Nitrogen-pumped Dye Laser Research Articles

Motor nerve transection and time-lapse imaging of glial cell behaviors in live zebrafish.

The nervous system is often described as a hard-wired component of the body even though it is a considerably fluid organ system that reacts to external stimuli in a consistent, stereotyped manner, while maintaining incredible flexibility and plasticity. Unlike the central nervous system (CNS), the peripheral nervous system (PNS) is capable of significant repair, but we have only just begun to understand the cellular and molecular mechanisms that govern this phenomenon. Using zebrafish as a model system, we have the unprecedented opportunity to couple regenerative studies with in vivo imaging and genetic manipulation. Peripheral nerves are composed of axons surrounded by layers of glia and connective tissue. Axons are ensheathed by myelinating or non-myelinating Schwann cells, which are in turn wrapped into a fascicle by a cellular sheath called the perineurium. Following an injury, adult peripheral nerves have the remarkable capacity to remove damaged axonal debris and re-innervate targets. To investigate the roles of all peripheral glia in PNS regeneration, we describe here an axon transection assay that uses a commercially available nitrogen-pumped dye laser to axotomize motor nerves in live transgenic zebrafish. We further describe the methods to couple these experiments to time-lapse imaging of injured and control nerves. This experimental paradigm can be used to not only assess the role that glia play in nerve regeneration, but can also be the platform for elucidating the molecular mechanisms that govern nervous system repair.

Imaging fluorescence lifetime modulation of a ruthenium-based dye in living cells: the potential for oxygen sensing

Fluorescence lifetime measurements of long excited-state lifetime, oxygen-quenched ruthenium dyes are emerging as methods for intracellular oxygen sensing. Fluorescence lifetime imaging microscopy (FLIM) studies in cells have been reported previously. Many current FLIM systems use high repetition rate (∼107 Hz) lasers optimized for nanosecond lifetime measurements, making measurement of long, microsecond lifetime fluorophores difficult. Here, we present an experimental approach for obtaining a large temporal dynamic range in a FLIM system by using a low repetition rate (101 Hz), high output, nitrogen pumped dye laser and a wide-field, intensified CCD camera for image detection. We explore the feasibility of the approach by imaging the oxygen-sensitive dye tris(2,2′-bipyridyl)dichloro-ruthenium(II) hexahydrate (RTDP) in solution and in living cells. We demonstrate the ability of the system to resolve 60% variations in RTDP fluorescence lifetime upon oxygen cycling in solution. Furthermore, the FLIM system was able to resolve an increase in RTDP fluorescence lifetime in cultured human epithelial cells under diminished oxygen conditions. The technique may be useful in developing methods for quantifying intracellular oxygen concentrations.

A UV–Visible–NIR fluorescence lifetime imaging microscope for laser-based biological sensing with picosecond resolution

This article describes the design and characterization of a wide-field, time-domain fluorescence lifetime imaging microscopy (FLIM) system developed for picosecond time-resolved biological imaging. The system consists of a nitrogen-pumped dye laser for UV–visible–NIR excitation (337.1–960 nm), an epi-illuminated microscope with UV compatible optics, and a time-gated intensified CCD camera with an adjustable gate width (200 ps-10-3 s) for temporally resolved, single-photon detection of fluorescence decays with 9.6-bit intensity resolution and 1.4-μm spatial resolution. Intensity measurements used for fluorescence decay calculations are reproducible to within 2%, achieved by synchronizing the ICCD gate delay to the excitation laser pulse via a constant fraction optical discriminator and picosecond delay card. A self-consistent FLIM system response model is presented, allowing for fluorescence lifetimes (0.6 ns) significantly smaller than the FLIM system response (1.14 ns) to be determined to 3% of independently determined values. The FLIM system was able to discriminate fluorescence lifetime differences of at least 50 ps. The spectral tunability and large temporal dynamic range of the system are demonstrated by imaging in living human cells: UV-excited endogenous fluorescence from metabolic cofactors (lifetime ∼1.4 ns); and 460-nm excited fluorescence from an exogenous oxygen-quenched ruthenium dye (lifetime ∼400 ns).

