Microspectrofluorometry by digital image processing: measurement of cytoplasmic pH

An interface of our microspectrofluorometer with an image processing system performs microspectrofluorometric measurements in living cells by digital image processing. Fluorescence spectroscopic parameters can be measured by digital image processing directly from microscopic images of cells, and are automatically normalized for pathlength and accessible volume. Thus, an accurate cytoplasmic "map" of various spectroscopic parameters can be produced. The resting cytoplasmic pH of fibroblasts (3T3 cells) has been determined by measuring the ratio of fluorescein fluorescence exited by two successive wavelengths (489 and 452 nm). Fluorescein-labeled dextran microinjected into the cells is used as a pH indicator, since it is trapped in the cytoplasm but is excluded from the nucleus and other organelles. The average cytoplasmic pH is 6.83 (+/- 0.38). However, cytoplasmic pH exhibits a nonunimodal distribution, the lower mean pH being 6.74 (+/- 0.23). When 3T3 cells pinocytose medium containing fluorescein dextran, pinosomes peripheral to the nucleus exhibit a lower pH than those closer to the ruffling edge of the cell. The present image processing system is analyzed for linearity of detection, light scattering artifacts, signal to noise ratio, standard curves, and spatial resolution. The results obtained from digital image analysis are shown to be comparable to the results from standard microspectrofluorometry. We also discuss several other applications of this ratio imaging technique in cell biology.     

Low-angle light-scattering instrument for DNA solutions

A light-scattering instrument capable of measurements on native DNA at angles as low as 10° is described. The major features of the instrument, which make it capable of low-angle measurements, are the use of an intense light source in which the incident beam is monitored directly and the use of a long, rectangular sample cell in which the scattered light can be measured at low angles with no interference from the incident beam. Methods for calibrating the instrument and for determining scattering correction factors are described. Procedures for the preparation and use of various calibration standards are given.     

Compact, high performance surface plasmon resonance imaging system

We report the construction and characterization of a new compact surface plasmon resonance imaging instrument. Surface plasmon resonance imaging is a versatile technique for detection, quantification and visualization of biomolecular binding events which have spatial structure. The imager uses a folded light path, wide-field optics and a tilted detector to implement a high performance optical system in a volume 7 in. x 4 in. x 2 in. A bright diode light source and an image detector with fast frame rate and integrated digital signal processor enable real-time averaging of multiple images for improved signal-to-noise ratio. Operating angle of the imager is adjusted by linear translation of the light source. Imager performance is illustrated using resolution test targets, refractive index test solutions, and competition assays for the antiepileptic drug phenytoin. Microfluidic flowcells are used to enable simultaneous assay of three sample streams. Noise level of refractive index measurements was found to decrease proportional to the square root of the number of pixels averaged, reaching approximately 5 x 10(-7) refractive index units root-mean-square for 160 x 120 pixels image regions imaged for 1s. The simple, compact construction and high performance of the imager will allow the device to be readily applied to a wide range of applications.     

Cytomat-R: a computer-controlled multiple laser source multiparameter flow cytophotometer system.

A multiple illumination wavelength multiparameter flow cytophotometer system, using laser sources and controlled by a small, general-purpose digital computer, has been produced for use in the development of new flow cytometric techniques. Three different laser wave-lengths can be used simultaneously to illuminate different regions of the flow chamber; as many as five measurements of light scattering at various angles, extinction, and fluorescence at one or more wavelengths can be made at each illuminated station. Cells in suspension may be examined at rates of 1000 cells/sec, with seven correlated optical measurements being recorded for each cell. A library of programs for data manipulation and statistical analysis make it possible to use the system to develop and implement cell characterization, counting and classification procedures for basic and clinical research applications.     

