Lorenz-Mie light scattering in cellular biology.

The Lorenz-Mie light scattering is discussed as a tool allowing living cell characterization. The scattered light carries information about the size, shape, internal structure and refractive index of the cell. The advantages of light scattering methods consist in high speed, nondestructive, sensitive and relatively easy measurements. Light scattering methods are compatible with other methods. In light scattering in both static and flow systems. For sphere-like cells reliable size and refractive index information can be extracted. On the empirical basis, light scattering pattern can be used for the cell identification and separation purposes. The full utilization of the light scattering information is limited due to the lack of theoretical knowledge about the complex scatterer properties and efficient inversion schemes. The rapid progress in computer technique and in single-particle scattering experiments may significantly improve the interpretation of light scattering patterns of the biological particles.     

Characterization of yeast cultivations by steric sedimentation field-flow fractionation.

The characterization and quantification of biomass is often time consuming and dependent on the cultivation media and gives no detailed information between cell size and shape and their productivity. By monitoring the bioprocess with steric sedimentation field-flow fractionation (Sd/StFFF) in combination with laser light scattering, not only cell growth, but also the variation of cell size and shape during the cultivation, can be observed. In this work, the feasibility of separating and characterizing cell populations by steric sedimentation field-flow fractionation is demonstrated by its application to three different yeast cultivation broths. For this purpose samples which were collected at different cultivation times were injected into an FFF system. Fractograms were obtained in less than 4 min. Due to the relatively high resolution of the method, a cell sample could be fractionated in several subpopulations differing in their size as well as in their number of buds.     

Shape, size, and distribution of cell structures by 3-D graphics reconstruction and stereology. I. The regulatory volume decrease of astroglial cells

This report discusses fundamental limitations in attempting to derive cell size, shape, or distribution from the two-dimensional images provided by conventional electron microscopy. Morphometric or stereologic measurement of random thin sections is a convenient way to obtain some information of this type. However, it cannot provide complete, objective information about real size, shape, or connectivity of cells containing irregular or unevenly distributed structures or nonuniform populations of cells. Anisotropic structures require analysis of a complete set of serial sections. The analysis may utilize either stereo, mono, or tilted optical slices, and subsequent integration of this information into a single 3-D computer data set. In this study, we analyze stereo pairs of high-voltage electron micrographs of serial thick sections (0.5 micron) and critical-point-dried whole-cell mounts of rat brain astroglial cell cultures. The Z-axis resolution is increased by digitizing contours at discrete levels within each stereo view. This is accomplished with a new type of stereoscopic contouring device. We calculated area and volume changes accompanying hypo-osmolar swelling and spontaneous reversal of the swelling. (Regulatory Volume Decrease-RVD). An understanding of the mechanism of swelling of astroglial cells is important for improving the treatment of brain injury. The total cell-volume results are comparable with results previously obtained using nonmetabolized, radioactively tagged compounds that diffuse into various cell compartments. Our serial-section and whole-cell data also provide new information about the relative swelling of nucleus, cytoplasm, and individual organelles such as mitochondria. The basic biological problem being approached is whether homeostasis of cell function is accompanied by surface area and volume regulation of enzyme-rich membranes and organelles. Conversely, it is proposed to explore the possibility that abnormal organelle areas and volumes are indicators of perturbations of cell division, metabolism, or gene expression.     

Shape effects in elastic scattering by orientationally random ensembles of simple model viruses

Recent theoretical work has shown that the complete set of polarized elastic light scattering studies should yield information about particle structure that has so far hardly been utilized. We present calculations of such light-scattering properties for a number of model structures, exploring particularly the size limit at which the new effects should become visible. The particles are assumed to be randomly oriented in aqueous suspension, and all identical to each other. We compare several particle models of differing geometrical shape, but with identical forward scattering power and identical radii of gyration. We find that one of the ten observables shows particularly desirable properties as a general large-particle characterization parameter: it is nonzero for all structures, it approaches zero as particle size decreases, and it shows an angular dependence that distinguishes among models of different shape. Assuming incident light at 350 nm, it differentiates between different shaped particles with radii of gyration as small as 50 nm. Such particles are well below the optical resolution limit and about the size of many types of viruses.     

