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At least 109 records · Page 6Linked to original sources
PubMed1979

Effect of phosphate ions on the fluorescence of tryptophan derivatives. Implications in fluorescence investigation of protein-nucleic acid complexes.

In order to test the ability of phosphate groups to quench the fluorescence of tryptophan in protein-nucleic acid complexes we have studied the effect of various phosphate ions on the fluorescence of tryptophan derivatives. Unsubstituted and monoalkyl monoanions (H2PO4- and CH3OPO3H-) quench the fluorescence of all investigated indole derivatives while the dimethyl anion (CH3O)2 PO2- does not. This suggests that quenching of tryptophan fluorescence by phosphate monoanions requires the presence of an acidic OH group and could be due to a proton transfer from the phosphate ion to the indole chromophore. Trianions (PO4 3-4) which are strong proton acceptors quench the fluorescence of all tryptophan derivatives except N(1)methyl tryptophan. This result strongly supports our proposal that quenching of tryptophan fluorescence by phosphate trianions occurs through deprotonation of the NH indole group. Bianions (HPO '4(7), and CH3O PO3 2-3) quench the fluorescence of several indole derivatives including N-acetyl tryptophanamide but have no effect on tryptophan or N(1)-methyl tryptophan. From our results we conclude that phosphate groups of nucleic acids are not able to quench the fluorescence of tryptophyl residues in protein-nucleic acid complexes except if an accessible residue is located near a phosphorylated polynucleotide chain end.

Nucleic Acids↗
PubMed1984

Static and time-resolved fluorescence studies of fluorescent phosphatidylcholine bound to the phosphatidylcholine transfer protein of bovine liver.

Phosphatidylcholine analogues containing a cis- parinaroyl chain at the sn-1, sn-2, or both sn-1 and sn-2 positions (1-PnA-PC, 2-PnA-PC, and diPnA -PC, respectively) have been used to investigate the lipid binding site of the phosphatidylcholine transfer protein (PC-TP) from bovine liver by fluorometric techniques. Binding of these fluorescent lipids to the protein was registered by measuring the enhancement of parinaroyl fluorescence and the quenching of the tryptophanyl fluorescence. The fluorescence intensity of 1-PnA-, 2-PnA- and diPnA -PC bound to PC-TP was proportional to the chromophore content. The energy-transfer efficiency between the tryptophan residues and the bound chromophores was approximately 40% for 1-PnA- and 2-PnA-PC and 60% for diPnA -PC. Quenching of the tryptophanyl fluorescence was, in part, accounted for by a decrease of the fluorescence lifetimes. The orientation of the 1 and 2 fatty acyl chains of the PnA-PC analogues on the transfer protein was analyzed by time-resolved fluorescence anisotropy measurements. The fluorescence anisotropy decayed according to a single exponential function yielding a rotational correlation time of 26 ns for 1-PnA-PC, 11 ns for 2-PnA-PC, and 15 ns for diPnA -PC. These correlation times indicate that both fatty acyl chains are immobilized at different positions on the protein. From the difference in correlation time we propose that the shape of the phosphatidylcholine transfer protein is ellipsoidal (axial ratio congruent to 2.5) with the 1 fatty acyl chain oriented parallel to the long symmetry axis and having an angle of 60-90 degrees with the 2 fatty acyl chain.

Androgen-Binding Protein↗
PubMed1986

Time-resolved fluorescence and 1H NMR studies of tyrosine and tyrosine analogues: correlation of NMR-determined rotamer populations and fluorescence kinetics.