Narrow-angle forward light scattering from individual algal cells: implications for size and shape discrimination in flow cytometry

Single-cell forward light scattering patterns have been examinedfor four algal species (one pennate diatom, two green flagellatesand one filamentous cyanobacterium), mounted statically in afocused laser beam. In all cases, the distribution of lightintensity at narrow angles (within the first scattering lobe)is well described by diffraction theory. Narrow-angle forwardscattering measurements can therefore be used in principle todeduce the size and approximate shape of algal cells. The feasibilityof using this technique in flow cytometry has been tested usingan instrument which orientates elongated cells uniformly inthe flow stream, and uses fibre optics to make azimuthally resolvedforward scatter measurements at sub-degree polar angles. Withthis instrument it is possible to discriminate between specieswith similar volume and fluorescence characteristics using forwardlight scattering as a shape-sensitive parameter.     

Optical plankton analyser: a flow cytometer for plankton analysis, II: Specifications

An analysing flow cytometer, the optical plankton analyser (OPA), is presented. The instrument is designed for phytoplankton analysis, having a sensitivity comparable with commercially available flow cytometers, but a significantly extended particle size range. Particles of 500 microns in width and over 1,000 microns in length can be analysed. Sample flow rates of up to 55 microliters/s can be used. Also, the dynamic range of the instrument is significantly increased for particles larger than about 5 microns. The optics, hydraulics, and electronics of the instrument are described, including the best form for a low fluid shear cuvette. The new pulse quantification technique we call digital integration is presented. This technique is essential for the instrument to handle both short and very long particles with a large dynamic range. Test measurements demonstrating particle size range and dynamic range are presented. Dynamic ranges of 10,000 and 100,000 were typically observed, measuring field samples with Microcystis aeruginosa colonies, whereas one sample showed a dynamic range of 10(6). A simple method for interpretation of time of flight (TOF) data in terms of particle morphology is presented. The specifications of the instrument are given.     

Qualitative und quantitative Fluoreszenzmikrospektrographie mit dem Leitz-Mikrospektrographen

Zusammenfassung Ausgehend vom Leitz-Mikrospektrographen wird gezeigt, wie dieses Gerät für mikrospektrofluorimetrische Untersuchungen und für die quantitative Fluoreszenzcytophotometrie ausgerüstet werden kann. Die im Zusammenhang mit der Anwendung des Gerätes auftretenden Fragen der Eichung und Korrektur der Spektralkurven werden erörtert.

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Qualitative and quantitative fluorescence cytophotometry at the Leitz-Microspectrograph

Summary The use and additional equipment of the Leitz-Mikrospectrograph for microspectrofluorometry and fluorescence cytophotometry are described in detail.Fluorescence excitation with incident light together with the use of dichromatic beam splitters for optimum separation of the excitation beam from emitted radiation are the characteristic features of this instrument.The fluorescence light is spectrally dispersed by a glass-prism (Special glass for visible light, quarz for UV).A motor driven oscillating mirror moves along the spectrum and reflects the light of continuously changing wavelength on to the photo-cathode of the multiplier, that transfers the optical impulse into an electrical signal. The wavelength dependend change of spectral intensity is registered by a light beam oscillograph fitted with four fixed potentiometers indicating the 400, 500, 600 and 700 nm wavelength position by crosspoints with an oblique line drawn by the wavelength potentiometer, while the sixth potentiometer records the spectral curve.Problems of spectral calibration and correction are discussed.When the prism and the oscillating mirror are removed the instrument may be used as fluorescence cytophotometer. For this purpose of quantitative fluorescence cytophotometry a digital measuring unit with a print-out instrument is presented.

     

A solid-state,portable instrument for measurement of chlorophyll luminescence induction in plants

A newly developed compact instrument is described for the measurement of chlorophyll luminescence induction in plants. The instrument operates with a pulsed light emitting diode (LED) as light source and a photodiode as luminescence detector. A special emitter-detector geometry provides for high irradiance of the sample and efficient collection of luminescence by the detector. With insertion of appropriate filters the same probe is also suited for measuring prompt chlorophyll fluorescence. The instrument shows considerable flexibility with respect to pulse frequency, relative lengths of light/dark intervals and luminescence sampling periods. Due to a selective amplifier system only that part of luminescence is processed which is induced by the individual excitation pulses. By this approach, the problem of slow phase accumulation, encountered with conventional phosphoroscopes, is eliminated. Some examples are given for system operation, demonstrating satisfactory performance in measurements with intact leaves and isolated chloroplasts.     

