Laser-Based Fluorescence Eem Instrument for In-Situ Groundwater Monitoring

I. ABSTRACT

A fluorescence excitation-emission matrix (EEM) instrument is described which employs laser excitation and optical fiber light delivery for remote sensing applications. Ultraviolet laser light is generated at a variety of discrete wavelengths by pumping a Raman shifter with the third or fourth harmonic of a Nd:YAG laser, and this Raman-shifted light is used for fluorescence excitation to generate EEM spectra. Seven-meter optical fibers are used for laser beam delivery and fluorescence light collection, and a diode array detector is used to detect fluorescence emission spectra. As a test of the capability of this instrument, the composition of a two-component mixture of the groundwater pollutants carbazole and p-cresol is determined quantitatively by least-squares analysis of the mixture's fluorescence EEM. Detection limits were in the ppb range and the linear dynamic range was better than 3 orders of magnitude.     

On-Line Computer Control of a Luminescent Spectrometer with Off-Line Computer Data Processing and Display

SUMMARY

A luminescent spectrometer has been interfaced with a general purpose digital computer operated in a time-share mode. The computer controls both monochromators and logs the data on magnetic tape. By means of its constituent mode subroutines, the computer controls the spectrometer in any of six modes for the acquisition of luminescent information. It will record the excitation, emission or detailed three-dimensional spectra, integrate luminescent intensity at fixed wavelength coordinates, locate the wavelength region and general form of an unknown spectrum, and record luminescent intensity at fixed wavelength coordinates at selected time intervals, or oscillate either one or both monochromators between two fixed points so that the change of luminescence at either one or two points of the emission and excitation wavelengths can be monitored as a function of time.

The data are processed off-line with a CDC 6600 computer that permits the data to be portrayed in several graphic forms. The raw data can be corrected for instrumental response, smoothed, differentiated; data arrays can be either magnified or eliminated by masking.     

A Phosphorimeter for Measurements of Triplet Diffusion

ABSTRACT

A phosphorimeter is described which is intended to be used for delayed fluorescence measurements in which the excitation light is introduced in a spatially nonhomogeneous manner. The excitation optics produce a reduced image of the space intermittency pattern at a 1 mm layer of the test solution. The delayed emission is isolated from prompt fluorescence by an adjustable phasing between excitation and emission choppers. Solutions of anthracene and 9, 10-diphenyl anthracene in ethanol both show changes in delayed fluorescence intensity with changing dimensions of the space intermittency pattern. The effects observed are in agreement with theoretical expectations.     

A Phosphorimeter for Studying Delayed Fluorescence from Samples Immersed in a Magnetic Field

SUMMARY

The design and construction of a phosphorimeter especially suited for investigating the effect of a magnetic field on delayed luminescence processes is described. Although it is designed for the light levels and time domains usually pertinent to fluid-solution delayed fluorescence studies, it can be easily adapted to phosphorescence work. In order to avoid the influence of the applied field on the excitation and detection elements, these components are removed from the field and are heavily shielded. A medium-pressure mercury arc is used as an excitation source, and either frontal or right-angular illumination of the sample can be employed. Filters in both the excitation and emission beams provide the means of wavelength selection. The use of a single, rotating, slotted disk to time both beams gives the instrument a compact design and enhances its physical stability. Tests of instrument performance show that the phosphorimeter is useful for measurements at fields up to 8000 G.     

Two-photon excitation and emission spectra of the green fluorescent protein variants ECFP, EGFP and EYFP

Two-photon (TP) excitation (820–1150 nm) and emission (280–700 nm) spectra for the fluorescent proteins (FPs) ECFP ³ , EGFP ³ and EYFP ³ produced in human tumour cells were recorded. TP excitation spectra of pure and highly enriched samples were found to be more differentiated in comparison with their one-photon (OP) spectra. They exhibited more pronounced main and local maxima, which coincided among different purity grades within small limits. TP and OP emission spectra of pure and enriched samples were identical. However, in crude samples, excitation was slightly blue-shifted and emission red-shifted. The data indicate that both OP and TP excitation routes led to the same excited states of these molecules. The emission intensity is dependent on the pH of the environment for both types of excitation; the emission intensity maximum can be recorded in the alkaline range. Reconstitution of emission intensity after pH quenching was incomplete, albeit that the respective spectral profiles were identical to those prequenching. When emission data were averaged over the whole range of excitation, the resulting emission profile and maximum coincided with the data generated by optimal excitation. Therefore, out-of-maximum excitation, common practice in TP excitation microscopy, can be used for routine application.     

Time Correlated Single-Photon Counting (Tcspc) Using Laser Excitation

Abstract

The basic methods of time-correlated single-photon counting are outlined briefly, and the advantages of rapid repetition-rate pulsed lasers as light sources in this experiment are outlined. Application of the method to time-resolved fluorescence anisotropy is described.

The basic principles of time-correlated single-photon counting have been the subjects of many reviews^1-6. The method relies on the basic concept that the probability distribution for emission of a single-photon following excitation gives the actual intensity against time distribution of all photons emitted, thus by sampling the time of single-photon emission following a large number of excitation pulses, the probability distribution is created.