Application of optical diffractometry in studies of cell fine structure. Comparison of arterial smooth muscle cells in contractile and synthetic state

Arterial smooth muscle cells in contractile and synthetic state were analyzed by optical diffractometry. Cell sections (80-90 nm) were photographed in an electron microscope and diffraction patterns of the plates (negatives) were produced using a helium-neon laser. Radial and angular distributions of light intensity in the diffractograms were measured and digitized using an electronic detector plate consisting of ring- and wedge-shaped photosensitive elements; radial distributions provide information about size of structures and distances between them and angular distributions about spatial orientation of structures in the images. Micrographs of nuclei and cytoplasm were analyzed separately (40-50 plates in each group). Computerized statistical analysis of radial distributions of light intensity showed that the nuclear chromatin pattern differed between cells in contractile and synthetic state. The probability that the observed difference could have arisen purely by chance was less than 10(-5). Computer-aided classification to the a priori known cell group was correct in 96.5% of the cases. Analysis of radial distributions of light intensity similarly showed marked differences in cytoplasmic structure between cells in contractile state (dominated by bundles of myofilaments) and synthetic state (dominated by cisternae of rough endoplasmic reticulum). The probability that the observed difference could have arisen purely by chance was less than 10(-5). Computer-aided classification to the a priori known cell group was correct in 92.0% of the cases. In contrast, analysis of angular distributions of light intensity did not indicate any statistically significant differences between contractile and synthetic state cells. A likely reason is that both myofilaments and cisternae of rough endoplasmic reticulum were arranged in parallel. The results demonstrate that optical diffractometry is a useful method for image analysis in studies of cell fine structure. It provides information about size and orientation of structures with poorly defined shape and is particularly well suited for studies on cell differentiation and effects of pharmacological and other experimental treatments on cell fine structure. It represents an alternative and a complement to stereology for quantitative and objective evaluation of morphological data.     

The influence of photometer design on optical-conformational changes.

When cells and large subcellular structures suffer a change in volume or internal structure, their light-scattering properties are normally altered. These optical-conformation changes are potential sources of information about conformation and processes which alter it. Classical light-scattering theory for spherical particles is used to determine how the transmittance or extinction of a cell suspension should respond when such a conformational change occurs and the measurements are made with a conventional photometer. This extends an earlier study of transmittances measured with an “ideal” photometer. The photocell of an ideal instrument collects only the directly transmitted light. In a conventional instrument it also collects the light scattered at small angles, which is usually most of the scattered light. Extinction (optical density, absorbance) of suspensions of spherical cells was computed for several photometer designs. It is found that γ, the angle of acceptance of the photocell, has a significant influence on the extent and even the nature of the photometric response to a given conformational change. Earlier, it was shown that a decrease in cell volume or increase in internal structure will increase extinction for cells of many sizes. Now it is found that a large γ-value increases these effects. An approach to the interpretation of transmitted light fluxes in terms of theoretical predictions is outlined.     

Heterogeneity among dog red blood cells

A phthalate density-separation technique has been used to study the heterogeneity of dog red blood cells that becomes manifest when they are suspended in KCl media. It is demonstrated that the proportions of cells that separate into light and dense fractions can be varied by altering the tonicity of the KCl medium. This results from the fact that the Na and K permeabilities of each cell are continuous functions of cell volume. It was found that quinidine inhibits selectively the volume dependence of Na permeability. In the presence of this drug, the heterogeneity demonstrated by KCl incubation disappears. The notion that dog red blood cells are heterogeneous in their permeabilities to Na and K is thus upheld, but the heterogeneity is not an abruptly discontinuous one, as has been claimed. A sample of dog blood does not contain two discrete populations of red cells.     