A portable, microprocessor operated instrument for measuring chlorophyll fluorescence kinetics in stress physiology

A portable instrument for measuring chlorophyll fluorescence induction kinetics is described and examples of measurements are given. The instrument is centered around a statistically-mixed bifurcated optical fiber. One fiber branch guides the actinic light to the sample, whereas the other branch carries the emitted chlorophyll fluorescence to the photodetector. Scattered actinic light is cut out from the detector by a red interference filter. The instrument measures fast as well as slow fluorescence induction kinetics, but is particularly well designed for analyzing fast kinetics. The high time resolution and strong, variable actinic light mean that both F_o (non-variable fluorescence) and F_m (maximal fluorescence at the P-peak) are well defined. A built in microprocessor unit with attached memory stores the fluorescence induction curve and calculates key fluorescence parameters such as F_o, F_m, F_v (variable fluorescence equals F_m?F_o), F_v/F_m (the photochemical efficiency of photosystem II) and t_1/2 (half rise time from F_o, to F_m). These values are digitally displayed after each recording and they (or the whole induction curve) can be stored in a memory and later retrieved. Because of a flexible setting of the instrument it can be used with high accuracy both for optically thick leaves and for diluted suspensions of algae or chloroplasts. A simple, light weight clamp cuvette for dark adaptation of leaves has been developed. It is equipped with a gate allowing the optical fiber to be inserted without daylight reaching the dark adapted portion of the leaf. The instrument has been developed for rapid monitoring of changes in activities and organization of the photosynthetic apparatus in vivo when plants are exposed to environmental stress both in the field and in the laboratory. Examples of measurements are given for differently treated leaves of Pinus sylvestris, Salix sp., Betula verrucosa, Zea mays, Epilobium angustifo-lium and for chloroplast thylakoids isolated from Spinacia oleracea.     

A microprocessor-controlled photometer for monitoring microbial growth in multi-welled plates

Multi-welled microtitre plates provide a convenient means of handling 'large block' multifactorial experiments with microbial cultures. An inexpensive instrument, termed a 'Biophotometer', has been designed to monitor microbial growth in each well, by transmitted light measurements. Optional microcomputer control is employed to facilitate scanning of plates and data handling. A unique method for agitating cultures is incorporated into the system. Typical results are presented to illustrate the versatility of this system.     

A microprocessor-controlled photometer for monitoring microbial growth in multi-welled plates

Multi-welled microtitre plates provide a convenient means of handling 'large block' multifactorial experiments with microbial cultures. An inexpensive instrument, termed a 'Biophotometer', has been designed to monitor microbial growth in each well, by transmitted light measurements. Optional microcomputer control is employed to facilitate scanning of plates and data handling. A unique method for agitating cultures is incorporated into the system. Typical results are presented to illustrate the versatility of this system.     

Microspectrofluorometric identification of formaldehyde induced fluorescence in hypothalamic nuclei of Xenopus laevis tadpoles