Detection of high-grade dysplasia in Barrett's esophagus by spectroscopy measurement of 5-aminolevulinic acid-induced protoporphyrin IX fluorescence

Background: Preliminary studies with qualitative detection methods suggest that 5-aminolevulinic acid-induced protoporphyrin IX fluorescence might improve the detection of dysplastic Barrett's epithelium. This study used quantitative methods to determine whether aminolevulinic acid-induced protoporphyrin IX fluorescence can differentiate between Barrett's mucosa with and without dysplasia. Methods: Patients were given 10 mg/kg of aminolevulinic acid orally 3 hours before endoscopy. Quantitative fluorescence spectra were acquired by using a nitrogen-pumped dye laser (l 400 nm) spectrograph system. The protoporphyrin IX fluorescence intensity at 635 nm was compared with the histopathologic diagnosis for mucosal biopsy specimens taken immediately after the fluorescence measurements. Results: Ninety-seven spectra were obtained from 20 patients. The mean (± standard error) standardized protoporphyrin IX fluorescence intensity was significantly greater ( p < 0.05) for high-grade dysplastic Barrett's epithelium (0.29 ± 0.07, n = 13) than for nondysplastic Barrett's epithelium (0.11 ± 0.02, n = 43). By using protoporphyrin IX fluorescence alone, high-grade dysplasia was distinguished from nondysplastic tissue types with 77% sensitivity and 71% specificity. Decreased autofluorescence was particularly found in nodular high-grade dysplasia. By using the fluorescence intensity ratio of 635 nm/480 nm, nodular high-grade dysplasia could be differentiated from nondysplastic tissue with 100% sensitivity and 100% specificity. Conclusion: Protoporphyrin IX fluorescence may be useful for identifying areas of high-grade dysplasia in Barrett's esophagus and for targeting of biopsies. (Gastrointest Endosc 2002;56:479-87.)

An introduction to laser modeling studies with a nitrogen-pumped dye laser

The following exercises, which are suitable for an intermediate physics or physical chemistry class, provide an introduction to the use of computer modeling in the study of laser dynamics. The first exercise is performed with a tunable nitrogen-pumped dye laser to investigate empirically the effects that changes in gain and internal loss have on laser pulse characteristics. The second exercise employs a numerical model, based on the laser rate equations, to perform a quantitative study of the same effects. Since the model is constructed with the parameters that characterize the laser used in the first exercise, it can be exploited to gain deeper insight into the dynamics of the system. Together, these exercises expose students to the fundamentals of laser dynamics and techniques used to understand the buildup of a laser pulse.

Computer-controlled laser ablation: a convenient and versatile tool for micropatterning biofunctional synthetic surfaces for applications in biosensing and tissue engineering.

This paper describes laser-based methods for preparing micropatterns of bioactive molecular species in self-assembled monolayers (SAMs) and micropatterns of proteins and other biological molecules immobilized on solid substrates. Applications of these micropatterned surfaces in multianalyte biosensing and tissue engineering are emphasized. The focus of the paper is on the use of a computer-controlled laser ablation system comprising a research-grade inverted optical microscope, a pulsed nitrogen-pumped dye laser emitting at 390 nm, a programmable sample stage, and the computerized control system. The laser system can be implemented in a typical biosensor or tissue culture laboratory to enable the facile and reproducible fabrication of micropatterned surfaces by several methods. Various methods for patterning are discussed with examples given and emphasis placed on (1) laser ablation in the fabrication of photolithography masks, (2) electrochemical patterning of SAMs, and (3) laser desorption of SAMs. The relative merits of each technique are discussed with respect to application in fabrication of active surfaces for biosensing and tissue culture applications.

Early diagnosis of upper aerodigestive tract cancer by autofluorescence.

To explore the potential of autofluorescence spectroscopy as a tool for early detection of upper aerodigestive tract cancer. Autofluorescence spectral characteristics of 19 untreated oral and oropharyngeal lesions in 13 patients were studied with excitation wavelengths of 370 and 410 nm generated by a nitrogen pumped dye laser. Ten healthy volunteers were recruited to characterize the fluorescence spectra of normal mucosa at different oral sites and to study individual variations. Fluorescence intensity and line shape of the spectra from lesions were compared with the same parameters from the contralateral control site in the same individual. Otolaryngology Research Center, Department of Otolaryngology-Head and Neck Surgery, New England Medical Center, Boston, Mass. The ratio of peak fluorescence intensities of the neoplastic lesions to contralateral normal control mucosa were consistently different compared with these ratios in benign lesions or normal mucosa. These differences were seen in 2 distinct regions of the fluorescence spectrum with both of the excitation wavelengths, but were more obvious with the excitation wavelength of 410 nm. Using these differences, we were able to correctly diagnose 17 of the 19 lesions studied, with 2 false-positive results. Neoplastic oral mucosa shows consistent differences in autofluorescence spectral intensity and line shape when compared with the normal mucosa in the same individual. These early results show that fluorescence spectroscopy may represent a useful technique for noninvasive early diagnosis of cancer of the upper aerodigestive tract.

