Microdensitometric Analysis Research Articles

Roughness spectrum and surface plasmons for surfaces of silver, copper, gold, and magnesium deposits

The roughness spectrum $g(\stackrel{\ensuremath{\rightarrow}}{\mathrm{k}})$ (or spectral density function) for surfaces of silver, copper, gold, and magnesium deposits is deduced from surface profiles determined by using microdensitometer analysis of micrographs of shadowed-surface carbon replicas. A process based on computation of autocovariance function via the fast-Fourier-transform algorithm is used. It is shown that the $g(\stackrel{\ensuremath{\rightarrow}}{\mathrm{k}})$ function is not a perfect Gaussian function as is usually assumed. An analytical model is given that provides a rather suitable fit to the computed $g(\stackrel{\ensuremath{\rightarrow}}{\mathrm{k}})$ functions. Results are compared with those obtained from optical experiments (mainly the attenuated-total-reflection method). Discrepancies between the values of $\ensuremath{\delta}$ (rms roughness) and $\ensuremath{\sigma}$ (autocorrelation length) determined by the use of our procedure and corresponding values deduced from optical experiments are elucidated.

Roughness spectrum for surfaces of silver deposits

The roughness spectrum g( k) (or spectral density function) for surfaces of silver deposits is deduced from surface profiles determined by using microdensitometer analysis of micrographs of surface shadowed carbon replicas. We used a process based on the computation of autocovariance function (ACF) via the fast Fourier transform (FFT) algorithm. Results are compared with those provided by attenuated total reflexion (ATR) method. It is shown that the g( k) function is not perfectly gaussian as it is usually assumed.

Height and slope distributions for surfaces of rough metallic deposits

This paper deals with the accurate determination of height and slope distributions for surfaces of rough metallic deposits (magnesium, copper, silver, and gold). These distributions are computed using a microdensitometer analysis of electron micrographs of surface replicas. It is shown that most of the surfaces examined have reasonable Gaussian height and slope distributions. Apart from magnesium surfaces, the rms roughnesses determined from these distributions agree (within the accuracy range of their measurements) with rms roughnesses deduced from the autocovariance functions computed previously. Within the framework of scalar scattering theory, some emphasis is laid on the value of slopes to draw certain conclusions about the validity of the assumptions under which the scalar scattering theory is derived.

Autocovariance functions, root-mean-square-roughness height, and autocovariance length for rough deposits of copper, silver, and gold

Autocovariance functions (ACF's) for rough surfaces of copper, silver, and gold deposits are deduced from surface profiles determined by the use of microdensitometer analysis of surface-shadowed carbon replicas. It is shown that the initial portions of the ACF's have a Gaussian form. The root-mean-square (rms) surface roughness $\ensuremath{\delta}$ and the autocorrelation length $\ensuremath{\sigma}$ are deduced for each surface. Values of $\ensuremath{\sigma}$ agree with corresponding values deduced from the ACF's of the surface slopes. Results of this study are compared with those previously obtained for rough magnesium deposits. We conclude that the Gaussian model is suitable to represent in general the ACF's of rough metallic surfaces. Lastly, it is shown that $\ensuremath{\sigma}$ increases with $\ensuremath{\delta}$, and within the accuracy range of their measurements there is a linear relation between $\ensuremath{\delta}$ and $\ensuremath{\sigma}$.

The determination of the center of gravity of a planet from photographic plates

The difficulty of the determination of the center of gravity of a planet depends on the fact that it has usually a large apparent diameter. On a photographic plate, the size of the image of a planet depends on the exposure time and, mainly, on the focal length. Moreover, the phase angle (Sun-Planet-Earth) makes the image of the planet dissymetric and the surface features-when existing-make its brightness not uniform. For a planet the position of the satellites of which is well known (for example, Jupiter), tests for the determination of the center of gravity have been made using different methods. These methods need a microdensitometric analysis of the plates and allow a better accuracy for the position of the center of gravity of the planet (referred to near stars) than the classical methods using manual measuring machines.

Validity of surface-roughness study using microdensitometer analysis of electron micrographs of surface replicas

We reconstruct the micrograph images from surface profiles previously obtained1 by using microdensitometer analysis of electron micrographs of surface replicas. These micrograph images compare favorably with the original images.