Shape,size, and distribution of cell structures by 3-D graphics reconstruction and stereology

This report discusses fundamental limitations in attempting to derive cell size, shape, or distribution from the two-dimensional images provided by conventional electron microscopy. Morphometric or stereologic measurement of random thin sections is a convenient way to obtain some information of this type. However, it cannot provide complete, objective information about real size, shape, or connectivity of cells containing irregular or unevenly distributed structures or nonuniform populations of cells. Anisotropic structures require analysis of a complete set of serial sections. The analysis may utilize either stereo, mono, or tilted optical slices, and subsequent integration of this information into a single 3-D computer data set. In this study, we analyze stereo pairs of high-voltage electron micrographs of serial thick sections (0.5 μm) and critical-point-dried whole-cell mounts of rat brain astroglial cell cultures. The Z-axis resolution is increased by digitizing contours at discrete levels within each stereo view. This is accomplished with a new type of stereoscopic contouring device. We calculated area and volume changes accompanying hypo-osmolar swelling and spontaneous reversal of the swelling. (Regulatory Volume Decrease-RVD). An understanding of the mechanism of swelling of astroglial cells is important for improving the treatment of brain injury. The total cell-volume results are comparable with results previously obtained using nonmetabolized, radioactively tagged compounds that diffuse into various cell compartments. Our serial-section and whole-cell data also provide new information about the relative swelling of nucleus, cytoplasm, and individual organelles such as mitochondria. The basic biological problem being approached is whether homeo-stasis of cell function is accompanied by surface area and volume regulation of enzyme-rich membranes and organelles. Conversely, it is proposed to explore the possibility that abnormal organelle areas and volumes are indicators of perturbations of cell division, metabolism, or gene expression.     

Segmentation of stained blood cell images measured at high scanning density with high magnification and high numerical aperture optics

In hematological morphology, it is necessary to resolve and analyze the smallest possible cellular details appearing in the light microscope. A prerequisite for computer-aided analysis of subtle morphological features is measuring the cells at a high scanning density with high magnification and high numerical aperture optics. Contrary to visual observations, the information content in a measured picture can be increased by setting the condensor's numerical aperture (NA) greater than the objective's NA. The complexity and heterogeneity of such cell images necessitate a new segmentation method that conserves the morphological information required in the subsequent image analysis, feature extraction, and cell classification. In our segmentation strategy, characteristic color difference thresholds for each nucleus and cytoplasm are combined with geometric operations, probability functions, and a cell model. All thresholds are repeatedly recalculated during the successive improvements of the image masks. None of the thresholds are fixed. This strategy segments blood cell images containing touching cells and large variations in staining, texture, size, and shape. Biological inconsistencies in the calculated cell masks are eliminated by comparing each mask with the cell model criteria integrated into the entire segmentation process. All 20,000 leukocyte images from 120 smears in our leukemia project were segmented with this method.     

Multiangle light scattering flow photometry of cultured human fibroblasts: comparison of normal cells with a mutant line containing cytoplasmic inclusions.

Multi-angle light scattering flow photometry was used to study the light scattering properties of normal cultured fibroblasts and a mutant fibroblast line containing cytoplasmic lysosomal inclusions. The effect of glutaraldehyde fixation on the light scattering properties of the cells was also examined and correlated with their ultrastructure. Normal fibroblasts showed uniform organelle distribution with few vacuoles or dense bodies in the cytoplasm while the mutant line showed abnormal cytoplasmic inclusions of varying morphology, density and lucency. As predicted by light scattering theory, the mutant cells containing the cytoplasmic inclusions scattered more light at large angles (greater than theta = 1.85 degrees) than did the normal cells. Glutaraldehyde fixation decreased light scattering at small angles (less than theta = 1.85 degrees), increased light scattering at larger angles (greater than theta = 1.85 degrees) in both normal and mutant cells and enhanced resolution of the light scattering signatures. The mutant line scattered 2-3 times more light at a wide angle (greater than theta = 12.74 degrees) than did the normal cells. These data suggest that abnormal lysosomal storage inclusion bodies in the cytoplasm of the cells can be detected by differential light scattering methods.     

A quasi-elastic light scattering and cinematographic investigation of motile Chlamydomonas reinhardtii.