An Introductory Dye Laser Experiment

Students measure the visible absorption spectrum, the fluorescence spectrum, and the dye laser emission curve of one or more laser dyes. The absorption and fluorescence spectra are measured using commercial spectrometers and the laser emission curve is measured with a nitrogen-pumped dye laser and an energy meter. While this experiment produces qualitative results, its investigative flavor allows students to figure out for themselves the nature of the energy levels of the dye molecule and how the dye laser works. They must draw on a variety of spectroscopic and kinetic concepts to understand the meaning of the three spectra and the relationship between them. In addition, the students particularly enjoy maximizing the dye laser energy by making adjustments to the optics in the laser cavity. This allows them to tinker with the inner workings of an instrument in a more direct way than they have experienced before.

In vivo cancer diagnosis of the esophagus using differential normalized fluorescence (DNF) indices

We report the use of new diagnostic parameters based on the differential normalized fluorescence (DNF) signals for malignant tumor diagnosis. Over 200 measurements of endogenous fluorescence from normal and malignant esophageal tissues were performed during routine endoscopy in 48 patients. A pulsed nitrogen-pumped dye laser was used to provide in situ excitation at 410 nm. Direct collection of the fluorescence signal emitted by the tissue was achieved using an intensified photodiode array detector equipped with a fiberoptic probe. The fluorescence signals were normalized with respect to the total fluorescence signal area. The cancer diagnosis indices were defined by the difference between the normalized fluorescence signal of a tumor and the mean value of a reference set of normal tissues. The results of the DNF approach were compared with endoscopic examinations and histopathology interpretations of the biopsy samples. Excellent correlation in the classification of normal and malignant tumors for the samples was found. The data indicated that the DNF approach has a significant potential to provide a direct, real-time, and in-situ technique for cancer diagnosis of the esophagus without requiring biopsy of the tumors and time-consuming histopathology tests.

A Modified Fast Analog Technique for Determining Luminescence Decay Times of Scintillators

ABSTRACTWe have modified our previous fast analog technique to determine the luminescence decay times of scintillators following an excitation with a Sr90β3-source. In the original technique [1], the sample was excited with a nitrogen-pumped dye laser, and the fluorescence pulses (consisting of typically 50 to 1000 photons) were detected by a multi-channel plate photomultiplier tube (MCP-PMT). The output from the MCP-PMT was directed to a fast waveform digitizer triggered externally by the exciting laser. In the modified technique, the digitizer acquires the fluorescence decay in the internal trigger mode, as no corresponding external trigger pulse is available from the Sr90source. For efficient light collection from scintillators, an ellipsoidal mirror assembly has been tested. The fluorescence decays are acquired as multi-photon pulses and are subsequently corrected for the temporal instrument response by using a deconvolution technique. The overall time resolution of the technique is about 100 ps. The fluorescence decay time obtained using this technique for a commercial scintillator (SCSN-81) agrees well with literature. We also discuss our results on new epoxy-polymer based scintillators prepared in our laboratory. The primary motivation for this work was development of new scintillators with shorter fluorescence decay times for high collision rate experiments.

Fluorescence lifetime data analysis using simplex searching and simulated annealing

Simulated annealing, a nonlinear parameter estimation method, is used to address the problem of biexponential decay law determinations. With a laboratory-constructed fluorometer using a nitrogen-pumped dye laser as a source of very short light pulses and a photomultiplier tube whose output is sampled by a boxcar averager, the distribution of noise in the data may not be assumed to be normal and the noise level may be relatively high. Perhaps for these reasons the traditional means of data analysis sometimes falls

Development of a fibre optic luminescence lifetime spectrometer

A spectrometer for measuring luminescence lifetimes has been developed. The design includes a bifurcated fibre optic cable for delivering the light from the source to the sample and from the sample to the detector. It uses a fast photomultiplier tube as the detector, a boxcar averager for data acquisition and a PC clone for data analysis. A nitrogen pumped dye laser is used as the light source and this has a pulse width of 1·2 ns. The spectrometer has been used to measure lifetimes from a minimum of 0·75 ns (rose bengal) to 597 μs (solid uranyl nitrate hexahydrate). A study has been made of the effects of concentration and temperature on the decay of uranyl ions (UO 2 2+ ) in aqueous solution. The decay is found to be biexponential, except at temperature above 60°C, where a three-component decay analysis gives the best results for matching the data to an Arrhenius Plot. The nature of the third emissive state, appearing at high temperatures is unknown and no spectroscopic state assignment has yet been made.