Study of surface roughness using microdensitometer analysis of electron micrographs of surface replicas II: Autocovariance functions

Autocovariance functions (ACF’s) for rough surfaces of magnesium and silver deposits are deduced from surface profiles previously determined by using microdensitometer analysis of micrographs of surface-shadowed carbon replicas. It is shown that initial portions of the ACF’s for magnesium and silver surfaces have a Gaussian form, whereas the ACF for a grating is perfectly periodic. The rms roughness height δ and the autocovariance length σ are deduced for each surface. There is a linear relation between δ and σ for rough deposits of magnesium. For this metal, values of δ and σ are compared with corresponding values of Gesell et al. [ Phys. Rev. B7, 5141 ( 1972)], who have fitted their experimental data to a theoretical expression given by Elson and Ritchie [ Phys. Lett.33A, 255 ( 1970)]. Good agreement is obtained. Finally, the ACF’s of the surface slopes are computed, and it is shown that results obtained are consistent with results deduced from ACF’s for surface profiles.

Study of surface roughness using a microdensitometer analysis of electron micrographs of surface replicas: I Surface profiles

A new method for studying surface roughness is presented that uses a microdensitometer to analyze electron micrographs of shadowed surface replicas. First we give a preliminary description of the replication technique and the microdensitometer analysis. Then we proceed to show that the micrograph density (or transmittance) is approximately proportional to the slope of the surface elements, which enables us to determine the surface profile by integration of the microdensitometer data. Bidimensional and monodimensional median-filtering algorithms are used in the data reduction, and their validity is analyzed. With the help of a computer program that plots a perspective view of rough surfaces, we find that a close approach to the real profiles is obtained. This shows that our method may have other important applications in addition to the study of alterable metallic surfaces.

The early ontogeny of hematopoietic cells studied by grafting cytogenetically labeled tissue anlagen: Localization of a prospective stem cell compartment

The relative contributions of ventral blood island mesoderm and dorsal anterior mesoderm to differentiated lineages of hematopoietic cells was assessed by reciprocal grafting of cytogenetically labeled tissues between 67- and 72-hr-old frog embryos (Shumway stages 15–16). Diploid (2 N) and triploid (3 N) cell populations from hematopoietic organs were distinguished by Feulgen-DNA microdensitometric analysis. Ventral blood island mesoderm appears to contribute an embryonic erythrocyte population that progressively declines during larval development. Dorsal anterior mesoderm appears to contribute a population of precursor cells that gives rise to differentiated lineages of hematopoietic cells found in the thymus, pronephros, mesonephros, spleen, and blood. Histological examination of the developing dorsal anterior area indicates that extensive vascularization is a prominent characteristic of this region. The dorsal aortae and vasculature surrounding the pronephros may be sites where at least one population of hematopoietic cells matures and subsequently enters circulation.

Dye Film Dosimetry for Radiation Processing

Commercially available plastic films containing dyes or dye precursors are convenient dosimeters and imaging media for electron beams or photons used for industrial radiation processing. As grainless imaging systems having thicknesses down to a few micrometers, they provide high spatial resolution for determining detailed absorbed dose distributions through microdensitometric analysis. The radiation absorption properties of these systems are adjusted by changing film composition so that the dosimeter materials can be made to simulate the material of interest undergoing irradiation (e.g., tissue, bone, various insulating materials, semiconductor devices, and a wide assortment of polymers). Other advantages include long-term stability, dose-rate independence, and ease of use and calibration. Radiochromic dye films with thicknesses varying from 0.005 to 1 mm are presently used to monitor electron-beam or gamma-ray doses from 10 to 105, Gy (103 to 107 rad), typical of those encountered in medical applications, radiation curing of polymeric composites, wire and cable insulation, shrinkable plastic tubing and film, as well as sterilization of medical supplies and treatment of municipal and industrial wastes. An NBS calibration service to industry involves the traceability of standard 60Co gamma ray absorbed dose measurements by means of these films employed as transfer standards.

Cellular mechanisms for increased fetal hemoglobin production in culture. Evidence for continuous commitment to fetal hemoglobin production during burst formation.