Quasi-elastic light scattering and cinematographical techniques were used to investigate the motility of Chlamydomonas reinhardtii (wild type). It was found that quantitative information on the trajectory of motion was required for a meaningful interpretation of the autocorrelation functions. Two models for describing the oscillatory motion of the cell were developed; one based on the instantaneous forward-and-backward motion of the cell, and the other based on a sinusoidal perturbation to the average forward motion. Both models gave satisfactory agreement with the shape of the experimentally measured autocorrelation function, thus making it possible to use this measurement to determine mean progressive swimming velocities in a population of greater than 200 cells.     

Cellular Heterogeneity in the Late-onset Form of Hereditary Melanomas in the Xiphophorus Fish Hybrids

The late-onset form of melanomas occurring in the Xiphophorus , fish hybrids carrying a macro-melanophore gene Sp was investigated for its cellular heterogeneity. The melanoma tissues were dissociated enzymatically and cultured for a short term. The cultured melanoma cells were characterized according to cell size, cell shape, pigmentation, and response to epinephrine. The melanoma cells were considerably heterogeneous in these phenotypic traits. Various combinations of these heterogeneous cells gave a great heterogeneity to individual melanomas. The stability of the phenotypic traits was followed during the course of tumor growth. Cell size and cell shape were stable, but pigmentation and response to epinephrine varied. The results are discussed in relation to cell differentiation and tumor progression.     

Growth behavior of microorganisms using UV-Vis spectroscopy: Escherichia coli

Multiwavelength transmission spectra of microorganisms and cell suspensions consist of combined absorption and scattering phenomena resulting from the interaction of light with microorganisms or cells typically suspended in a nonabsorbing media. The distribution of intensities as a function of wavelength depends on the size, shape, and optical properties of the sample. The optical properties are functions of the chemical composition and the state of aggregation, or association, of the chromophoric groups contained in the microorganisms. This article explores the growth behavior of Escherichia coli from the perspective of multiwavelength UV-Vis spectroscopy. Experimentally, it is demonstrated that the spectral signatures of the microorganism evolve as a function of time. It is also demonstrated that the spectral changes observed during growth are consistent with data reported elsewhere. From the theoretical point of view, it is demonstrated that the spectral signatures can be adequately represented with an interpretation model based on light-scattering theory. The parameters from the interpretation model reflect changes in size and chemical composition known to take place in the microorganisms during growth.     

Quantitative spectroscopy analysis of prokaryotic cells: vegetative cells and spores

Multiwavelength ultraviolet/visible (UV-Vis) spectra of microorganisms and cell suspensions contain quantitative information on properties such as number, size, shape, chemical composition, and internal structure of the suspended particles. These properties are essential for the identification and classification of microorganisms and cells. The complexity of microorganisms in terms of their chemical composition and internal structure make the interpretation of their spectral signature a difficult task. In this paper, a model is proposed for the quantitative interpretation of spectral patterns resulting from transmission measurements of prokaryotic microorganism suspensions. It is also demonstrated that different organisms give rise to spectral differences that may be used for their identification and classification. The proposed interpretation model is based on light scattering theory, spectral deconvolution techniques, and on the approximation of the frequency dependent optical properties of the basic constituents of living organisms. The quantitative deconvolution in terms of the interpretation model yields critical information necessary for the detection and identification of microorganisms, such as size, dry mass, dipicolinic acid concentration, nucleotide concentration, and an average representation of the internal scattering elements of the organisms. E. coli, P. agglomerans, B. subtilis spores, and vegetative cells and spores of Bacillus globigii are used as case studies. It is concluded that spectroscopy techniques coupled with effective interpretation models are applicable to a wide range of cell types found in diverse environments.     

Cellular senescence and the analysis of generation time distributions.

Generation time analysis by time lapse cinematography is an important method for investigating cellular senescence in culture, but its interpretation is complicated by several types of bias and artifact, including small sample size, cut-off bias, changes in global growth rate, and phase of the population growth cycle. When these factors are considered, interpretation of the data base used by previous investigators changes considerably, and does not reveal any differences in growth behavior between middle and late passage WI-38 cells. Nor does it support the transition probability theory either of cell cycle transit or of culture senescence.     

