The reaction of protein tryptophanyl residues with reduced nicotinamide adenine dinucleotide. Structure of a model adduct.

This paper describes in detail a simple, light producing and handling system which differs greatly from the commercial unit presently used by most investigators, and which overcomes a number of its disadvantages. The apparatus is designed so that it can easily be substituted for the light producing, dispersing, and collimating section of the commercial photoelectric scanners, with no further changes in the commercial scanners except the substitution of optical flats for the collimating and condensing lenses in the bottom and top, respectively, of the rotor chamber. The most outstanding feature of this new system is that the use of a faster monochromator ($\text{f}\text{75.3}$) and a cylindrical lens theoretically increases light intensity by up to 100 times that of the commercial scanner, under otherwise identical conditions. Other advantages include the following: (1) For collecting and collimating light, two pairs of mirrors give a precell optical system focused in the radial direction at all wavelengths of light; (2) these components and the lamp are located on a table outside the centrifuge, so virtually any size of lamp can be used; (3) the entire precell optical system is a self-contained unit which, within reasonable limits, may be moved to any desired location without markedly affecting the quality of the collimated light; and (4) the optical path length has been shortened, providing less dissipation of light energy. The principles behind the selection and design of the key components are discussed. New alignment procedures and apparatus developed to aid in fast, easy, accurate alignment are described and discussed. Several components for use with a system using a computer-controlled stepping motor scanner for collection of data are also described. This system is simple enough and is documented in sufficient detail so that other interested workers, even those with little or no optics experience, can duplicate and use the system.     

Evaluation of a LED-based flatbed document scanner for radiochromic film dosimetry in transmission mode

Flatbed scanners are the most frequently used reading instrument for radiochromic film dosimetry because its low cost, high spatial resolution, among other advantages. These scanners use a fluorescent lamp and a CCD array as light source and detector, respectively. Recently, manufacturers of flatbed scanners replaced the fluorescent lamp by light emission diodes (LED) as a light source. The goal of this work is to evaluate the performance of a commercial flatbed scanner with LED based source light for radiochromic film dosimetry. Film read out consistency, response uniformity, film-scanner sensitivity, long term stability and total dose uncertainty was evaluated. In overall, the performance of the LED flatbed scanner is comparable to that of a cold cathode fluorescent lamp (CCFL). There are important spectral differences between LED and CCFL lamps that results in a higher sensitivity of the LED scanner in the green channel. Total dose uncertainty, film response reproducibility and long-term stability of LED scanner are slightly better than those of the CCFL. However, the LED based scanner has a strong non-uniform response, up to 9%, that must be adequately corrected for radiotherapy dosimetry QA. The differences in light emission spectra between LED and CCFL lamps and its potential impact on film-scanner sensitivity suggest that the design of a dedicated flat-bed scanner with LEDs may improve sensitivity and dose uncertainty in radiochromic film dosimetry.     

Gafchromic film dosimetry: Four years experience using FilmQA Pro software and Epson flatbed scanners

Purposes: To assess performance of FilmQA Pro software for pre-treatment patient-specific quality assurance (QA), using radiochromic films and two commercial flatbed scanners. To evaluate a novel multichannel approach compared to the classical red channel evaluation.Material and methodsPatient films (mostly EBT2 films, one box of EBT3) were digitalized using successively two flatbed scanners: the A4-size Epson V750 and the A3-size Epson 10000XL. Prior to patient dose verification, basic characteristics of films and scanners were investigated. Patient films were analyzed using FilmQA Pro software, which enables to use the signal from all three colour channels (Red, Green, Blue).ResultsCompared to the red channel evaluation, multichannel evaluation presents better passing rates with regard to local gamma index. As expected, we obtained better results using A3-size scanner compared to A4-size scanner, especially when considering large region of interest. An observation of great interest was made for both scanners: after intensive use, a tilting in the blue transmittance profiles appeared in the lamp direction, making multichannel analysis unsuitable for accurate dose evaluation.     

A new optical multichannel microspectrofluorometer.

A new microspectrofluorometer has been developed that combines a photometric fluorescence microscope with an optical multichannel analyzer. This instrument provides fluorescence emission spectra of biological materials by detecting the entire spectrum simultaneously in real time. These spectra are subsequently recorded and corrected so as to identify the fluorescent reaction products or to test whether fluorescent cytochemical probes bind to the expected substrate within cells. The procedures and advantages of optical multichannel analysis are described, and an application of microspectrofluorometry to acriflavine-Feulgen cytochemistry is given.     