Using microscopic immunodiffusion assays and microdensitometric analysis of pericellular immunoprecipitate, the percentage of nucleated erythrocytes containing fetal hemoglobin (FNRBC) and the mean picograms of fetal or adult hemoglobin per nucleated erythrocyte (picograms HbF/NRBC, picograms HbA/NRBC) were assayed in 14-d-old colonies (bursts) derived from peripheral blood erythroid progenitors. In the peripheral blood of 11 normal adults only 2.2+/-0.5% (mean+/-SE) erythrocytes contained HbF whereas pooled bursts from the same subjects revealed a 13-fold increase in the percentage of FNRBC (29.6+/-3.9%). In culture both the picograms HbF/NRBC (5.2+/-0.4) and the picograms HbA/NRBC (27.7+/-1.5) are increased approximately 20% above the mean in vivo levels in NRBC from normal bone marrow aspirates. Analysis of each of 58 bursts from one subject demonstrated that FNRBC are present in all bursts and range from 5.0 to 95.0% of the total NRBC per burst. The percent FNRBC in each burst was neither correlated with picograms HbF/NRBC per burst nor with picograms HbA/NRBC per burst. Individual subcolonies from one burst in each of two subjects demonstrated between 3 and 81% FNRBC. These findings indicate that first, the increase in HbF production in culture is primarily due to increased production of the number of cells containing HbF, not to increased picograms HbF/NRBC. Second, all 14-d bursts contain some FNRBC. Third, just as seen in vivo, the picograms HbF/cell and the number of cells that contain HbF are independently regulated in culture. Fourth, commitment to produce HbF in vitro continues after subcolony formation in 14-d-old bursts. Augmentation of HbF production in culture therefore closely resembles that seen in acute erythroid stimulation in vivo.

Flow Cytometric Analysis of Megakaryocyte Ploidy. Comparison With Feulgen Microdensitometry and Discovery That 8N is the Predominant Ploidy Class in Guinea Pig and Monkey Marrow

Flow cytometry (FCM) was adapted to measure ploidy values in megakaryocytes isolated from normal guinea pigs and African green monkeys. The FCM approach was validated by microscopic identification of electronically sorted 8N and higher ploidy cells and by comparisons with Feulgen microdensitometry on the same samples. Sorted 8N populations contained only megakaryocytes and were completely free of cell clumps; clumping was also absent in cell suspensions examined prior to FCM. Discrete megakaryocyte peaks were found at 8N, 16N, and 32N but not at intermediate values. The ploidy patterns of isolated megakaryocytes were similar by both methods (approximately 15% 8N, 58% 16N, and 27% 32N in guinea pigs). Unexpectedly, FCM analysis of unseparated marrow cell suspensions found that the most frequent ploidy class of megakaryocytes was 8N (50%). In contrast, by microdensitometry, the 8N class accounted for only 21% of the megakaryocytes in unseparated marrow cell suspensions. Since the traditional method depends on measurement only of easily recognizable megakaryocyte nuclei, a microdensitometric analysis of unselected marrow cells was carried out. Many more smaller 8N megakaryocytes (48%) were found by this approach, confirming the FCM data and the bias of the traditional sampling method. The decrease in 8N megakaryocytes (50%–15%) with enrichment was accounted for by the consistent finding that cells lost during isolation were predominantly 8N. with relative sparing of the 32N class. FCM provides more rapid and objective analysis of ploidy patterns in much larger samples of megakaryocytes than previous methods.

Flow cytometric analysis of megakaryocyte ploidy. Comparison with Feulgen microdensitometry and discovery that 8N is the predominant ploidy class in guinea pig and monkey marrow

