thyroglobulin, serum albumin, or lysozyme also promotes fluorescence from the drug. By contrast, the highly rigid analogue of colchicine, imerubine, fluoresces in water and is unaffected by viscosity changes. We concluded that a major contribution to colchicine fluorescence stems from immobilization of colchicine in the site and that this response to immobilization depends, in part, on the partially flexible nature of the drug. Since certain other flexible molecules such as auramine O, reduced flavines, and diarylalkanes also require increased viscosity or binding to macromolecules to fluoresce at room temperature, we propose that immobilization-enhanced fluorescence may be more common than heretofore believed.

Animals↗

PubMed1993

Fluorescence postlabeling assay of DNA damage induced by N-methyl-N-nitrosourea.

We have used a new technique to assay DNA adduct by combining enzymatic digestion of DNA to nucleotides and fluorescence postlabeling. The assay relies on the selectivity of nucleotide chromatography by high-performance liquid chromatography and the sensitivity of fluorescence detection. This report describes the fluorescence postlabeling assay of N7-(N7-methyldGuo) and O6-methyl-2'-deoxyguanosine (O6-methyldGuo) in calf thymus DNA exposed to N-methyl-N-nitrosourea. Using a conventional fluorescence detector, fluorescence postlabeling assay detected 1 modified nucleotide/10(6) normal nucleotides in 100 micrograms DNA. Laser induced fluorescence detection offers a linear response of the chromatographic signal from 10(-13)-10(-16) mol fluorescent nucleotides (correlation coefficient, 0.998) improving the detection limit of the assay to 1 modified nucleotide/10(8) normal nucleotides in 10 micrograms DNA. Fluorescence postlabeling analysis of the authentic markers shows that both N7- and O6-methyldGuo can be assayed with similar detection sensitivity. However, the inherent nature of the instability of N7-methyldGuo, even under physiological conditions, makes its quantitation difficult by any postlabeling technique. O6-MethyldGuo, on the other hand, can be detected with sufficient selectivity and sensitivity by a fluorescence postlabeling assay. Quantitative efficiency of enzymatic excision and chemical postlabeling of O6-methyldGuo were validated by fluorescence postlabeling analysis of synthetic model DNA. Fluorescence postlabeling assay complements 32P-postlabeling assay for O6-methyldGuo without requiring the handling and disposal of radiolabel. Fluorescence postlabeling assay has potential, therefore, to monitor the level of O6-alkyldGuo in human DNA exposed to both environmental and chemotherapeutic alkylating agents.

Animals↗

PubMed2006

Design of peptide substrates for nanosecond time-resolved fluorescence assays of proteases: 2,3-diazabicyclo[2.2.2]oct-2-ene as a noninvasive fluorophore.

Fluorescence protease assays were investigated with peptide substrates containing a 2,3-diazabicyclo[2.2.2]oct-2-ene-labeled asparagine (Dbo) as a fluorescent amino acid. The special characteristic of the fluorophore Dbo is its exceedingly long fluorescence lifetime (ca. 300 ns in water under air), which allows the use of nanosecond time-resolved fluorescence (Nano-TRF) detection to efficiently suppress shorter-lived background emission. In addition, the natural amino acids tryptophan and tyrosine can be employed as intramolecular fluorescence quenchers, which facilitates substrate design. Fourteen synthetic peptide substrates (composed of 2-19 amino acids) and five enzymes (trypsin, pepsin, carboxypeptidase A, leucine aminopeptidase, and chymotrypsin) were investigated and, in all 28 examined combinations, enzymatic activity was detected by monitoring the increase in steady state fluorescence with time and determining the reaction rates as kcat/Km values, which ranged from 0.2 to 80x10(6) M-1 min-1. The results suggest an excellent compatibility of the very small and hydrophilic fluorescent probe Dbo with solid-phase peptide synthesis and the investigated proteases. For all 14 peptides the fluorescence lifetimes before and after enzymatic cleavage were measured and Nano-TRF measurements were performed in 384-well microplates. The fluorescence lifetimes of the different peptides provide the basis for the rational design of Dbo-based fluorescent substrates for protease assays. Measurements in Nano-TRF mode revealed, in addition to efficient suppression of background fluorescence, an increased differentiation between cleaved and uncleaved substrate. The Dbo-based assays can be adapted for high-throughput screening.