The time-resolved fluorescence properties of phenol and straight-chained phenol derivatives and tyrosine and simple tyrosine derivatives are reported for the pH range below neutrality. Phenol and straight-chained phenol derivatives exhibit single exponential fluorescence decay kinetics in this pH range unless they have a titratable carboxyl group. If a carboxyl group is present, the data follow a two-state, ground-state, Henderson-Hasselbalch relationship. Tyrosine and its derivatives with a free carboxyl group display complex fluorescence decay behavior as a function of pH. The complex kinetics cannot be fully explained by titration of a carboxyl group; other ground-state processes are evident, especially since tyrosine analogues with a blocked carboxyl group are also multiexponential. The fluorescence kinetics can be explained by a ground-state rotamer model. Comparison of the preexponential weighting factors (amplitudes) of the fluorescence decay constants with the 1H NMR determined phenol side-chain rotamer populations shows that tyrosine derivatives with a blocked or protonated carboxyl group have at least one rotamer exchanging more slowly than the radiative and nonradiative rates, and the fluorescence data are consistent with a slow-exchange model for all three rotamers, the shortest fluorescence decay constant is associated with a rotamer where the carbonyl group can contact the phenol ring, and in the tyrosine zwitterion, either rotamer interconversion is fast and an average lifetime is seen or rotamer interconversion is slow and the individual fluorescence decay constants are similar.

Hydrogen-Ion Concentration↗
PubMed1985

Evidence for plasma membrane impermeability to small ions in acrosome-intact mouse spermatozoa bound to mouse zonae pellucidae, using an aminoacridine fluorescent pH probe: time course of the zona-induced acrosome reaction monitored by both chlortetracycline and pH probe fluorescence.

Previous studies have shown that capacitated mouse spermatozoa bind to zonae pellucidae of mouse eggs with acrosomes apparently intact. The question addressed in this study was the following: are the membrane permeability barriers of the apparently acrosome-intact sperm still retained or is there a preliminary stage of the acrosome reaction in which these barriers are lost and the intracellular space becomes accessible to extracellular substrates? The experimental approach was to use the fluorescent pH probe 9-amino-3-chloro-7-methoxyacridine, which accumulates in intracellular spaces of lower pH than the suspending medium with the result that the cells become fluorescent. Freshly capacitated mouse spermatozoa bound to isolated zonae showed uniform fluorescence over the head and midpiece with this fluorescent probe at early times of binding. The fluorescence was abolished by NH4+ and nigericin, agents that equilibrate H+ across cell membranes. At these early times of binding, the acrosomes were fully intact as judged by chlortetracycline fluorescence pattern, which itself was unaffected by either N4+ or nigericin. The time course of the loss of this chlortetracycline pattern characteristic of acrosome-intactness was closely paralleled by loss of fluorescence of 9-amino-3-chloro-6-methoxyacridine over the first 90 min; thereafter, loss of the chlortetracycline pattern was somewhat more rapid. This result shows that acrosome-intact sperm bound to zonae pellucidae retain the permeability barriers of the plasma membrane to small cations; no evidence was found for an early stage of membrane "leakiness" preceding the acrosome reaction. The ionophore A23187 induced a very rapid acrosome reaction in sperm bound to isolated zonae, as judged with both fluorescence probes. This rapid reaction was partially inhibited by 3-quinuclidinyl benzilate, which, in the absence of ionophore, completely blocks the occurrence of the acrosome reaction in sperm bound to zonae. This suggests involvement of a specific calcium entry mechanism in the acrosome reaction of mouse sperm induced by mouse zonae pellucidae.

Acrosome↗
PubMed2006

Monitoring protein interactions in the living cell through the fluorescence decays of the cyan fluorescent protein.