Flow cytometry (FCM) was adapted to measure ploidy values in megakaryocytes isolated from normal guinea pigs and African green monkeys. The FCM approach was validated by microscopic identification of electronically sorted 8N and higher ploidy cells and by comparisons with Feulgen microdensitometry on the same samples. Sorted 8N populations contained only megakaryocytes and were completely free of cell clumps; clumping was also absent in cell suspensions examined prior to FCM. Discrete megakaryocyte peaks were found at 8N, 16N, and 32N but not at intermediate values. The ploidy patterns of isolated megakaryocytes were similar by both methods (approximately 15% 8N, 58% 16N, and 27% 32N in guinea pigs). Unexpectedly, FCM analysis of unseparated marrow cell suspensions found that the most frequent ploidy class of megakaryocytes was 8N (50%). In contrast, by microdensitometry, the 8N class accounted for only 21% of the megakaryocytes in unseparated marrow cell suspensions. Since the traditional method depends on measurement only of easily recognizable megakaryocyte nuclei, a microdensitometric analysis of unselected marrow cells was carried out. Many more smaller 8N megakaryocytes (48%) were found by this approach, confirming the FCM data and the bias of the traditional sampling method. The decrease in 8N megakaryocytes (50%–15%) with enrichment was accounted for by the consistent finding that cells lost during isolation were predominantly 8N. with relative sparing of the 32N class. FCM provides more rapid and objective analysis of ploidy patterns in much larger samples of megakaryocytes than previous methods.

A computer-assisted image-processing system for the analysis of autoradiographs of cerebral metabolic activity.

A computerized image-processing system has been developed for quantitative analyses of the autoradiographs obtained with the [14C]deoxyglucose method. By means of this system, these cerebral metabolic images can be digitized and the resultant data can be manipulated for image construction, enhancement, enlargement, and microdensitometric analysis. It is also possible to generate quantitative color-coded metabolic maps that display the distribution of the actual rates of local glucose utilization throughout the entire central nervous system with a resolution conservatively estimated to be equivalent to that of a full-width-half-maximum of approximately 100 micron.

Computerized densitometry and color coding of [14C] deoxyglucose autoradiographs.

A computerized image processing system has been developed for quantitative analyses of autoradiographs obtained with the [14C]deoxyglucose method. The system is composed of standard, commercially available components and includes a scanning microdensitometer, computer, image memory and display system, and monochrome and color monitors. The associated computer programs are written in PASCAL. Autoradiographs are automatically scanned, and the optical density of each spot is digitized at a maximum resolution of 65,536 readings per 6.4 x 6.4 mm area and stored in memory. Images can be reconstructed from the data in memory, displayed on the monitors, and utilized for microdensitometric analyses or manipulated for image enhancement, enlargement, or weighted averaging of selected regions. The digitized data can also be utilized to solve the operational equation of the [14C]deoxyglucose method, and color-coded images of autoradiographs can be reconstructed so that each color represents a narrow range of the rate of glucose utilization. By means of this system, it is possible to generate quantitative metabolic maps that display the distribution of actual rates of local glucose utilization throughout the entire central nervous system in regions as small as 100 micrometers or less.

Experimental Studies on Hemopoiesis in the Pronephros of Rana pipiens

Embryogenesis of hemopoietic cell populations in the pronephros of Rana pipiens was examined during embryonic and early larval development. Differential cell counts of Wright-Giemsa-stained cell suspensions demonstrated that granulopoiesis is the predominant hemopoietic activity in the pronephros, erythropoiesis accounts for a minor component of the hemopoietic activity (less than 10%), and lymphopoiesis within the organ is negligible. Microdensitometric analysis of Feulgen-DNA stained granulocyte populations in pronephroses from larvae that had received chromosomally labeled pronephric analgen transplants between 84 and 96 h of development demonstrated that hemopoiesis in this organ is dependent on colonization by an extrinsic hemopoietic stem cell. A similar analysis of pronephric hemopoiesis in larvae which had received chromosomally labeled, presumptive ventral blood island transplants between 62 and 67 h of development, indicates that granulopoietic cells are not derived from the embryonic blood islands. It is proposed that the pronephros may be the initial site of granulocyte differentiation during early embryogenesis. Although the embryonic origin of the hemopoietic stem cell is unknown, indirect evidence from this study indicates a dorsal stem cell compartment.

