In vivo fluorescence imaging using two excitation and/or emission wavelengths for image contrast enhancement

Steady-state fluorescence imaging can be used in conjunction with selective exogenous or endogenous fluorescent compounds for the diagnosis of skin lesions, for example cancer. Depending on the excitation and emission properties of the fluorescent compound used, various excitation and/or emission wavelengths can be chosen in order to allow fluorescence imaging. Unwanted background signals such as autofluorescence and scattering can decrease the image quality and, hence, the diagnosis potential of this imaging method. We have used an inexpensive dual excitation and/or emission wavelength approach in order to suppress the unwanted background signal and allow contrast enhanced fluorescence imaging. One excitation and/or emission wavelength is at the corresponding maximum of the fluorescent compound, while the second is at a nearby excitation/emission minimum. The first image contains the emission from the fluorescent compound used combined with the signal from the unwanted background. The second image provides an image of just the unwanted background signal. The difference of both images taken, thus gives a contrast enhanced image of the skin lesion. The method relies on the assumption that the background signal does not change significantly due to the small changes in wavelength for excitation or emission. Image ratio methods have already been applied towards diagnosis of basal cell carcinomas after administration of aminolevulinic acid-induced protoporphyin IX. In this study, we describe in vivo measurements in mice where the second image, usually the background signal only, contains new unwanted image data. This simple method can successfully resolve the desired image, thus demonstrating the versatility of the image processing procedure.     

Atomic fluorescence spectrometry with laser excitation

A laser atomic fluorescence spectrometry for the detection of trace concentrations of the elements is described. The detection limits for Pb, Fe, Na, Pt, Ir, Eu, Cu, Ag, Co and Mn in aqueous solutions obtained at present are the best ones for the rapid spectral analytical methods. The analytical potentials of the laser spectrometer are exemplified by the analysis of real samples of different chemical composition.     

Laser-induced fluorescence of formaldehyde in combustion using third harmonic Nd:YAG laser excitation

Formaldehyde (CH2O) is an important intermediate species in combustion processes and it can through laser-induced fluorescence measurements be used for instantaneous flame front detection. The present study has focussed on the use of the third harmonic of a Nd:YAG laser at 355 nm as excitation wavelength for formaldehyde, and different dimethyl ether (C2H6O) flames were used as sources of formaldehyde in the experiments. The investigations included studies of the overlap between the laser profile and the absorption lines of formaldehyde, saturation effects and the potential occurrence of laser-induced photochemistry. The technique was applied for detection of formaldehyde in an internal combustion engine operated both as a spark ignition engine and as a homogenous charge compression ignition engine.     

Computer modeling of fluorescence dip spectroscopy with pulsed laser excitation

This article is an electronic publication in Spectrochimica Acta Electronica (SAE), the electronic section of Spectrochimica Acta Part B (SAB). The hard copy text is accompanied by a disk with a demonstration program, block libraries, demonstration files, and ancillary files. The article discusses the computer modeling of fluorescence dip spectroscopy, a relatively new and powerful atomic diagnostic technique. The models were found to agree essentially perfectly with previously published photostationary results and to extend those results to the more realistic laser temporal profiles encountered in actual experiments.

The fluorescence dip parameters were found to be robust so long as the system under study approached saturation conditions. Short, noisy laser pulses were found to be acceptable, for determination of the fluorescence dip parameters, provided the laser pulses were sufficiently intense to cause near saturation of the three level system. Conversely, steady state excitation was not found to be crucial to the determination of accurate fluorescence dip parameters. For a five level model of the silver atomic system, “negative fluorescence dips” were readily found in the simulations, even though some of the relevant parameters were rough estimates of the actual, unknown parameters.     

Two-color two-photon excitation using femtosecond laser pulses

The use of two-color two-photon (2c2p) excitation easily extends the wavelength range of Ti:sapphire lasers to the UV, widening the scope of its applications especially in biological sciences. We report observation of 2c2p excitation fluorescence of p-terphenyl (PTP), 2-methyl-5-t-butyl-p-quaterphenyl (DMQ) and tryptophan upon excitation with 400 and 800 nm wavelengths using the second harmonic and fundamental wavelength of a mode-locked Ti:sapphire femtosecond laser. This excitation is energetically equivalent to a one-photon excitation wavelength at 266 nm. The fluorescence signal is observed only when both wavelengths are spatially and temporally overlapping. Adjustment of the relative delay of the two laser pulses renders a cross correlation curve which is in good agreement with the pulse width of our laser. The fluorescence signal is linearly dependent on the intensity of each of the two colors but quadratically on the total incident illumination power of both colors. In fluorescence microscopy, the use of a combination of intense IR and low-intensity blue light as a substitute for UV light for excitation can have numerous advantages. Additionally, the effect of differently polarized excitation photons relative to each other is demonstrated. This offers information about different transition symmetries and yields deeper insight into the two-photon excitation process.     