The experiment is carried out as follows, with reference to Figure 1. A trigger T. which could be a photomultiplier, an antenna pick-up, or a logical synchronising pulse from the electronics pulsing the excitation source, generates an electrical pulse at a time exactly correlated with the time of generation of the optical pulse. The trigger pulse is routed, usually through a discriminator, to start input of the time-to-amplitude converter, (TAC) which initiates charging of a capacitor. In the meantime the optical pulse excites the sample, which subsequently fluoresces. An aperture is adjusted so that at most one photon is “detected” for each exciting event. The signal resulting from this photon stops the charging ramp in the TAC, which puts out a pulse, the amplitude of which is proportional to the charge in the capacitor, and hence to the time difference between START and STOP pulses. The TAC output pulse is given a numerical value in the analogue-to-digital converter and a count is stored in the data storage device in an address corresponding to that number. Excitation and data storage are repeated in this way until the histogram of number of counts against address number in the storage device has enough data so that it represents, to some required precision, the decay curve of the sample. If deconvolution is necessary, the time profile of the excitation pulse is collected in the same way by replacing the sample by a light scatterer.     

A Wavelength Programmed Luminescence Spectrometer

ABSTRACT

Electrical control of the excitation and emission wavelengths of a luminescence spectrometer may be conveniently obtained by coupling the grating drive shafts directly to limited-rotation torque motors. The relationship between the wavelength and the applied voltage is very nearly linear, and the reproducability is ±1 nm or better. The torque motors are driven by operational amplifier circuits which provide convenient and wide-range control of scan parameters. Stereo-fluorograms may be recorded automatically on an X-Y recorder.     

Fortran Programs to Acquire and Process Isotopic Mass Spectrometry Data: A Conceptual Description

ABSTRACT

New FORTRAN computer programs have been written to process isotopic mass spectrometric data. A major goal of these programs was to achieve maximum flexibility in all phases of the analytical process: data acquisition, data processing, and report writing. Magnetic disk files are exploited to this end. The programs can acquire and process data from any element taken under any scanning scheme and with any desired ratios calculated. Results are stored on magnetic disks and results of similar samples can be listed in a single compact report.     

Two-photon excitation fluorescence pH detection using 2,3-dicyanohydroquinone: a spectral ratiometric approach

The use of 2,3-dicyanohydroquinone (DCHQ) as an emission ratiometric probe of pH in vitro and in fibroblast cells was evaluated using two-photon excitation fluorescence microscopy (TPEFM). In addition, methods for spectrally calibrating the Zeiss LSM510 META spectroscopy system for TPEFM were also developed. The emissions of both the acid and base forms of DCHQ were detectable when using an 800-nm excitation in TPEFM, thereby allowing ratiometric determination of pH. These data suggest that, in contrast to most other emission ratiometric probes, both acid and base forms of DCHQ have similar two-photon cross-sectional areas at 800 nm. Acid (maximum at ∼457 nm) and base (maximum at ∼489 nm) DCHQ TPEFM emission spectra were similar to previously reported one-photon excitation emission spectra. Calibration curves for pH were successfully constructed using the ratio of DCHQ emission difference maxima at 460 nm and 512 nm in vitro and in cells. To our knowledge, DCHQ is currently the only effective emission ratiometric pH indicator for two-photon microscopy and may serve as a useful starting point for the development of other TPEFM ratiometric dyes for quantitative measurement of other cell parameters such as Ca²⁺, Mg²⁺ or Na⁺.     

Imaging of three-dimensional objects in emission electron microscopy

Under investigation by emission electron microscopy, the shape and size of three-dimensional objects are distorted because of the appearance of a characteristic potential relief and a possible contact potential difference between the particles and the substrate. An estimation of these effects for spherical particles is made. It is shown that the apparent size of particles observed in an emission electron microscope (EEM) could be increased as well as decreased depending on the relation between the work functions of the particle and the substrate. The corresponding formulae are given and several possibilities are shown which permit us to determine from the EEM image the real size of particles and their work function relative to the substrate.     

Mechanism of atomic removal in elastic emission machining

Elastic emission machining (EEM) can be thought of as a machining method utilizing the chemical activity of a particle surface, rather than a liquid echant as in chemical etching. Selecting the combinations of silicon as workpiece material and SiO₂, Al₂O₃ and ZrO₂ as the powder particles, the material removal rate during machining was examined. Different combinations of workpiece materials and powders affected removal rates strongly, so that the machining process was considered from the standpoint of the atomic interactions at the interface between the workpiece and the powder particles. The surface voltage of the workpiece with adsorbed powder particles was measured as one of the parameters representing the interfacial interactions, and correlations with removal rates were obtained.     