Summary To identify the monoamine (s) produced in the paraventricular organ (PVO) and the nucleus infundibularis dorsalis (NID) of Xenopus laevis tadpoles, formaldehyde-induced fluorescence in these hypothalamic structures was analysed by microspectrofluorometric techniques. Reference values were obtained by recording excitation and emission spectra of fluorescence in monoamine containing protein models. The maxima of the excitation and emission spectra, both under normal conditions and after treatment with HCl vapour, indicate the presence of dopamine. Based on a number of emission spectra, the PVO and NID might also contain serotonin. With regard to the functional significance of dopamine produced in the hypothalamic nuclei, it may be concluded that the catecholamine is probably identical with the melanotropin inhibiting factor (MIF).The authors wish to thank Prof. Dr. P.G.W.J. van Oordt for his stimulating interest and support. The skillful assistance and valuable contributions of Miss A. G. Fennema and Miss M.G.A. de Bruyn are gratefully acknowledged. Many thanks are due to Prof. Dr. A. Oksche for the opportunity of performing measurements on fluorescence, during a visit to his institute (Zentrum für Anatomie und Cytobiologie, Gießen, BRD). The measurements were carried out by Dr. H.-G. Hartwig, whose help and advice are highly appreciated.     

An Instrument for Measuring Crop Density by Light Absorbance

A portable instrument is described which measures simultaneouslythe light intensity above and below a leaf canopy, calculatesthe absorbance (extinction) and displays it continuously ona meter. It measures the capacity of the crop to absorb lightand so provides an estimate of its density and, by inference,the approximate photosynthetic potential. The measurements,which are simple and non destructive, have been shown to correlatewith seed-rate, sowing date, seed-vigour and final yield inwheat. Although originally designed for cereals, the instrumentmay also find application with other crops. crop density measurement, creals, light abrbanace     

Use of dynamic light scattering to determine second virial coefficient in a semidilute concentration regime

The present work discusses an alternative procedure to obtain static light scattering (SLS) parameters in a dilute and semidilute concentration regime from a dynamic light scattering (DLS) instrument that uses an avalanche photodiode (APD) for recording the scattered intensity signal. An APD enables one to perform both SLS and DLS measurements by photon counting and photon correlation, respectively. However, due to the associated recovery time, the APDs are susceptible to saturation (above 1000 kcps), which may limit the measurements in systems that scatter too much light. We propose an alternative way of obtaining the SLS parameters with instruments that use APD for recording signal intensities.     

A scanning inverted microfluorometer with electronic shutter control for automatic measurements in micro-test plates

A microfluorometer is described for automated measurements with a fixed excitation period in small volumes (5–10 μl) using standard Terasaki test plates. The Leitz inverted microfluorometer has been equipped with a scanning stage that has been linked electronically to electric shutters to control the excitation and measuring periods. The excitation of the sample takes place from below through the bottom of the test plate. The fluorescence signal is measured with an average value current meter which integrates the anode current of the photomultiplier during 1 sec. The result is printed on a digital recorder. The total measuring time for a 60-well Terasaki tray is about 135 s. The sensitivity of the instrument and the linearity and reproducibility of the measurements have been determined with samples of increasing concentrations of 4-methyl umbelliferone (4-MU). The coefficient of variation was smaller than 5%. The blank corresponded to 0.025 μm 4-MU (in 10 μl) which is equivalent to 0.25 pmol of 4-MU. The applicability of the instrument is illustrated by the measurements of β-hexosaminidase activity in diluted homogenates of cultured human fibroblasts.     

Evaluation of a technique for the measurement of chlorophyll fluorescence from leaves exposed to continuous white light

Abstract An instrument for the generation and measurement of modulated chlorophyll fluorescence signals from leaves exposed to continuous, highintensity white light is described. Modulated fluorescence is generated in the leaf by pulsed diodes emitting low-intensity yellow radiation and is detected with a photodiode whose output is fed to an amplifier locked in to the frequency of the lightemitting diodes. Comparisons are made between the modulated fluorescence signals measured with this instrument and the continuous fluorescence signals emitted from dark-adapted leaf tissue and isolated thylakoids when photosynthetic activity is induced by exposure to a range of intensities of continuous broad-band, blue-green light. The modulated fluorescence signals were similar to the continuous fluorescence signals, but they were not always identical. The small differences between the two signals are mainly attributable to differences in the populations of chloroplasts being monitored in the two measurements as a result of differential penetration of the modulated and actinic light sources into the sample.     