Scanning-integrating microdensitometric analysis of age-related changes in RNA content of cerebrocortical neurons in mice subjected to auditory stimulation

Cytophotometric analyses were made of auditory cortical RNA changes in 10-week and 40-week-old mice subjected to a 30-min interval of continuous noise stimulation (100 dB re 2 × 10 −5 N/m 2, 0.35 to 20 kHz). Treatment groups consisted of young and old noise-exposed mice killed at 0-, 1-, and 4-h intervals after cessation of noise stimulation. Corresponding groups of control mice were not subjected to noise stimulation. Quantification of neuronal RNA changes was made on an individual cell basis using scanning-integrating microdensitometry of azure B-stained brain sections. In the noise-stimulated groups we found that auditory cortical neurons from older mice synthesized and accumulated RNA to the same extent if not more than those of younger mice. The similarity in the pattern and extent of neuronal RNA augmentation in old and young mice after sensory stimulation suggests there is no marked age-related impairment in the capacity of cerebrocortical cells to synthesize RNA. Prior to stimulation we found that older mice had appreciably more neurons with very low RNA values compared to younger mice; older mice also exhibited a more rapid restoration of brain RNA toward control values at the 4-h postexposure interval.

Experimental analysis of hematopoietic cell development in the liver of larval Rana pipiens

Hematopoietic cell development in the liver of Rana pipiens throughout the larval period was examined. Differential counts of Wright/Giemsa-stained cell suspensions demonstrated that hepatic hematopoiesis includes lymphoid, myeloid, and erythroid cell lineages. Hematopoietic activity appeared to be initiated near the end of embryonic development (Shumway stages 24–25) and reached a substantial level by 30 days of development (Taylor and Kollros stage IV). Although lymphopoiesis became highly variable during later larval stages, granulopoiesis remained stable and erythropoiesis increased throughout the larval period. Erythropoiesis is the predominant hematopoietic activity in the larval liver. Histological examination of plastic embedded tissue demonstrated that hematopoietic activity is localized in discrete foci within the endothelial-lined sinusoids. These hematopoietic foci are found in both the subcapsular region and deeper portions of the organ. Microdensitometric analysis of experimental embryos which received chromosomally labeled presumptive ventral blood island transplants at Shumway stage 15 indicated that the derivatives of this embryonic region are limited to the circulating erythrocyte population of the early larvae. The hematopoietic cell populations of the liver appear to be derived from an extrinsic stem cell source which may be located in the dorsal region of the early embryo.

A second look at the ultrastructure of the Langerhans cell of the human epidermis

The ultrastructure of the Langerhans cells of the normal human epidermis was investigated by applying conventional techniques and four standardized zinc iodide—osmium (ZIO) procedures. One of these methods stained the Langerhans cell granule (LCG) selectively. This made it possible to study semithick sections (0.5 to 1 μ m) under the electron microscope, which, in turn, allowed a better understanding of the internal structure of the LCG. This procedure together with a microdensitometric analysis led to the development of a tridimensional model of the LCG. Two components were easily distinguished in the LCG, one disk shaped (LCD) and one a vacuole (LCV). A second ZIO procedure stained tubular formations which were seen to be connected to the LCD. This method also stained the content of the LCV and the vesicles of the Golgi complexes. A third ZIO procedure stained, in addition, the cisternae of the Golgi complexes. The histochemical and microdensitometric analysis of the membrane limiting the LCG revealed that this is a unique type of membrane within the Langerhans cell. The application of the four ZIO procedures to material processed for light microscopy demonstrated histochemical differences between Langerhans cells, melanocytes, and melanin granules of the keratinocytes. The selective staining of the Langerhans cells by one of the ZIO methods was useful to establish the gross morphology of these cells as well as their relationships with the other cell types of the epidermis. The general arrangement of the Langerhans cells suggests that they are functionally polarized and closely related to events taking place in the free surface of the epidermis.

Age related differences in binding of Concanavalin a to plasma membranes of isolated neurons

Neurons isolated from the lateral vestibular nucleus of young adult and senescent Fischer-344 rats were incubated with fluorescamine-labelled Concanavalin A (fl-Con A) alone, or following incubation in trypsin or Vibrio cholerae neuraminidase. They were then observed and photographed. Microdensitometric analysis of fluorescence micrographs showed that senescent rat neurons were significantly more fluorescent than those from young adult rats. Additionally, either patches or caps of fl-Con A were seen on the surface of neurons from senescent rats, while most young adult rat neurons bound fl-Con A uniformly. Pretreatment with trypsin or neuraminidase had no effect on the amount of fluorescence on the surface of senescent rat neurons, and only a slight effect on the surface distribution. Trypsin and neuronal plasma membranes of young adult rats and a rearrangement of the binding pattern in the majority of neurons observed.