粗糙集合理论在旋转机械故障诊断技术中的应用

粗糙集合理论基于严格的集合分析方法,通过对数据集合进行等价关系、近似空间、分类等运算,发现隐含在数据中的规律与性质,从而完成知识发现。在人工智能机器故障诊断系统中,如何获得故障诊断等知识成为关键技术。论文提出将粗糙集合理论应用于加工中心故障诊断技术,使得故障诊断技术中的知识获取瓶颈问题得以有效解决。     

Automatic Control of the Arc Gap in DC Arc-Emission Spectrographic Analysis

SUMMARY

In emission spectrograpliy the result of a determination is read from the “analytical curve,” i.e., the working curve, which shows the ratio of the intensity of the analytical line chosen for the element to that of an internal standard line as a function of the concentration of the element. It is a well-known fact [1] that in actual practice such an analytical curve may shift; i.e., when the intensity ratios necessary for drawing the curve are redetermined after a certain period, their values deviate more from the original data than can be expected from the repeatability of the method.     

A Microprocessor Based Energy Dispersive X-Ray Diffractometer

ABSTRACT

A microprocessor based Energy Dispersive X-ray Diffraction system is described. The system is built around three 8085 microprocessors and is suitable for performing rapid structural analysis. It can acquire four spectra in programmable memory banks and can do online data analysis. The software includes a nonlinear least square fitting program, which can fit the complete diffraction pattern at a time. To highlight the data processing capabilities of this system, we have presented the results for a simple solid.     

A Microprocessor Controlled System For Photoelectrochemical Measurements

ABSTRACT

An automatic system for measuring photocurrent of electrodes as a function of excitation wavelength and electrode potential is described. Applications to a chemically modified transparent Sn0₂ electrode and an oxide-covered tantalum electrode are shown.     

Two‐photon microscopy assessment of the overall energy metabolism alteration of amoeba in hypertonic environment

In this study, a two‐photon fluorescence microscopic imaging technique is reported for assessment the effect of dynamic hypertonic environment on the overall energy metabolism alteration and adaptation of soil‐living amoeba Dictyostelium discoideum. For that purpose the fluorescence intensity of mitochondrial reduced nicotinamide adenine dinucleotide (NADH) was monitored and quantified in order to evaluate the corresponded metabolic state of monolayer cultured cells. The two‐photon excitation of NADH with 720 nm near infrared irradiation produced blue fluorescence emission with maximum wavelength centered at 460 nm. The benefits of reported noninvasive microscopic technique are the significantly less cellular damage and avoiding the excitation of other biomolecules except of NADH. It enabled to acquire data for NADH levels of the observed cells on agar plate specimen and hypertonic nutrition media in a Petri dish. The method demonstrated also good sensitivity, reproducibility and the obtained results revealed that D. discoideum species form aggregation in hypertonic environment within several minutes with aim to survive. The formed aggregate had amorphous shape and it consisted from dozen amoeba cells, which kept their NADH amount in constant level for few hours. The reported imaging method might be applicable in various studies for characterization of metabolic events and assessment of the cell energy balance in hypertonic environment.     

A Strategy to Probe Air-Liquid Interfaces by Confocal Fluorescence Microscopy

Abstract

Confocal fluorescence microscopy has been seldom applied to air-liquid interfaces due to technical difficulties. Satellite lines of an excitation laser beam can be used as an inherent reference for a confocal microscope to align and calibrate the setup. This strategy is especially useful and important to a liquid surface, and makes it possible to observe very weak fluorescence without time-consuming alignment procedures.     

新型数字输出式三轴过载传感器研究

介绍一种基于过采样数据采集和数字信号处理及补偿技术的新型三轴过载传感器。该传感器由电源模块、电容式MEMS加速度敏感头、信号调理模块、数据采集、处理与传输模块组成,敏感头的输出信号经过放大、滤波,确保了输出的模拟信号准确可靠;数据采集与传输模块采用高速单片机,用12位高精度AD进行数据采集,并通过通信芯片将数据发送给上位机。此外,还介绍了过采样理论。该传感器经过用户试验,取得了比较满意的结果。     

Some New Types of Sample Cells for Raman Spectroscopy with Laser Excitation in Molten Salts

SUMMARY

The introduction of the laser as an excitation source in Raman spectroscopy offers numerous new possibilities of investigations but demands the design of new types of sample cells specially suited for laser application. Two cells for work with molten salts corrosive to glass and quartz and a simple cell for melts noncorrosive to glass are described. The best results are obtained with a cell which provides total utilization of the laser energy for excitation by multiple reflection of the laser beam. In addition, some necessary accessories for molten salt work, i.e., a gas heater and filtration equipment to free the melt of suspended impurities which might cause an undesirable level of Tyndall and Rayleigh scattering, are mentioned.     

A Digital Signal Processor Based Instrument for Faradaic Admittance Measurements

ABSTRACT

An instrument suited for FTFAM (Fourier Transform Faradaic Admittance Measurements) has been constructed. It is controlled by two DSP:s (Digital Signal Processors, TMS32010) and a PC (Personal Computer) is used as main controller. The signal processors share program and data memory with the PC which permits fast data transfer and program downloading. One DSP generates the excitation waveform while the other acquires the potential and current signals. Frequencies up to 10 kHz are used in real time multifrequency analysis of electrochemical cells.