Light-scattering polarization measurements as a new parameter in flow cytometry

Polarization measurement of orthogonal light scattering is introduced as a new optical parameter in flow cytometry. In the experimental setup, the electrical field of the incident laser beam is polarized in the direction of the sample flow. The intensity of the orthogonal light scattering polarized along the direction of the incoming laser beam is called depolarized orthogonal light scattering. Theoretical analysis shows that for small values of the detection aperture, the measured depolarization is caused by anisotropic cell structures and multiple scattering processes inside the cell. Measurements of the orthogonal depolarized light scattering in combination with the normal orthogonal light scattering of human leucocytes revealed two populations of granulocytes. By means of cell sorting it was shown that the granulocytes with a relatively high depolarization are eosinophilic granulocytes. Similar experiments with human lymphocytes revealed a minor subpopulation of yet-unidentified lymphocytes with a relative large orthogonal light-scattering depolarization. The results were obtained with an argon ion laser tuned at different wavelengths as well as with a 630-nm helium neon laser. These results show that measurement of depolarized orthogonal light scattering is a useful new parameter for flow-cytometric cell differentiation.     

Cell cycle studies of murine leukemia cell L5l78Y by polarization effects of light scattering

The structural changes during the life cycle of a synchronized population of mouse leukemia cell line L5l78Y have been described by polarized light scattering measurements. Exponentially growing cells were synchronized by an automatic excess thymidine-colcemid treatment technique. Samples were removed from the suspension culture and fixed with glutaraldehyde at hourly intervals throughout the life cycle. The effect these cell samples had in changing right-hand circularly polarized light to 45° linearly polarized light during the scattering process was measured at angles 6–l60° to the incident beam. The reproducibility of the light scattering signals for each time interval was statistically evaluated and found to have good intertrial correlation for each time period in the angular range 6–60° to the incident beam. Statistically significant changes were seen between cell samples during the synchronous life cycle. Therefore, the system developed has applications as an extremely sensitive measure of cell structure, and of structural changes caused by low-level chemical, physical or biological agents.     

声光可调谐滤波器的细胞生物学研究应用

波长选择在荧光光谱仪和显微镜等光学应用中发挥了至关重要的作用。声光可调谐滤波器(AOTF)作为一种电光器件可实现多光源入射波长、功率的同时调制。在声光可调谐滤波器中,压电换能器结合于二氧化碲或石英晶体产生高频声波,改变晶体折射率形成周期性分布。该现象在晶体中生成衍射光栅,使以布拉格角正交入射的光束被高效衍射至一阶光束。当改变施加到晶体的信号频率时将改变折射率变化周期,因此,衍射光的波长随之改变。同时,衍射光强度由施加到晶体的信号振幅决定。本文从声光可调谐滤波器原理和特点出发,总结了声光可调谐滤波器在细胞生物学研究系统中的应用模型。得益于作用时间短、波长分辨率高、无振动部件等特性,声光可调谐滤波器提升了多波长光源功率调制能力,使细胞计数系统具备了细胞高光谱成像能力。所以不仅限于传统细胞生物学研究,包含声光可调谐滤波器件的系统还将在多参数高内涵成像分析、扫描荧光显微术、药物毒理研究等领域成为有力的研究工具。     

Multiple wavelength illumination in flow cytometry using a single arc lamp and a dispersing element

The principle of a multiple wavelength illumination method for flow cytometers, based upon a combination of a helium-neon laser and an arc lamp as illumination sources is described. By using a prism, the light from the arc lamp is dispersed and the different colors are imaged at different places on the sample stream. The small angle light scattering from the helium-neon laser light is measured as a relevant parameter and serves as a trigger signal for subsequent measurements of fluorescence or scattering of light from the arc lamp. Two experimental systems are described utilizing this principle: a system where the emission is detected orthogonally with respect to the direction of the illumination beams, and an epi-illumination system. With the orthogonal set-up multiple wave-length right angle scattering measurements are possible. This is illustrated by showing that the orthogonal scattering from erythrocytes is strongly dependent on the illumination wavelength. It is further shown that the apparatus is suitable for the measurement of intracellular pH using the pH dependence of the excitation spectrum of fluorescein. The epi-illumination system allows excitation of two (or more) fluorescent dyes with different excitation spectra. In this case the emission spectra of the fluorescent dyes may overlap substantially. This is shown by simultaneous measurement of DNA and protein of Chinese hamster lung cells using mitramycin and tetramethyl rhodamin isothiocyanate (TRITC).     