Lifetime Analysis of Weak Emissions and Time-Resolved Spectral Measurements with a Subnanosecond Dye Laser and Gated Analog Detection

A relatively inexpensive UV fluorometer has been developed for the temporal and spectral characterization of weak emissions, with the use of a frequency-doubled nitrogen-pumped dye laser for excitation wavelengths down to 260 nm. Gated detection of the photomultiplier pulse provides 0.1-ns resolution of the 1.4-ns FWHM instrument response function. On-line statistical measurements characterize the waveforms, with NATA, tryptophan, and NADH as model compounds providing emission signals of the order of the water Raman scatter signal. Time-decay waveforms are analyzed with the use of nonlinear least-squares fitting, with scatter or p-terphenyl as the reference, and a time shift. Good agreement with the literature is found for the subnanosecond and nanosecond decay times, with resolution of multiple decays down to several percent fractional emission. With multichannel data acquisition, a set of time-windowed emission spectra can be obtained in the same period as is required for an additional time-decay waveform. Time-windowed spectra and the derived decay-associated spectra provide evidence concerning the decay model in addition to spectral characteristics. Practical limitations include the peak current linearity of the photomultiplier and the wavelength variation of the response function.

Laser induced cell fusion in combination with optical tweezers: The laser cell fusion trap

A single-beam gradient force optical trap was combined with a pulsed UV laser microbeam in order to perform laser induced cell fusion. This combination offers the possibility to selectively fuse two single cells without critical chemical or electrical treatment. The optical trap was created by directing a Nd:YAG laser, at a wavelength of 1.06 microns, into a microscope and focusing the laser beam with a high numerical aperture objective. The UV laser microbeam, produced by a nitrogen-pumped dye laser (366 nm), was collinear with the trapping beam. Once inside the trap, two cells could be fused with several pulses of the UV laser microbeam, attenuated to an energy of approximately 1 microJ/pulse in the object plane. This method of laser induced cell fusion should provide increased selectivity and efficiency in generating viable hybrid cells.

Experimental investigation of the radiative transport of the resonance lines of sodium and lithium

Measurements are reported of the radiative transfer in the 589 nm Na line and the 670.7 nm Li line. The experiments were carried out in an atomic beam as well as in a cylindrical sodium resonance cell. A nitrogen pumped dye laser with 14 ns pulse duration was used for resonance excitation. The decay times were derived from the fluorescence signals recorded using a boxcar averager. The atomic particle density was measured by Doppler-free two- photon absorption. This measurement was carried out immediately after resonance excitation thus yielding the density value for each decay time. Our measurements verify the diffusion model for low optical depths 0.2 ⩽ k o l < 4 and Holstein's transport theory for intermediate and high opacities 4 ⩽ k o l < 50. If Holstein's transport equation is modified for k o l >50 to take into account partial redistribution in frequency, our experimental results again agree with theory.

Study of the reaction Li + H2O over the temperature range 850–1000 K by time-resolved laser-induced fluorescence of Li(22PJ–22S1/2)

The first direct study of the reaction between lithium atoms and water vapour is presented. Lithium atoms were produced by the pulsed photolysis of LiOH vapour in the presence of an excess of H2O and N2 both gas. The Li-atom concentration was monitored by laser-induced fluorescence of the metal atoms at λ= 670.7 nm using a pulsed nitrogen-pumped dye laser and boxcar integration of the fluorescence signal. Absolute second-order rate constants were obtained at T= 850, 900, 925, 950, 973 and 1000 K. A fit of these data to the Arrhenius form yields (2σ errors)k(T)=(5.6+ 4.3–2.4)× 10–10 exp [–(7970 ± 544)/T] cm3 molecule–1 s–1.The activation energy of 66.3 ± 4.5 kJ mol–1 may be compared to estimates for the standard enthalpy change (between 850 and 1000 K) of this reaction which rage from 62 to 73 kJ mol–1. To be compatible with our experimental data, and within the range of values derived from studies in flames, D°0(Li—OH) probably lies in the range 437–441 kJ mol–1.The reverse reaction between LiOH and H is important in both the chemistry of Li in flames (T > 1500 K) and in describing the atmospheric chemistry of Li in the mesosphere (T= 200 K). Estimates for this rate constant were obtained in both temperature regimes through detailed balancing.