Fluorescence-based staining for tartrate-resistant acidic phosphatase (TRAP) in osteoclasts combined with other fluorescent dyes and protocols.

Osteoclasts are the only bone-resorbing cells. In addition to other specific properties, osteoclasts are characterized by their expression of tartrate-resistant acidic phosphatase (TRAP), which is usually detected using a histochemical method for light microscopy. Using ELF97 phosphatase substrate, this study describes a new fluorescence-based method for TRAP detection. The fluorescence-based ELF97 TRAP stain not only results in a better resolution of the TRAP-positive granules, because confocal microscopy can be applied for image acquisition and analysis, but it reveals additional and more specific information about osteoclasts because it can be combined with other fluorescence-based methods.     

Endocrine cells in the gastric mucosa of elasmobranch fishes. An ultrastructural study

The endocrine cells of gastric mucosa of two elasmobranch species were studied by light and electron microscopy. Five cell types were identified in the fundic mucosa, four of which are of "open type". All of them show pleomorphic granules of variable size, except those of the type V cell which are round in shape and of comparatively small diameter. Six different cell types are found in the pyloric mucosa, all of "open type" except for type XI cells which appear to be "closed". Pyloric types VIII, IX, X and XI cells show similar structural characteristics as fundus types I, V, II and IV respectively. Silver impregnation was also used at both light and electron microscopical levels. No functional classification or analogies with other vertebrate gastric endocrine cells were attempted as these would be too speculative on the basis of ultrastructural characteristics only.     

Research progress on black phosphorus hybrids hydrogel platforms for biomedical applications

Cell migration is a pivotal biological process, whose dysregulation is found in many diseases including inflammation and cancer. Advances in microscopy technologies allow now to study cell migration in vitro, within engineered microenvironments that resemble in vivo conditions. However, to capture an entire 3D migration chamber for extended periods of time and with high temporal resolution, images are generally acquired with low resolution, which poses a challenge for data analysis. Indeed, cell detection and tracking are hampered due to the large pixel size (i.e., cell diameter down to 2 pixels), the possible low signal-to-noise ratio, and distortions in the cell shape due to changes in the z-axis position. Although fluorescent staining can be used to facilitate cell detection, it may alter cell behavior and it may suffer from fluorescence loss over time (photobleaching).

Here we describe a protocol that employs an established deep learning method (U-NET), to specifically convert transmitted light (TL) signal from unlabeled cells imaged with low resolution to a fluorescent-like signal (class 1 probability). We demonstrate its application to study cancer cell migration, obtaining a significant improvement in tracking accuracy, while not suffering from photobleaching. This is reflected in the possibility of tracking cells for three-fold longer periods of time. To facilitate the application of the protocol we provide WID-U, an open-source plugin for FIJI and Imaris imaging software, the training dataset used in this paper, and the code to train the network for custom experimental settings.

     

Vesicle size distributions measured by flow field-flow fractionation coupled with multiangle light scattering.

The separation method, flow field-flow fractionation (flow FFF), is coupled on-line with multiangle laser light scattering (MALLS) for simultaneous measurement of the size and concentration of vesicles eluting continuously from the fractionator. These size and concentration data, gathered as a function of elution time, may be used to construct both number- and mass-weighted vesicle size distributions. Unlike most competing, noninvasive methods, this flow FFF/MALLS technique enables measurement of vesicle size distributions without a separate refractive index detector, calibration using particle size standards, or prior assumptions about the shape of the size distribution. Experimentally measured size distributions of vesicles formed by extrusion and detergent removal are non-Gaussian and are fit well by the Weibull distribution. Flow FFF/MALLS reveals that both the extrusion and detergent dialysis vesicle formation methods can yield nearly size monodisperse populations with standard deviations of approximately 8% about the mean diameter. In contrast to the rather low resolution of dynamic light scattering in analyzing bimodal systems, flow FFF/MALLS is shown to resolve vesicle subpopulations that differ by much less than a factor of two in mean size.