Flow cytometric characterization of chronic lymphocyte leukaemias using orthogonal light scattering and quantitative immunofluorescence

Light scattering properties and antigen distribution of lymphocytes labeled with the monoclonal antibodies CD 5 and CD 20 were determined for 19 patients with a chronic B-cell derived leukaemia. The density of the antigen detected by the monoclonal antibody CD 5 appeared to be considerably lower on malignant B-lymphocytes of the patients as compared with T lymphocytes. A large variation was observed in the amount of receptors for the monoclonal antibodies CD 5 and CD 20 on the malignant cells of the different patients. B-cell chronic lymphocytic leukaemia (B-CLL) patients were clearly distinguishable from leukaemic follicular non Hodgkin lymphoma patients (LF-NHL, formerly lymphosarcoma cell leukaemia) and from a patient with a prolymphocytoid transformation (PLT) of the B-CLL according to the amount of the antigens for CD 5 and CD 20. Within the B-CLL patient population, no relation of progression of the disease with distribution of these antigens could be observed. In one patient the extraordinary phenotype CD 20+, CD 11+, leu 8+, CD 5- of the malignant lymphocytes was observed. An experimentally simple method to differentiate between the various chronic lymphocytic leukaemias (CLL) appeared to be the determination of orthogonal light scattering properties of lymphocytes. In healthy donors one can always distinguish two populations of lymphocytes in the orthogonal light scatter histograms. Lymphocytes of B-CLL patients show one uniform population with a relatively small orthogonal light scattering signal, lymphocytes of our patients with PLT of B-CLL or with LF-NHL show one uniform population with a relatively large orthogonal light scattering signal.     

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.     

Acousto-optic laser-scanning cytometer

An instrument has been developed that uses a computer-controlled rapidly scanning laser beam to make cytometric measurements on cells or particles and which can measure low levels of fluorescence when using low-power lasers (Gershman, Hoffman, and O'Connell, "Methods and Apparatus for Analysis of Particles and Cells.") The method used is based upon acousto-optic principles of light diffraction. A vertically polarized 5-mW He-Ne laser is directed into an acousto-optic Bragg cell in which a portion of the incident light undergoes a small angular variation or deflection. Suitable optics focus the beam to a 25 microns diameter spot, at the 1/e2 point, in a sample cuvette while translating the angular variation into a linear scan. The cuvette enclosing the sample is slowly moved (approximately 1 micron/ms) via a stepper drive into the scanning beam while the forward angle light scatter sensor is monitored for the presence of valid signal events. When an event occurs, appropriate software optimizes the position of the focused laser beam onto the cell. Subsequently, scanning is stopped to allow for cell interrogation times that last for milliseconds or longer.     

Laser light-scattering studies of bull spermatozoa. I. Orientational effects.

Calculations based on the known dimensions of bull spermatozoa show that the scattered light intensity is strongly dependent upon the relative orientation of the particle to the incident beam. The magnitude of this effect of apparently much greater than for other systems where motility has been investigated by dynamic light scattering. The calculations show that the scattering source can be approximated by a small spinning mirror, and consequently the greatest light intensity at the detector results from cells swimming in a direction perpendicular to the scattering vector. The calculations are in substantial agreement with photographic observations, as well as direct measurements of the scattered intensity. Previous treatments of dynamic light scattering from swimming bull spermatozoa based on point scattering models are shown to be incorrect.     