Biphotonic laser excitation of upper singlet state fluorescence in organic dyes

Fluorescence spectra are presented, originating from upper excited singlet states in three xanthene dyes. The experimental method, using biphotonic pulsed laser excitation, is shown to have advantages over single-photon ultraviolet irradiation. It is further shown that the observed spectra depend on the nature and timing of the excitation frequencies.     

Femtosecond fluorescence dynamics imaging using a fluorescence up-conversion microscope

Femtosecond fluorescence dynamics imaging microscopy was performed. Femtosecond fluorescence dynamics images were constructed based on the "mean" fluorescence decay or rise time constants that were evaluated by the time-resolved intensity sampling using a fluorescence up-conversion microscope. This dynamics imaging microscopy was carried out for the organic microcrystals alpha-perylene and tetracene-doped anthracene microcrystal, and ultrafast dynamics in the organic microcrystals were clearly imaged in the two-dimensional manner. For the alpha-perylene microcrystal, the obtained dynamics images showed that the crystal edges exhibited relatively shorter free exciton and the Y-state lifetimes compared to the crystal center, reflecting the higher concentration of defects. For the tetracene-doped anthracene microcrystal, the image was constructed based on the time constant of excitation energy transfer from anthracene to tetracene. By experiments changing the doping ratio of tetracene in anthracene, it was concluded that the inhomogeneity observed in the dynamics image arises from the difference in the local concentration of tetracene in the mixed crystal.     

Two-photon excitation of fluorescence for three-dimensional optical imaging of biological structures

Techniques based on two-photon excitation (TPE) allow three-dimensional (3D) imaging in highly localized volumes, of the order of magnitude of a fraction of a femtolitre up to single-molecule detection. In TPE microscopy a fundamental advantage over conventional widefield or confocal 3D fluorescence microscopy is given by the use of infrared (IR) instead of ultraviolet (UV) radiation to excite those fluorophores requiring UV excitation, hence causing little damage to the specimen or to fluorescent molecules outside the volume of the TPE event and allowing a deeper penetration within the sample compared with conventional one-photon excitation of fluorescence. In our laboratory, within the framework of a national INFM project, we have realized a TPE fluorescence microscope, part of a multipurpose architecture also including lifetime imaging and fluorescence correlation spectroscopy modules. The core of the architecture is a mode-locked Ti:sapphire infrared pulsed laser pumped by a high-power (5 W, 532 nm) solid-state laser and coupled to an ultracompact scanning head. For the source we have measured a pulse width from 65 to 95 fs as a function of wavelength (690-830 nm). The scanning head allows conventional and two-photon confocal imaging. Point spread function measurements are reported with examples of applications to the study of biological systems.     

Time-resolved fluorescence study of 4-dimethylaminobenzonitrile in nonhydrogen-bonding polymers,using picosecond dye laser pulses as excitation source

It is shown that the time-resolved fluorescence (TRF) spectra and the red edge effect (REE) phenomena are useful tools to observe the twisted intramolecular charge transfer (TICT) band, of 4-dimethylaminobenzonitrile (DMABN) in nonhydrogen-bonding polymers. The TRF results show that the relative intensity of the TICT band increases with the increase of time after excitation in all polymers used. The highest TICT relative intensity is observed in poly(butyl methacrylate) (PBMA) polymer matrix, which has a medium polarity and medium free volume compared with either poly(methylmethacrylate) (PHMA), which is more polar and more rigid, or poly(hexylmethacrylate) (PHMA), which is less polar and less rigid than PBMA polymer matrix. The lifetimes are rationalized to the opposite roles of local polarity and local free volume of polymer sites on TICT emission. © 1995 John Wiley & Sons, Inc.     

Attogram detection limits using laser induced fluorescence

Laser induced fluorescence has produced outstanding detection limits in liquid phase analysis. This paper presents a general method for optimizing detection limits as a function of sample volume.     

发光二极管诱导荧光检测器的研制

探讨了以亮度发光二极管为诱导荧光检测激发光源的可行性，考察了直流驱动和脉冲驱动发光二极管(LED)对输出光强的影响以及LED塑料保护层厚度对输出光强的影响。发现脉冲驱动比直流驱动能提高光强3倍，无塑料保护层相对有保护层可提高光强2．5倍。采用毛细管电泳柱上检测方式对检测系统进行了评价，最小检出浓度为0．18μmol／L。结果表明该装置可以满足普通分析需求。     

Atomic and ionic fluorescence spectrometry with pulsed dye laser excitation in the inductively-coupled plasma

The atomic and ionic fluorescences of iron, tin, barium and indium excited by flash-lamp-and nitrogen laser-pumped pulsed dye lasers in the inductively-coupled plasma (ICP) are studied. Noise sources are investigated and detection limits are compared to the techniques of ICP-emission and laser-excited atomic fluorescence spectrometry.     