Using fluorescence lifetime microspectroscopy and imaging techniques, we have studied the fluorescence of cyan fluorescent protein (CFP) transiently expressed in HEK-293 cells, in the presence or absence of its fluorescence resonance energy transfer (FRET) partner, yellow fluorescent protein (YFP). When the two proteins are attached through a 27-amino-acid linker, a 33 % average efficiency of intramolecular energy transfer is accurately determined inside the cell. Additionally, we observe a systematic quenching of the CFP fluorescence with increasing levels of protein expression. This quenching cannot be accounted for by formation of the previously described dimer of GFP-related proteins, since its magnitude is unchanged when the fluorescent proteins carry the mutation A206K shown to dissociate this dimer in vitro. Even when the intracellular protein concentration largely exceeds the in vitro dissociation constant of the dimer, self-association remains undetectable, either between free proteins or intramolecularly within the CFP-YFP construct. Instead, the detailed concentration effects are satisfactorily accounted for by a model of intermolecular, concentration-dependent energy transfer, arising from molecular proximity and crowding. In the case of CFP alone, we suggest that self-quenching could result from a pseudo-homo FRET mechanism between different, spectrally shifted emissive forms of the protein. These phenomena require careful consideration in intracellular FRET studies.

Bacterial Proteins↗
PubMed1979

Simultaneous use of retrograde fluorescent tracers and fluorescence histochemistry for convenient and precise mapping of monoaminergic projections and collateral arrangements in the CNS.

A procedure is described for the use of fluorescent retrograde tracers in conjunction with monamine fluorescence histochemistry for single or double labeling studies of the projections of identified catecholamine or indoleamine neurons in the CNS. A number of fluorescent tracers, including Evans Blue, bisbenzimide, DAPI, 'True Blue', 'Granular Blue' and propidium iodide, have been tested and characterized microspectrofluorometrically. Of these, 'True Blue', propidium iodide and Evans Blue were found to have the most suitable properties to be studied concomitant with the monoamine fluorphores. A combination of two of these tracers--'True Blue' fluorescing blue and propidium iodide or Evans Blue fluorescing orange to red--and the catecholamines and indoleamines fluorescing yellow-green and yellow to brownish yellow, respectively, makes possible simultaneous use of four different fluorescent markers in one and the same section. The present procedure has great practical advantages over available HRP techniques and should in modified form also be applicable to other types of transmitter-specific neuronal tracing based on immunocytochemistry or enzyme histochemistry.

Animals↗
PubMed2000

One- and two-photon excited fluorescence lifetimes and anisotropy decays of green fluorescent proteins.

We have used one- (OPE) and two-photon (TPE) excitation with time-correlated single-photon counting techniques to determine time-resolved fluorescence intensity and anisotropy decays of the wild-type Green Fluorescent Protein (GFP) and two red-shifted mutants, S65T-GFP and RSGFP. WT-GFP and S65T-GFP exhibited a predominant approximately 3 ns monoexponential fluorescence decay, whereas for RSGFP the main lifetimes were approximately 1.1 ns (main component) and approximately 3.3 ns. The anisotropy decay of WT-GFP and S65T-GFP was also monoexponential (global rotational correlation time of 16 +/- 1 ns). The approximately 1.1 ns lifetime of RSGFP was associated with a faster rotational depolarization, evaluated as an additional approximately 13 ns component. This feature we attribute tentatively to a greater rotational freedom of the anionic chromophore. With OPE, the initial anisotropy was close to the theoretical limit of 0.4; with TPE it was higher, approaching the TPE theoretical limit of 0.57 for the colinear case. The measured power dependence of the fluorescence signals provided direct evidence for TPE. The general independence of fluorescence decay times, rotation correlation times, and steady-state emission spectra on the excitation mode indicates that the fluorescence originated from the same distinct excited singlet states (A*, I*, B*). However, we observed a relative enhancement of blue fluorescence peaked at approximately 440 nm for TPE compared to OPE, indicating different relative excitation efficiencies. We infer that the two lifetimes of RSGFP represent the deactivation of two substates of the deprotonated intermediate (I*), distinguished by their origin (i.e., from A* or B*) and by nonradiative decay rates reflecting different internal environments of the excited-state chromophore.

Amino Acid Substitution↗
PubMed2001

Red fluorescent protein from Discosoma as a fusion tag and a partner for fluorescence resonance energy transfer.