Two-photon irradiation of an intracellular singlet oxygen photosensitizer: Achieving localized sub-cellular excitation in spatially-resolved experiments

Abstract

The response of a given cell to spatially-resolved sub-cellular irradiation of a singlet oxygen photosensitizer (protoporphyrin IX, PpIX) using a focused laser was assessed. In these experiments, incident light was scattered over a volume greater than that defined by the dimensions of the laser beam as a consequence of the inherent inhomogeneity of the cell. Upon irradiation at a wavelength readily absorbed by PpIX in a one-photon transition, this scattering of light eliminated any advantage accrued to the use of focused irradiation. However, upon irradiation at a longer wavelength where PpIX can only absorb light under non-linear two-photon conditions, meaningful intracellular resolution was achieved in the small spatial domain where the light intensity was high enough for absorption to occur.     

Two-photon irradiation of an intracellular singlet oxygen photosensitizer: achieving localized sub-cellular excitation in spatially-resolved experiments

The response of a given cell to spatially-resolved sub-cellular irradiation of a singlet oxygen photosensitizer (protoporphyrin IX, PpIX) using a focused laser was assessed. In these experiments, incident light was scattered over a volume greater than that defined by the dimensions of the laser beam as a consequence of the inherent inhomogeneity of the cell. Upon irradiation at a wavelength readily absorbed by PpIX in a one-photon transition, this scattering of light eliminated any advantage accrued to the use of focused irradiation. However, upon irradiation at a longer wavelength where PpIX can only absorb light under non-linear two-photon conditions, meaningful intracellular resolution was achieved in the small spatial domain where the light intensity was high enough for absorption to occur.     

基于旋转偏振角的线偏振扫描成像方法研究

提出一种基于旋转偏振角的新的偏振光成像方法:改变入射线偏振光偏振角和检偏角,采集样品图像系列,总结出背向散射光2个正交偏振分量的光强差关于入射线偏振光偏振角和检偏角的函数关系式.通过对模拟散射介质,猪肉脂肪,猪肉骨骼肌和牛肉骨骼肌等样品进行实验,论证了偏振差函数式中各个参数与样品光学特性之间的联系,并从中提取出2个相互独立的参数,分别表征样品的纤维方向角和光学各向异性度,从而实现对样品浅表层光学特性进行定量测量.和普通光强图像相比,用这些独立的光学信息生成不同基色的图像,能更直观明了地区分组织结构差异,具有潜在的临床医学应用价值,如成为一种皮肤疾病、皮肤损伤的检测方法.     

A fiber-optic retroreflective turbidimeter for continuously monitoring cell concentration during fermentation

A fiber-optic retroreflective turbidimeter has been developed to automatically and continuously assay the cell concentration in a fermentor by measuring the turbidity of the solution as a function of the light scattered at 180° to the incident light. The output signal is nearly directly proportional to the cell concentration in a fermentor when the sample stream contains from 0 to more than 50 g of cells per liter (wet weight). The device consists of a bifurcated fiber-optics light pipe with its distal end inserted into a flow cell through which the material to be analyzed passes. A light source on one proximal branch of the light pipe illuminates the sample stream; light that is back-scattered from participates in the stream re-enters the light pipe and is returned to a photodetector on the other proximal branch of the light pipe. A signal conditioning system connected to the optical head by a cable provides gain and zero adjustment.     

Eliminating Unwanted Far-Field Excitation in Objective-Type TIRF. Part II. Combined Evanescent-Wave Excitation and Supercritical-Angle Fluorescence Detection Improves Optical Sectioning