A scanner for the absorption optical system of the analytical ultracentrifuge based on a vidicon television camera

This paper describes a photoelectric scanner, based on a television camera. This scanner, although designed for use in an anlytical ultracentrifuge, could be readily incorporated into other types of optical measuring instruments.The camera fitted with an ultraviolet-sensitive vidicon is capable of operating down to 225 nm.     

Instrumentation for differential fluorescence spectroscopy

Instrumentation is described for monitoring small differences in two strongly fluorescing samples by time sharing an essentially single-beam optical system. Details are provided for an understanding of the optical and electrical principles of operation. Overall performance is illustrated with fluorescence-emission difference spectra of bovine serum albumin.     

Microfluorometry analysis II. A Bayesian approach.

In flow microfluorometry (FMF) analysis cells stained with a fluorescent dye that binds specifically to DNA are passed through the instrument. The number of cells in the population having a fluorescence intensity is recorded in a single channel of a multichannel pulse height analyzer. The result is a DNA fluorescence histogram for the population.A method for decomposing an FMF histogram into its G₁, S and G₂ + M components, corresponding to similarly designated phases of the cell cycle is given. This technique can also be applied to find the parameters in all of the previous approaches. The parameters are calculated by iteration which eliminates the need for non-linear optimization procedures.     

Simultaneous measurements of fast optical and proton current kinetics in the bacteriorhodopsin photocycle using an enhanced spectrophotometer

A one-of-a-kind high speed optical multichannel spectrometer was designed and built at NIH and described in this journal in 1997 [J.W. Cole, R.W. Hendler, P.D. Smith, H.A. Fredrickson, T.J. Pohida, W.S. Friauf. A high speed optical multichannel analyzer. J Biochem Biophys Methods 1997;35:16–174.]. The most unique aspect of this instrument was the ability to follow an entire time course from a single activation using a single sample. The instrument has been used to study rapid kinetic processes in the photon-driven bacteriorhodopsin photocycle and electron transport from cytochrome c to cytochrome aa₃ and from cytochrome aa₃ to oxygen. The present paper describes a second generation instrument with a number of important enhancements which significantly improve its capabilities for multichannel kinetic studies. An example application is presented in which the kinetics of photon-induced proton flow across the biological membrane is measured simultaneously with the individual steps of the photocycle determined optically. Matching the time constants for the two processes indicates which molecular transformations are associated with major proton movements.     

New multichannel kinetic spectrophotometer–fluorimeter with pulsed measuring beam for photosynthesis research

A multichannel kinetic spectrophotometer–fluorimeter with pulsed measuring beam and differential optics has been constructed for measurements of light-induced absorbance and fluorescence yield changes in isolated chlorophyll-proteins, thylakoids and intact cells including algae and photosynthetic bacteria. The measuring beam, provided by a short (2 μs) pulse from a xenon flash lamp, is divided into a sample and reference channel by a broad band beam splitter. The spectrum in each channel is analyzed separately by a photodiode array. The use of flash measuring beam and differential detection yields high signal-to-noise ratio (noise level of 2 × 10⁻⁴ in absorbance units per single flash) with negligible actinic effect. The instrument covers a spectral range between 300 and 1050 nm with a spectral resolution of 2.1, 6.4 or 12.8 nm dependent on the type of grating used. The optical design of the instrument enables measuring of the difference spectra during an actinic irradiation of samples with continuous light and/or saturation flashes. The time resolution of the spectrophotometer is limited by the length of Xe flash lamp pulses to 2 μs.     

Biological desulfurization in an optical-fiber photobioreactor using an automatic sunlight collection system

Biological desulfurization using C. thiosulfatophilum has many more advantages over conventional physico-chemical methods due to low operational cost and no production of secondary pollutants. However, it requires effective and economical supply of light energy, which is a key factor in determining the success of commercialization. In this study, optical-fiber photobioreactor with internal illumination system was applied to increase the light availability. Furthermore, sunlight was used as the main light energy in the daytime and metal-halide lamp was applied as an additional light energy at night. Most UV light was eliminated by the chromatic aberration of the aspherical lenses in the solar light collector and 60% of infrared light intensity was eliminated. Physical scratching optical fibers enhanced the light availability about five times as much as that with unscratched ones in the previous study, but it resulted in the adsorption problem of elementary sulfur particles deteriorating light diffusivity considerably in a long operation. In order to solve this problem, scratched optical fibers were inserted into pyrex-glass tubes, which made light diffusivity nearly the same as that without glass tubes. Removal rate per unit cell concentration, using sunlight in the daytime and a metal-halide lamp at night, was 0.41 <0.73 micromol H(2)S min(-1)/(mg protein l(-1)) using a 400 W metal-halide lamp day and night, since the automatic sunlight collection system can transmit the light intensity as only 10% of that with a metal-halide lamp.     