The biochemical and biophysical properties of a red fluorescent protein from a Discosoma species (DsRed) were investigated. The recombinant DsRed expressed in E. coli showed a complex absorption spectrum that peaked at 277, 335, 487, 530, and 558 nm. Excitation at each of the absorption peaks produced a main emission peak at 583 nm, whereas a subsidiary emission peak at 500 nm appeared with excitation only at 277 or 487 nm. Incubation of E. coli or the protein at 37 degrees C facilitated the maturation of DsRed, resulting in the loss of the 500-nm peak and the enhancement of the 583-nm peak. In contrast, the 500-nm peak predominated in a mutant DsRed containing two amino acid substitutions (Y120H/K168R). Light-scattering analysis revealed that DsRed proteins expressed in E. coli and HeLa cells form a stable tetramer complex. DsRed in HeLa cells grown at 37 degrees C emitted predominantly at 583 nm. The red fluorescence was imaged using a two-photon laser (Nd:YLF, 1047 nm) as well as a one-photon laser (He:Ne, 543.5 nm). When fused to calmodulin, the red fluorescence produced an aggregation pattern only in the cytosol, which does not reflect the distribution of calmodulin. Despite the above spectral and structural complexity, fluorescence resonance energy transfer (FRET) between Aequorea green fluorescent protein (GFP) variants and DsRed was achieved. Dynamic changes in cytosolic free Ca2+ concentrations were observed with red cameleons containing yellow fluorescent protein (YFP), cyan fluorescent protein (CFP), or Sapphire as the donor and RFP as the acceptor, using conventional microscopy and one- or two-photon excitation laser scanning microscopy. Particularly, the use of the Sapphire-DsRed pair rendered the red cameleon tolerant of acidosis occurring in hippocampal neurons, because both Sapphire and DsRed are extremely pH-resistant.

Animals↗
PubMed2004

Origin, nature, and fate of the fluorescent state of the green fluorescent protein chromophore at the CASPT2//CASSCF resolution.

Ab initio CASPT2//CASSCF relaxation path computations are employed to determine the intrinsic (e.g., in vacuo) mechanism underlying the rise and decay of the luminescence of the anionic form of the green fluorescent protein (GFP) fluorophore. Production and decay of the fluorescent state occur via a two-mode reaction coordinate. Relaxation along the first (totally symmetric) mode leads to production of the fluorescent state that corresponds to a planar species. The second (out-of-plane) mode controls the fluorescent state decay and mainly corresponds to a barrierless twisting of the fluorophore phenyl moiety. While a "space-saving" hula-twist conical intersection decay channel is found to lie only 5 kcal mol(-1) above the fluorescent state, the direct involvement of a hula-twist deformation in the decay is not supported by our data. The above results indicate that the ultrafast fluorescence decay observed for the GFP chromophore in solution is likely to have an intrinsic origin. The possible effects of the GFP protein cavity on the fluorescence lifetime of the investigated chromophore model are discussed.

Anions↗
PubMed2001

Use of fluorescent Ca2+ dyes with green fluorescent protein and its variants: problems and solutions.

We have studied the degree to which fluorescent Ca(2+) indicator dyes, and green fluorescent protein and its variants, can be used together. We find that the most commonly used fluorescent protein, enhanced green fluorescent protein (EGFP), seriously contaminates fura 2 signals. We suggest two alternative combinations for which there is no detectable contamination of the Ca(2+) indicator signal by the fluorescent protein. Blue fluorescent protein can be used with the Ca(2+) indicator Fura Red; EGFP can be used with the Ca(2+) indicator X-Rhod 1. The use of these combinations will permit the accurate measurement of Ca(2+) signals in cells transfected with fluorescent proteins.

Animals↗
PubMed1963

Adsorption and fluorescence of fat-soluble fluorescent dyes on class I and class III Saccharomyces cerevisiae.