Azimuthal beam scanning makes evanescent-wave (EW) excitation isotropic, thereby producing total internal reflection fluorescence (TIRF) images that are evenly lit. However, beam spinning does not fundamentally address the problem of propagating excitation light that is contaminating objective-type TIRF. Far-field excitation depends more on the specific objective than on cell scattering. As a consequence, the excitation impurities in objective-type TIRF are only weakly affected by changes of azimuthal or polar beam angle. These are the main results of the first part of this study (Eliminating unwanted far-field excitation in objective-type TIRF. Pt.1. Identifying sources of nonevanescent excitation light). This second part focuses on exactly where up beam in the illumination system stray light is generated that gives rise to nonevanescent components in TIRF. Using dark-field imaging of scattered excitation light we pinpoint the objective, intermediate lenses and, particularly, the beam scanner as the major sources of stray excitation. We study how adhesion-molecule coating and astrocytes or BON cells grown on the coverslip surface modify the dark-field signal. On flat and weakly scattering cells, most background comes from stray reflections produced far from the sample plane, in the beam scanner and the objective lens. On thick, optically dense cells roughly half of the scatter is generated by the sample itself. We finally show that combining objective-type EW excitation with supercritical-angle fluorescence (SAF) detection efficiently rejects the fluorescence originating from deeper sample regions. We demonstrate that SAF improves the surface selectivity of TIRF, even at shallow penetration depths. The coplanar microscopy scheme presented here merges the benefits of beam spinning EW excitation and SAF detection and provides the conditions for quantitative wide-field imaging of fluorophore dynamics at or near the plasma membrane.     

Filament interaction monitored by light scattering in skinned fibers

The intensity of light scattered by chemically skinned rabbit psoas fibers in relaxed, rigor, and activated states was monitored at 90 degrees to the incident beam. In the relaxed state, scattering varied in proportion to the volume of muscle in the beam. Scattering increased to 2.3 times the resting value when rigor was induced by withdrawal of MgATP or when the myofibrils were activated by the caffeine-induced release of Ca from the sarcoplasmic reticulum. The rigor-induced increase in scattering decreased monotonically when MgATP was reintroduced stepwise (0-100 microM). This decrease in scattering was accompanied by an increase in tension up to an optimum MgATP level of approximately 10 microM, and then tension decreased at higher concentrations (10-100 microM). The increase in scattering during both rigor and activation was dependent upon fiber length. At lengths when thick-thin filament overlap was near zero, the light signal due to rigor and activation fell to within 10% of the signal for the relaxed fiber at that length. The signal during rigor increased only minimally (approximately 10%) when stretch (approximately 1%) was applied. This increase in signal was small despite a measured 5- to 10-fold increase in tension and an estimated twofold increase in stiffness. Thus, the increased light scattering caused by rigor and activation depends on filament overlap and not tension, stiffness, or substrate binding.     

Quasi-elastic light scattering studies of membrane motion in single red blood cells.

Studies of red blood cells (RBCs) and RBC ghosts, using a quasi-elastic light scattering (QELS) microscope spectrometer, have identified the membrane as the primary source of the light scattering signal. This is the first report in which motion of the cell membrane has been demonstrated to be the primary source of the QELS signal from a cell. Cytoplasmic changes induced in the RBC by varying the osmotic strength of the medium were also detected using this technique. Comparison of the data from white blood cells (WBCs) with the RBC data demonstrated significant differences between different types of cells.     

AGGREGATELESS MUTANT DEFECTIVE IN SIGNALING AND RELAYING IN THE CELLULAR SLIME MOLD, DICTYOSTELIUM DISCOIDEUM

An aggregateless mutant (HT41), isolated from D. discoideum NP14, was examined for the parameters involved in cell aggregation. HT41 cells were induced to express some of these parameters by cyclic AMP pulses. Spontaneous oscillations in light scattering were not observed in the suspension of the mutant cells. A pulse of cyclic AMP caused a monophasic decrease in light scattering, which was similar to the response of pre-aggregation cells of the wild-type. When cyclic AMP pulses were applied on a cell layer, HT41 cells aggregated without forming the streams. These results suggest that HT41 is a mutant defective in signal emission and relay.     

On-line monitoring of marine cyanobacterial cultivation based on phycocyanin fluorescence.