A light-scattering pressure-jump kinetics apparatus.

We have developed an optical sample cell made of stainless steel and fitted with three quartz ultracentrifuge windows in standard holders, to follow the kinetics of macromolecular reactions by the pressure-jump technique. Photomultiplier response to transmitted white light is continuously subtracted from photomultiplier response to white light scattered at 90°C, the difference being displayed by an oscilloscope. The pressure is simultaneously monitored by a quartz pressure sensor in mechanical contact with the sample. Pressurization is accomplished by leading in gas from a commercial cylinder, as originally described by Ljunggren and Lamm, but the pressurization time has been reduced by a factor of 25, to 2 millisec, by valving off a fixed volume of helium and introducing it into the sample cell through a high-speed solenoid valve. Determinations may be repeated at will on a single sample, of total volume under 2 ml. This light-scattering pressure-jump apparatus has been used to observe the kinetics of a number of macromolecular interactions and to determine rate constants for the ribosome-subunit interaction of Escherichia coli.     

A new multiparameter flow cytometer: optical and electrical cell analysis in combination with video microscopy in flow

BACKGROUND: Flow cytometers, which are commercially available, do not necessarily meet all demands of actual biomedical research. This is the case for the investigation of mechanisms involved in cell volume regulation, which requires electrical volume measurement and ratiometric multichannel fluorescence analysis for the simultaneous assessment of different physiologic parameters (intracellular pH and the intracellular concentration of calcium ions, etc). METHODS AND RESULTS: We describe the construction of a new nonsorting flow cytometer designed for the simultaneous acquisition of seven parameters including fluorescence signals, forward and perpendicular light scatter, cell volume according to the electrical Coulter principle, and flow cytometric imaging. The instrument is equipped with three different light sources. A tunable argon-ion laser generates efficient excitation of the most standard fluorescent probes in the visible spectral range, and an arc lamp provides the means for ultraviolet excitation at low cost. Because of the spatial filtering by the excitation and detection optics, two independent sets of dual fluorescence measurements can be performed, a prerequisite for flexible ratiometric fluorescence analysis. A flow video microscope integrated into the optical system optionally generates either brightfield or phase images of selected flowing particles. Only particles whose individual datasets meet predefined gating conditions are imaged in real time. To avoid smear effects, the motion of the object to be imaged (speed approximately 8 m/s) is frozen on the target of a CCD camera by flash illumination. For this purpose, a high radiance gas discharge lamp with 25-mJ electric pulse energy provides an illumination time of 18 ns (full width half maximum). Test results obtained from latex spheres and cells are shown. CONCLUSIONS: Test results indicate that our instrument can perform Coulter measurements in combination with flexible optical analysis. Moreover, integration of an adapted video microscope into a flow cytometer is an approach to overcome the gap between flow and image cytometry.     

Characterization of noise and digitizer response variability in radiochromic film dosimetry. Impact on treatment verification

PurposeTo study how noise and scanner response variability affect radiochromic film dosimetry.MethodsFive treatment plans were analyzed in this work with two different multichannel protocols: the multichannel algorithm of Mayer et al. and the efficient protocol of Lewis et al.Results and conclusionThe multichannel protocol of Mayer et al. is not able to compensate variability in scanner response, which is an important issue for radiochromic film dosimetry. The efficient protocol compensates variations of scanner response, so dose values and gamma scores become more accurate and reproducible. The compensation of digitizer scan variability of the efficient protocol, together with time averaging improve radiochromic film dosimetry. Noise is related to selected resolution in the scanner, our results show that if high resolution measurements are required, de-noising should be considered.     

Construction and calibration of an optical trap on a fluorescence optical microscope

The application of optical traps has come to the fore in the last three decades. They provide a powerful, sterile and noninvasive tool for the manipulation of cells, single biological macromolecules, colloidal microparticles and nanoparticles. An optically trapped microsphere may act as a force transducer that is used to measure forces in the piconewton regime. By setting up a well-calibrated single-beam optical trap within a fluorescence microscope system, one can measure forces and collect fluorescence signals upon biological systems simultaneously. In this protocol, we aim to provide a clear exposition of the methodology of assembling and operating a single-beam gradient force trap (optical tweezers) on an inverted fluorescence microscope. A step-by-step guide is given for alignment and operation, with discussion of common pitfalls.     

Double-beam autocompensation for fluorescence polarization measurements in flow cytometry.