A kinetic study of the reaction lithium + nitrous oxide: non-Arrhenius behavior over the temperature range 363-900 K

A kinetic study is presented of the reaction between lithium atoms and nitrous oxide over the temperature range 363-900 K, using a new experimental system. Li atoms are produced in an excess of N/sub 2/O and He bath gas by pulsed photolysis of either LiI or LiCl vapor. The concentration of the metal atoms is then monitored in real time by the technique of laser-induced fluorescence of Li atoms at lambda = 670.7 nm using a pulsed nitrogen-pumped dye laser and box-car integration of the fluorescence signal. Second-order rate constants for the title reaction have been measured at T = 363, 411, 474, 545, 600, 657, 700, 800, and 900 K. When this data is plotted in the standard Arrhenius form, a strong departure from linearity above 600 K is evident with an activation energy increasing with temperature. The best analytical description of this temperature dependence over the temperature range studied is given by k/sub 1/(T) = (2.4 +- 0.5) x 10/sup -11/T/sup 1/2 exp(-(1468 +- 137)T) + (7.9 +- 4.7) x 10/sup -10/T/sup 1/2 exp(-(4157 +- 540)T). The first term of this expression describes a low-temperature Arrhenius behavior similar to that observed by other workers for the analogous reactionmore » of sodium atoms. The author interpret the form of the second term as describing the participation in the reaction at temperatures above 600 K of highly vibrationally excited N/sub 2/O, which enables the reaction to proceed by a close-range charge-transfer mechanism. This significantly enhances the reaction cross section. A simple two-channel model is discussed which accounts for the observed temperature dependence« less

A study of the reaction Li+HCl by the technique of time-resolved laser-induced fluorescence spectroscopy of Li (2 2P J–2 2S1/2, λ=670.7 nm) between 700 and 1000 K

A kinetic study is presented of the reaction between lithium atoms and hydrogen chloride over the temperature range 700–1000 K. Li atoms are produced in an excess of HCl and He bath gas by pulsed photolysis of LiCl vapor. The concentration of the metal atoms is then monitored in real time by the technique of laser-induced fluorescence of Li atoms at λ=670.7 nm using a pulsed nitrogen-pumped dye laser and box-car integration of the fluorescence signal. Absolute second-order rate constants for this reaction have been measured at T=700, 750, 800, and 900 K. At T=1000 K the reverse reaction is sufficiently fast that equilibrium is rapidly established on the time scale of the experiment. A fit of the data between 700 and 900 K to the Arrhenius form, with 2σ errors calculated from the absolute errors in the rate constants, yields k(T)=(3.8±1.1)×10−10 exp[−(883±218)/T] cm3 molecule−1 s−1. This result is interpreted through a modified form of collision theory which is constrained to take account of the conservation of total angular momentum during the reaction. Thereby we obtain an estimate for the reaction energy threshold, E0=8.2±1.4 kJ mol−1 (where the error arises from uncertainty in the exothermicity of the reaction), in very good agreement with a crossed molecular beam study of the title reaction, and substantially lower than estimates of E0 from both semiempirical and ab initio calculations of the potential energy surface.

Analysis and performance of a nitrogen pumped dye laser optimized for narrowband operation

The analysis and the operating characteristics of a nitrogen pumped dye laser using four prisms as a beam expander are reported. Three configurations with different expansion values M p and incidence angles on the grating θ 0, corresponding to a constant total magnification M p/cosθ 0, have been compared.

Single longitudinal mode interaction between a uranium atomic beam and a modified H�nsch-type pulsed dye laser

Laser-induced fluorescence is used as a diagnostic tool for testing the tuning between a longitudinal mode of a nitrogen-pumped dye laser and a uranium spectroscopic level in an atomic beam. According to a simple resonator model, the tuning instabilities can be ascribed to thermal drifts in the dye-laser system. Problems encountered in attempting a single mode scanning are also described.