Lycette, R. M. (Illinois Institute of Technology, Chicago) and L. R. Hedrick. Adsorption and fluorescence of fat-soluble fluorescent dyes on class I and class III Saccharomyces cerevisiae. J. Bacteriol. 85:1-6. 1963.-Class III cells of the brewery yeast Saccharomyces cerevisiae flocculate, but class I cells do not, when cultured in an organic YM medium. Class III cells adsorb the fat-soluble stilbyl-triazole fluorescent dye (STC) more readily and produce more fluorescence than do class I cells. This suggests that cells of the class III yeasts have more lipids or more phospholipids on their surface than do cells of class I yeasts. Fluorescence of STC in the absence of cells is pH-independent, but adsorption of the compound on the surface of cells, with the associated fluorescence, is pH-dependent. As the pH level was decreased within the range of pH 9 to 0, and as the temperature was elevated within the range of 0 to 40 C, there was an increase in adsorption and a corresponding increase in fluorescence. Maximal adsorption was attained in less than 15 min when the cells and dye were shaken at 26 C. Cells of class III adsorbed 2.5 mug of STC per 3.25 mg (dry wt) of cells. The kinetics of adsorption follow the Langmuir monomolecular model.

Adsorption↗
PubMed2003

A novel design method of ratiometric fluorescent probes based on fluorescence resonance energy transfer switching by spectral overlap integral.

A ratiometric measurement, namely, simultaneous recording of the fluorescence intensities at two wavelengths and calculation of their ratio, allows greater precision than measurements at a single wavelength, and is suitable for cellular imaging studies. Here we describe a novel method of designing probes for ratiometric measurement of hydrolytic enzyme activity based on switching of fluorescence resonance energy transfer (FRET). This method employs fluorescent probes with a 3'-O,6'-O-protected fluorescein acceptor linked to a coumarin donor through a linker moiety. As there is no spectral overlap integral between the coumarin emission and fluorescein absorption, the fluorescein moiety cannot accept the excitation energy of the donor moiety and the donor fluorescence can be observed. After cleavage of the protective groups by hydrolytic enzymes, the fluorescein moiety shows a strong absorption in the coumarin emission region, and then acceptor fluorescence due to FRET is observed. Based on this mechanism, we have developed novel ratiometric fluorescent probes (1-3) for protein tyrosine phosphatase (PTP) activity. They exhibit a large shift in their emission wavelength after reaction with PTPs. The fluorescence quenching problem that usually occurs with FRET probes is overcome by using the coumarin-cyclohexane-fluorescein FRET cassette moiety, in which close contact of the two dyes is hindered. After study of their chemical and kinetic properties, we have concluded that compounds 1 and 2 bearing a rigid cyclohexane linker are practically useful for the ratiometric measurement of PTPs activity. The design concept described in this paper, using FRET switching by spectral overlap integral and a rigid link that prevents close contact of the two dyes, should also be applicable to other hydrolytic enzymes by introducing other appropriate enzyme-cleavable groups into the fluorescein acceptor.

Cell Membrane Permeability↗
PubMed2006

A comparison of the fluorescence dynamics of single molecules of a green fluorescent protein: one- versus two-photon excitation.

We report on the dynamics of fluorescence from individual molecules of a mutant of the wild-type green fluorescent protein (GFP) from Aequorea victoria, super folder GFP (SFGFP). SFGFP is a novel and robust variant designed for in vivo high-throughput screening of protein expression levels. It shows increased thermal stability and is able to retain its fluorescence when fused to poorly folding proteins. We use a recently developed single-molecule technique which combines fluorescence-fluctuation spectroscopy and time-correlated single photon counting in order to characterize the photophysical properties of SFGFP under one- (OPE) and two- (TPE) photon excitation conditions. We use Rhodamine 110 as a model chromophore to validate the methodology and to explain the single-molecule results of SFGFP. Under OPE, single SFGFP molecules undergo fluorescence flickering on the time scale of micros and tens of micros due to triplet formation and ground-state protonation-deprotonation, respectively, as demonstrated by excitation intensity- and pH-dependent experiments. OPE single-molecule fluorescence lifetimes indicate heterogeneity in the population of SFGFP, indicating the presence of the deprotonated I and B forms of the SFGFP chromophore. TPE of single SFGFP molecules results in the photoconversion of the chromophore. TPE of single SFGFP molecules show fluorescence flickering on the time scale of micros due to triplet formation. A flicker connected with protonation-deprotonation of the SFGFP chromophore is detected only at low pH. Our results show that SFGFP is a promising fusion reporter for intracellular applications using OPE and TPE microscopy.