Wavelength dispersive X-ray fluorescence imaging using a high-sensitivity imaging sensor

A new wavelength-dispersive X-ray fluorescence (WD-XRF) imaging spectrometer equipped with a high-sensitivity imaging sensor was developed in our laboratory. In this instrument, a straight polycapillary optic was applied instead of a Soller slit as well as a 2D imaging X-ray detector instead of X-ray counters, which are used in conventional WD-XRF spectrometers. Therefore, images of elemental distribution were available after a short exposure time. Ni Kα images and Cu Kα images were clearly obtained at corresponding diffraction angles for a short exposure time of 10 s. By optimizing the spectrometer, the time required for imaging is reduced, leading to XRF image movies. It is difficult to distinguish two peaks (Ti Kα (4.508 keV) and Ba Lα (4.465 keV)) due to the poor energy resolution of EDXRS. However, Ti and Ba images could be successfully observed by the WD-XRF imaging spectrometer. The energy resolution of the developed spectrometer was 25 eV at the Ti Kα peak.     

Phosphorescence of 1,1,1-trifluoroacetone using pulsed nitrogen laser excitation

Phosphorescence decay times of 1,1,1-trifluoroacetone in the vapour phase have been measured as a function of pressure of ketone with low-power excitation from a pulsed N₂ laser. A strong pressure dependence is observed, yielding a rate constant for self-quenching of (4.6 ± 0.5) × 10⁶ ? mol^?1 s^?1, and an extrapolated zero-pressure lifetime of 60 μs, considerably shorter than that measured earlier in a higher-energy flash photolysis experiment. Reasons for these differences are discussed.     

Sub-picogram detection of lead by non-flame atomic fluorescence spectrometry with dye laser excitation

The frequency-doubled radiation of flashlamp-pumped dye laser has been compared with the radiation of an eleotrodeless discharge lamp (EDL) and a hollow cathode lamp (HCL) as excitation light sources in non-flame atomic fluorescence spectrometric (AFS) measurements. Detection limits and linear ranges of the analytical working curves have been studied. The aqueous Pb sample solutions used in this study were atomized in an electrically heated graphite rod. Direct line fluorescence at 405.8 nm has been observed. The limit of detection obtained with the laser as excitation source is about one order of magnitude lower than the detection limits obtained with the EDL and HCL. The absolute limit of detection of 0.2 pg is the lowest reported value to date attained in any atomic spectrometric method. The ultraviolet laser irradiance was found to be high enough to approach saturation conditions. As a result selfabsorption of the exciting laser beam is reduced and the linear range of the analytical working curve is extended to higher analyte concentrations.     

On the shape of atomic fluorescence analytical curves with a laser excitation source

The effect of high irradiance levels (approaching saturation) upon the shape of atomic fluorescence analytical curves is considered theoretically for both line and continuum sources. The linear range of the analytical curve may be greatly extended over that obtained with normal (low irradiance) sources, as is experimentally verified using a pulsed dye laser as an excitation source.     

Improved detectability and signal strength for rotating phase fluorescence immunoassays through image processing

Fluorescence immunoassays based on rotating solid phase have shown promise of lowered detection limits, among other advantages. However, intrinsic background distortion effects have limited their utility. Here, novel image processing strategies are used to minimize these effects and improve the estimate of concentration and lower the detection limit. This initial demonstration of a new processing capability is performed on data for a protein, myoglobin, which is a biomarker for acute myocardial infarction. For these data, compared with published results, the detection limit is improved by a factor of approximately one hundred (to 700 fM), which is competitive with or better than other immunoassay strategies (ELISA, for example) that are fully developed. This work suggests that image and video processing technologies can provide a valuable alternative approach to biochemical detection and concentration estimation.     

Two-photon excitation with pico-second fluorescence lifetime imaging to detect nuclear association of flavanols

Two-photon excitation enabled for the first time the observation and measurement of excited state fluorescence lifetimes from three flavanols in solution, which were ∼1.0 ns for catechin and epicatechin, but <45 ps for epigallocatechin gallate (EGCG). The shorter lifetime for EGCG is in line with a lower fluorescence quantum yield of 0.003 compared to catechin (0.015) and epicatechin (0.018).     

Hadamard transform fluorescence image microscopy using one-dimensional movable mask

With a 511-slit one-dimensional (1D) Hadamard mask and a highly sensitive linear charge-coupled device (CCD), spatial multiplexing is performed and a programmable Hadamard transform (HT) microscopic fluorescence imaging system was developed. The system can generate 511×512 pixel format images for small samples. Sensitivity, signal to noise ratio, imaging speed and spatial resolution of this system were discussed. The results show that the system can be applied for single-cell imaging sensitively in a short time. Spatial resolution up to 0.24 μm/pixel, which is close to the resolution limit of the conventional optical microscope, has been obtained under oil lens. The weak native fluorescence imaging for pollen cells can be realized within 1 min. The system has been applied for multi-parameter evaluation of tumor malignancy based on nuclear DNA ploidy measurements for one breast tumor specimen. The result indicates that the system has good application prospect in cell biology and medicine.