A microfluorometric study of masked metachromasia in cells of the endocrine polypeptide (APUD) series

Synopsis Masked metachromasia can be demonstrated by staining with a metachromatic basis dye after a Feulgen-type hydrolysis of suitably fixed tissue, and is believed to be indicative of polypeptides with a high concentration of side-chain acidic groups and a random-coil conformation. In this investigation, the metachromatic fluorochrome Coriphosphine O was used. After staining, the degree of metachromasia under various conditions and in several tissues was assessed by microspectrofluorometric measurements of the ratio of metachromatic fluorescence (red) to orthochromatic fluorescence (green). This technique was employed, in the first instance, to determine the optimum staining conditions; details of the final staining method are presented. Measurements of metachromasia in different tissues under standardized conditions showed that the degree of metachromasia varied between different cell types in the APUD series.     

Transducers,sensors, and instrumentation in clinical biomechanics

Successful measurements in any field are dependent on the availability of appropriate transducer materials and the associated instrumentation. In recent years there has been a most welcome advance in both these areas. If we consider first the transducer developments that have recently taken place, these have much to do with the discovery and application of new materials such as electroactive polymers, fibre optic devices and many others. Instrumentation has largely benefitted from the microelectronics revolution. Our ability to process and ultimately display data has improved to a remarkable extent. Indeed, the designer of instrumentation is under enormous pressure to convert the data into the digital domain as early as possible simply because the resulting instrument will usually be easier to design and construct, more accurate, more reliable, smaller and possibly more important still, cheaper. This paper reviews some of these developments, gives a number of examples of applications in the clinical biomechanics area and makes some predictions for future developments.     

A polychromator-based microspectrophotometer

A microspectrophotometer is a digital microscope used to measure absorption and fluorescence spectra. In this paper we describe a polychromator-based microspectrophotometer that performs in vivo absorption or emission measurements at the same time on different subcellular compartments such as photoreceptive and photosynthetic structures of algal cells. In this system, a flat field imaging concave grating polychromator is connected to the slit-shaped exit pupil of a light-guide probe mounted onto a microscope equipped with an epifluorescence module. The subcellular components, on which the spectra will be measured, are placed in the microscope field and finely adjusted. The outer bundle of the probe is used for centering the objects, while the central bundle of the probe, containing 19 light guides, is used for acquiring either transmitted or emitted light (i.e. fluorescence). The light transmitted or emitted by the subcellular components is collected by the probe mounted in the back focal plane of the ocular. The exit pupil of this probe, connected to a flat field imaging concave grating polychromator, produces a dispersion image that in turn is focused onto a digital slow scan cooled CCD camera. Absorption and emission spectra of algal subcellular compartments are presented.     

Imaging system for morphometric assessment of absorption or fluorescence in stained cells

An image acquisition and processing system has been developed for quantitative microscopy of absorption or fluorescence in stained cells. Three different light transducers are used in the system to exploit the best characteristics of these sensors for different biological measurements. A digital scanner, in the form of a linear array charge-coupled device (CCD), acquires data with high spatial and photometric resolution. A color (RGB) camera is employed when spectral information is required for the segmentation of cellular subcomponents. An image-intensified charged-injection device (CID) camera provides for very low light intensity measurements, primarily for fluorescence-labeled cells. Properties of these transducers, such as contrast transfer function, linearity, and photo-response nonuniformity, have been measured. Two dedicated image processing units were incorporated into the system. The front-end processor, based on a digital signal processor, provides functions such as object detection, raw image calibration, compression, artifact removal, and filtering. The second image processor is associated with the frame memory and includes a histogram processor, a dedicated arithmetic logic unit for image processing functions, and a graphics module for one-bit overlay functions. An interactive program was developed to acquire cell images and to experiment with a range of segmentation algorithms, feature extractions, and other image processing functions. The results of any image operation are displayed on the video monitor. Once a desired processing sequence is determined, the sequence may be stored to become part of a command library and can be executed thereafter as a single instruction.