Amino Acid Sequence↗

PubMed2001

Development of a time-resolved fluorometric method for observing hybridization in living cells using fluorescence resonance energy transfer.

We previously showed that a specific kind of mRNA (c-fos) was detected in a living cell under a microscope by introducing two fluorescently labeled oligodeoxynucleotides, each labeled with donor or acceptor, into the cytoplasm, making them hybridize to adjacent locations on c-fos mRNA, and taking images of fluorescence resonance energy transfer (FRET) (A. Tsuji, H. Koshimoto, Y. Sato, M. Hirano. Y. Sei-Iida, S. Kondo, and K. Ishibashi, 2000, Biophys. J. 78:3260-3274). On the formed hybrid, the distance between donor and acceptor becomes close and FRET occurs. To observe small numbers of mRNA in living cells using this method, it is required that FRET fluorescence of hybrid must be distinguished from fluorescence of excess amounts of non-hybridizing probes and from cell autofluorescence. To meet these requirements, we developed a time-resolved method using acceptor fluorescence decays. When a combination of a donor having longer fluorescence lifetime and an acceptor having shorter lifetime is used, the measured fluorescence decays of acceptors under FRET becomes slower than the acceptor fluorescence decay with direct excitation. A combination of Bodipy493/503 and Cy5 was selected as donor and acceptor. When the formed hybrid had a configuration where the target RNA has no single-strand part between the two fluorophores, the acceptor fluorescence of hybrid had a sufficiently longer delay to detect fluorescence of hybrid in the presence of excess amounts of non-hybridizing probes. Spatial separation of 10-12 bases between two fluorophores on the hybrid is also required. The decay is also much slower than cell autofluorescence, and smaller numbers of hybrid were detected with less interference of cell autofluorescence in the cytoplasm of living cells under a time-resolved fluorescence microscope with a time-gated function equipped camera. The present method will be useful when observing induced expressions of mRNA in living cells.

3T3 Cells↗

PubMed1995

Fluorescence cystoscopy following intravesical instillation of 5-aminolevulinic acid: a new procedure with high sensitivity for detection of hardly visible urothelial neoplasias.

Methods have been sought for the in vivo marking of tiny papillary tumors of the bladder and flat urothelial lesions such as dysplasia or carcinoma in situ, which can easily be missed during conventional endoscopy under white light. A new procedure is reported for the fluorescence detection of urothelial dysplasia and early bladder cancer. The method is based on intravesical application of 5-aminolevulinic acid (ALA). ALA if applied exogenously induces accumulation of protoporphyrin IX (PPIX) in the urothelium of the bladder. PPIX is an intensively red fluorescing agent. The mean ratio of fluorescence intensity between urothelial cancer and normal epithelium was found to be 17:1. Fluorescence excitation was achieved by violet light from a krypton ion laser (lambda = 406.7 nm) or from a xenon arc lamp with a bandpass filter system (lambda = 375-440 nm). Both light sources proved to be of equal suitability for fluorescence excitation. Fluorescence microscopy revealed that the PPIX fluorescence is strictly limited to the urothelium. It could not be detected from the submucosa or muscle of the bladder. Bladder wall biopsies were taken from 90 patients with suspicion of bladder cancer under fluorescence view. The fluorescence detection proved to be of high sensitivity (98%). No serious side effects which would preclude further clinical testing, especially no cutaneous photoreaction, were observed. Tumor-associated fluorescence induced by topical ALA application offers new perspectives in the diagnosis and treatment of bladder cancer. In case of suspicious or positive urine cytologic findings, ALA fluorescence cystoscopy may be useful for detecting the precise site of the malignancy. The procedure might be helpful in complete resection or coagulation of urothelial neoplasms. Due to this, diminishing recurrence rates are expected. However, this hypothesis has to be studied in prospective clinical trials.

Administration, Intravesical↗