A novel on-line fluorescence monitoring system for marine cyanobacterial cultivation was developed. This method is based on the measurement of intracellular phycocyanin content, which is the major light harvesting protein. A fluorescence spectrophotometer, equipped with a flow cell connected with a culture liquid recycling tube was used. Experiments were carried out using a marine unicellular cyanobacteria Synechococcus sp. NKBG 042902 isolated from Japanese coastal sea water. We have optimized excitation wavelength to avoid the light scattering, using non-pigmented old cells which no longer contained phycocyanin. At an excitation wavelength of 590 nm, light scattering was minimized. Viable cell concentration could be measured in the range of 2 x 10(6) to 2 x 10(8) cells per ml, without pronounced light scattering. Continuous monitoring of marine cyanobacteria cultivation was performed. Cell concentrations were determined by both culture fluorescence and by using a hemacytometer. A good linear correlation was obtained. We conclude that on-line monitoring of cyanobacterial culture fluorescence based on phycocyanin is a rapid, efficient and also versatile method for determining viable cell concentration.     

Violet laser diodes as light sources for cytometry

BACKGROUND: Violet laser diodes have recently become commercially available. These devices emit 5-25 mW in the range of 395-415 nm, and are available in systems that incorporate the diodes with collimating optics and regulated power supplies in housing incorporating thermoelectric coolers, which are necessary to maintain stable output. Such systems now cost several thousand dollars, but are expected to drop substantially in price. Materials and Methods A 4-mW, 397-nm violet diode system was used in a laboratory-built flow cytometer to excite fluorescence of DAPI and Hoechst dyes in permeabilized and intact cells. Forward and orthogonal light scattering were also measured. RESULTS: DNA content histograms with good precision (G(0)/G(1) coefficient of variation 1.7%) were obtained with DAPI staining; precision was lower using Hoechst 33342. Hoechst 34580, with an excitation maximum nearer 400 nm, yielded the highest fluorescence intensity, but appeared to decompose after a short time in solution. Scatter signals exhibited relatively broad distributions. CONCLUSIONS: Violet laser diodes are relatively inexpensive, compact, efficient, and quiet light sources for DNA fluorescence measurement using DAPI and Hoechst dyes; they can also excite several other fluorescent probes.     

A simple light scattering method to assay magnetism in Magnetospirillum gryphiswaldense

Abstract A simple optical method was developed for assaying cellular magnetism in culture samples of magnetic spirilla. Cells are aligned parallel to the field lines in a magnetic field, resulting in a change in light scattering. The ratio of scattering intensities at different angles of magnetic field relative to the light beam ( C _mag) is used to characterize the average magnetic orientation of the cells. C _mag was found to be well correlated with the average number of particles in different magnetic cell populations. Thus, estimations of magnetosome content can be made using magnetically induced differential light scattering. The method provides a fast and sensitive tool for monitoring the magnetite formation in growing cultures of Magnetospirillum gryphiswaldense .     

Four-parameter white blood cell differential counting based on light scattering measurements

Measurement of the depolarized orthogonal light scattering in flow cytometry enables one to discriminate human eosinophilic granulocytes from neutrophilic granulocytes. We use this method to perform a four-parameter differential white blood cell analysis. A simple flow cytometer was built equipped with a 5-mW helium neon laser that measures simultaneously four light scattering parameters. Lymphocytes, monocytes, and granulocytes were identified by simultaneously measuring the light scattering intensity at angles between 1.0 degrees and 2.6 degrees and angles between 3.0 degrees and 11.0 degrees. Eosinophilic granulocytes were distinguished from neutrophilic granulocytes by simultaneous measurement of the orthogonal and depolarized orthogonal light scattering. Comparison of a white blood cell differentiation of 45 donors obtained by the Technicon H-6000 and our instrument revealed good correlations. The correlation coefficients (r2) found were: 0.99 for lymphocytes, 0.76 for monocytes, 0.99 for neutrophilic granulocytes, and 0.98 for eosinophilic granulocytes. The results demonstrate that reliable white blood cell differentiation of the four most clinically relevant leukocytes can be obtained by measurement of light scattering properties of unstained leukocytes.