Biophysical Phenomena↗
PubMed2004

Scanning fluorescent microscopy is an alternative for quantitative fluorescent cell analysis.

BACKGROUND: Fluorescent measurements on cells are performed today with FCM and laser scanning cytometry. The scientific community dealing with quantitative cell analysis would benefit from the development of a new digital multichannel and virtual microscopy based scanning fluorescent microscopy technology and from its evaluation on routine standardized fluorescent beads and clinical specimens. METHODS: We applied a commercial motorized fluorescent microscope system. The scanning was done at 20 x (0.5 NA) magnification, on three channels (Rhodamine, FITC, Hoechst). The SFM (scanning fluorescent microscopy) software included the following features: scanning area, exposure time, and channel definition, autofocused scanning, densitometric and morphometric cellular feature determination, gating on scatterplots and frequency histograms, and preparation of galleries of the gated cells. For the calibration and standardization Immuno-Brite beads were used. RESULTS: With application of shading compensation, the CV of fluorescence of the beads decreased from 24.3% to 3.9%. Standard JPEG image compression until 1:150 resulted in no significant change. The change of focus influenced the CV significantly only after +/-5 microm error. CONCLUSIONS: SFM is a valuable method for the evaluation of fluorescently labeled cells.

Fluorescent Dyes↗
PubMed2006

Single molecule fluorescence imaging of the photoinduced conversion and bleaching behavior of the fluorescent protein Kaede.

Photoconversion and photobleaching behavior of the fluorescent protein Kaede immobilized in polyacrylamide gel matrix at room temperature was studied by single molecule wide-field fluorescence microscopy. Photobleaching kinetics of Kaede molecules upon excitation at 488 nm showed slight heterogeneity, suggesting the presence of different protein conformations and/or the distribution of local environments in the gel matrix. Statistical analysis of intensity trajectories of single molecules revealed four major types of fluorescence dynamics behavior upon short illumination by a violet light pulse (405 nm). In particular, two types of photoswitching behavior were observed: the green-to-red photoconversion (4% of Kaede molecules) and the photoactivation of green fluorescence without emission of red fluorescence (13%). Two other major groups show neither photoconversion nor red emission and demonstrate photoinduced partial deactivation (43%) and partial revival (30%) of green fluorescence. The significantly lower green-to-red conversion ratio as compared with bulk measurements in aqueous solution might be induced by the immobilization of the protein molecules within a polyacrylamide gel. Contrary to Ando et al. (Proc Natl Acad Sci 2002;99:12651-12656), we found a significant increase in green fluorescence emission upon illumination with 405-nm light, which is typical for GFP and related proteins.

Animals↗
PubMed1994

Measurement of fluorescence changes of NAD(P)H and of fluorescent flavoproteins in saponin-skinned human skeletal muscle fibers.