The degree of depolarization of fluorescent light emitted from an organic dye, which is used as molecular probe, is a powerful tool in probing the microenvironment. By fluorescence depolarization the macromolecular structure can be investigated as well as the the mobility of the marker molecule itself or of the complex formed by the probe. Additional information such as energy transfer rates, donor-acceptor distances, and orientations are also measurable. These data are of particular interest if they can be measured from whole cells. Using flow cytometry, we can analyze a large number of cells with high statistical significance in a short period of time. We describe a newly developed double-beam epi-illumination arrangement for fluorescence polarization measurements that uses an autocompensation technique. This new technique permits the various depolarizing effects within the optical as well as the electronic components of the system to be continually compensated for on a cell by cell basis. Simultaneous measurements of other cell parameters for cell cycle analysis by total fluorescence intensity remains possible. The sensitivity of the system to measure polarization was determined as +/- 0.006 p (0 less than or equal to p less than or equal to 0.5 in isotropic media), which amounts to +/- 1.2% of the maximum p value. Polarization data for latex microspheres plotted in the histogram mode were measured with a standard deviation of 0.006, which proved the high resolution and the high performance of the system.     

A pyroelectric thermal imaging system for use in medical diagnosis

The value of infra-red thermography in a number of pathologies, notably rheumatology and vascular diseases, is becoming well established. However, the high cost of thermal scanners and the associated image processing computers has been a limitation to the widespread availability of this technique to the clinical community. This paper describes a relatively inexpensive thermographic system based on a pyroelectric vidicon scanner and a microcomputer. Sofware has been written with particular reference to the use of thermography in rheumatoid arthritis and vasospastic conditions such as Raynaud's phenomenon.     

Transient spectroscopy of bacterial rhodopsins with an optical multichannel analyzer. 1. Comparison of the photocycles of bacteriorhodopsin and halorhodopsin

We used a gated optical multichannel analyzer to measure transient flash-induced absorption changes in bacteriorhodopsin (BR) and halorhodopsin (HR) and developed criteria for calculating the absorption spectra of the photocycle intermediates and the kinetics of their rise and decay. The results for BR agree with data reported by a large number of other authors. The results for HR in the presence of chloride are consistent with earlier data and reveal an additional intermediate, not previously seen, in the submicrosecond time scale. Although an M412-like intermediate is not in the HR photocycle, a one-by-one comparison of the rest of the intermediates observed for BR and HR indicates a striking similarity between the photocycles of the two bacterial rhodopsins. This was previously not apparent, perhaps because the experimental approaches to the spectroscopy of the two pigments were different and the data were thus more fragmented.     

The automated analytical electrophoresis microscope

The components of an automated computer-controlled analytical electrophoresis microscope (AEMS) are described. Computer tracking of migrating cells projected under phase contrast onto a vidicon permits the rapid taking of multiple velocity measurements per cell so that reliable determinations of electrophoretic mobilities thereby result. The computer-controlled cell search and tracking algorithms allow high speed operation so that statistically valid profiles and data bases can be collected rapidly. Initial electrophoretic mobility evaluations have been carried out on populations of lymphocytes, erythrocytes, and platelets.     

Construction and performance of the Brice photoelectric differential refractometer

The design modification and performance of a photoelectric differential refractometer originally designed by the late Dr. B. A. Brice of the U. S. Department of Agriculture is described. The instrument consists of a mercury lamp light source, monochromatic filters, variable slit, beam splitter, light path consisting of a blackened tube, divided cell containing sample and solvent, lens, surface mirror reflecting the light back through the blackened tube to the beam splitter, and a matched pair of photocells mounted on a movable flat carriage. The slit image is detected by manipulating the carriage until the slit image is exactly between the two photocells. A nullmeter is used to determine this point. The main advantages of the photoelectric differential refractometer over previous designs is freedom from eyestrain, ease of operation, and linearity of operation.     

Spectra of cells in flow cytometry using a vidicon detector.

A TV type vidicon detector was interfaced to a flow cytometer (FCM) to obtain spectra of fluorophores in cells during flow. The normal operations of the FCM are undisturbed. A spectrograph spreads 320 nm of the fluorophore fluorescence emission across the 500 channels of the detector. Spectra of fluorescamine (a surface labeling agent) and of propidium iodide (a nuclear stain) were obtained from Balb 3T3 cells, and the chlorophyll and phycobilin peaks were resolved from flowing blue-green algae in the FCM. Under typical flow conditions, operation of the vidicon in the continuous mode gives for these fluorophores a S/N of several hundred to one in approximately 3 sec. The vidicon was also gated to obtain spectra of single cells and of cells in selected portions of the cell cycle. For example, the spectrum of fluorescamine was obtained from cells in the G1 phase of the growth cycle by using as a gate trigger the FCM discriminator output derived from the propidium iodide signal.