Saponin-skinned human muscle fibers from M. vastus lateralis were immobilized in a quartz capillary to detect the fluorescence changes of NAD(P)H and of fluorescent flavoproteins. To get sufficient intense fluorescence signals from a small amount of muscle tissue the NAD(P)H fluorescence was excited by means of an HeCd laser at 325 nm and the flavoprotein fluorescence by an argon-ion laser at 454 nm or by the second wavelength of a HeCd laser at 442 nm. Using this experimental setup the fluorescence spectra of NAD(P)H, of alpha-lipoamide dehydrogenase and of electron-transfer flavoprotein were detected in saponin-skinned human muscle fibers. These fibers behaved identically to isolated mitochondria: (i) The addition of substrates caused an increase in reduction of mitochondrial NAD+, (ii) the addition of ADP caused its reoxidation, and (iii) the addition of respiratory chain inhibitors led to an almost complete reduction of NAD+. It was observed that the redox state of the NAD(P) system and of the alpha-lipoamide dehydrogenase reached after addition of 1 mM ADP correlates with the rate of active state respiration with NAD-dependent substrates. Therefore, this fluorimetric method is suitable to compare the mitochondrial oxidation capacities of NAD-dependent substrates in less then 5 mg wet weight muscle tissue. Moreover, the maximal changes in fluorescence of NAD(P)H and flavoproteins correlate with the amount of mitochondrial marker enzymes per milligram muscle tissue. Using this method a myopathy caused by a diminished content of mitochondria per milligram muscle tissue was observed.

Adenosine Diphosphate↗
PubMed1977

Formaldehyde-induced fluorescence in the telencephalon and diencephalon of the eel (Anguilla anguilla l.). A fluorescence-microscopic and microspectrofluorometric investigation with special reference to the innervation of the pituitary.

In the telencephalon and diencephalon of the eel (Anguilla anguilla L.) formaldehyde-induced fluorescence was studied microscopically and microfluorometrically with special emphasis on the innervation of the pituitary. In the telencephalon fluorescent fibers contained predominantly noradrenaline fluorophores. Fluorescent nuclei could not be established. In the diencephalon fluorescent perikarya were found in: (1) the paraventricular organ (PVO), possessing either dopamine or, to a lesser extent, serotonin fluorophores; (2) the PVO-accompanying group, exhibiting spectral data resembling those of noradrenaline fluorophores; (3) the nucleus hypothalami anterior (NHA), a small paired group of catecholamine-containing cells posterior to the commissura transversa.--The nucleus lobi inferioris exhibited a high density of delicate, most probably dopamine-containing terminals, while fibers surrounding this nucleus contained noradrenaline fluorophores. A high density of fluorescent terminals containing dopamine and/or noradrenaline was found in the habenular complex. Fluorescent terminals in the pituitary contained fluorophores resembling either dopamine or noradrenaline. Fluorescent tracts entered the pituitary from different directions. A rostral, unpaired tract enters the neurointermediate lobe, as also verified experimentally. The rostral pars distalis receives two paired tracts, one from a rostral and one from a dorsal direction. The proximal pars distalis also receives two paired tracts, one from a dorsal and one from a posterior direction.

Anguilla↗
PubMed1996

Fluorescence lifetime imaging: an emerging technique in fluorescence microscopy.

Fluorescence microscopy is an important tool for biological research, in part because of the extremely high detection sensitivity that can be achieved, but also because fluorescent molecules can be used as probes on account of their environmental responsiveness, for example to measure intracellular pH or metal ion concentration. Unfortunately, the environmental sensitivity can sometimes be a source of problems because of enhancement or 'quenching', which can make it very difficult to relate emission intensity to the amount of fluorophore present. The measured intensity is essentially proportional to the product of the amount of fluorophore present in the sample and the local quantum yield of the fluorophore (the quantum yield can be thought of as the probability that an excited molecule decays by fluorescence emission rather than by other non-radiative processes). This is a particular difficulty in an environment such as a cell or tissue slice in which quantum yield and flurophore concentration can both vary within the sample. Ideally we would wish to be able to measure the quantum yield of fluorescence as well as the fluorescence intensity, as this would allow environmental effects to be compensated for. Unfortunately, this is not at all easy, and indirect means to achieve the same goal are more appropriate. A recently introduced technique, fluorescence lifetime imaging (Morgan et al. 1992, Wang et al. 1992), offers one such means to improve quantification of fluorescence microscopy. In addition, as will be explained, the technique offers the prospect of significantly improving detection sensitivity in appropriate circumstances.

Energy Transfer↗