Histogram Normalization Research Articles

Reduction of obscuration noise using multiple images

Noise that replaces, rather than perturbs, a signal is not amenable to the usual approaches of noise removal. Here, multiple images of a stationary scene are used to reduce random obscuration or dropout noise by the following method. For each image coordinate, a gray-level initial histogram is computed over all the images. The initial histograms are averaged for all image coordinates, and for each image coordinate a gray-level histogram corresponding to the object is derived by subtracting the averaged histogram from the initial one. The gray level of the object is obtained from the resulting object histogram. The effectiveness of this method is confirmed through experiments using a scene obscured by air bubbles in a water tank.< <ETX xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">&gt;</ETX>

Effect of drug efflux blockers on vital staining of cellular DNA with hoechst 33342

The present study shows that staining of certain live cells, e.g., adriamycin-resistant P388 cells, by Hoechst 33342 is difficult because of the presence of a rapid efflux pump, which reduces intracellular dye concentration. Coincubation of these refractory cells in the presence of efflux blockers such as phenothiazines (trifluoperazine) or Ca++ channel blockers (verapamil) enhances dye retention and thus leads to generation of normal DNA distribution histograms. Laser flow cytometric data is confirmed by fluorometric assays, which show that P388/R cells retain one-third the amount of Hoechst 33342, and coincubation with efflux blockers increases Hoechst retention to values similar to those of drug-sensitive P388 cells. DNA histograms of mouse splenocytes incubated with Hoechst 33342 alone have a bimodal distribution possibly because of the presence of subpopulations that do not retain the fluorochrome owing to rapid efflux. Coincubation with an efflux blocker results in the generation of unimodal DNA histograms from these cells. These preliminary studies suggest that reduced retention of Hoechst 33342 in certain cell types (because of rapid efflux) can be blocked by efflux blockers, thus leading to generation of typical DNA distribution histograms.

Wilms Tumors: Relationship of Nuclear Deoxyribonucleic Acid Ploidy to Patient Survival

Nuclear deoxyribonucleic acid ploidy studies with paraffin-embedded archival tumor specimens were performed by flow cytometry on extracted nuclei from 56 Wilms tumors. Before the era of chemotherapy 9 patients had a 33 per cent survival rate at 5 years. No significant correlation between deoxyribonucleic acid ploidy pattern and survival was seen in this early group of patients. Since 1960, 47 patients underwent radical nephrectomy and received chemotherapy. Deoxyribonucleic acid histograms in this group were normal in 13 tumors, while 23 tumors exhibited a tetraploid pattern (greater than 10 per cent nuclei in the G2 peak) and 11 showed an aneuploid pattern. Survival rates according to deoxyribonucleic acid ploidy pattern showed that 100 per cent of the patients with a normal or aneuploid histogram pattern were alive at 2 and 5 years. Patients with a deoxyribonucleic acid tetraploid pattern had a significantly worse survival of 74 and 69 per cent at 2 and 5 years (p less than 0.02 and less than 0.01), respectively. Tumors with a normal or aneuploid histogram pattern for all stages and a deoxyribonucleic acid tetraploid pattern for pathological stage I or II were associated with a 2 and 5-year survival rate of 100 and 97 per cent, respectively. However, patients with stages III and IV tumors with a deoxyribonucleic acid tetraploid pattern had a significantly worse survival of 25 per cent at 2 and 5 years (p less than 0.0001). Measurement of nuclear deoxyribonucleic acid ploidy identifies a patient group who are at high risk for treatment failure, that is stages III and IV tumors with a deoxyribonucleic acid tetraploid pattern.

Well Differentiated Clear Cell Renal Carcinoma: Significance of Nuclear Deoxyribonucleic Acid Patterns Studied by Flow Cytometry

AbstractNuclear deoxyribonucleic acid ploidy was determined in 206 samples of well differentiated clear cell renal carcinoma via a previously described technique on paraffin-embedded archival material Grade 2 tumors had a higher incidence of abnormal deoxyribonucleic acid patterns than grade 1 tumors. Of stage 1 well differentiated clear cell renal tumors 60 per cent had a normal deoxyribonucleic acid histogram pattern, whereas 67 per cent of higher stage tumors had an abnormal pattern. The existence of abnormal nuclear deoxyribonucleic acid histogram patterns in the primary tumor tissue had a significant correlation with subsequent development of metastatic disease, independent of tumor grade and stage. The 10-year actuarial survival rate was 62 per cent for patients with normal deoxyribonucleic acid histograms and 37 per cent for patients with abnormal patterns. These results demonstrate that nuclear deoxyribonucleic acid ploidy measured by flow cytometry is an important variable in the classification and determination of prognosis for patients with clear cell renal carcinoma.

Flow Cytometry of Renal Oncocytoma: Common Occurrence of Deoxyribonucleic Acid Polyploidy and Aneuploidy

Flow cytometry was performed on 51 typical specimens of renal oncocytoma. Nuclei were extracted from paraffin-embedded archival material and isolated nuclei were stained with propidium iodide. Of the 51 available tissue blocks 86 per cent were evaluable and 50 per cent of these samples showed a deoxyribonucleic acid (DNA) histogram that was approximately the same as normal renal parenchyma. Of the oncocytoma samples 39 per cent showed a marked increase (more than 10 per cent of the nuclei) in the tetraploid DNA peak, while 11 per cent showed a distinct DNA aneuploid peak. Among 21 evaluable grade 2 oncocytic renal tumors 33 per cent showed a normal DNA histogram, 43 per cent showed a marked increase in the DNA tetraploid peak and 24 per cent showed a DNA aneuploid peak. The common presence of polyploid nuclei containing double quantities of chromosomal DNA may correlate with the long-standing pathological observation that oncocytic tumors often contain a distinct population of large nuclei. Indeed, 86 per cent concurrence was seen between the detection of an abnormal DNA content by flow cytometry and the histopathological presence of large abnormal nuclei in these specimens. Since renal oncocytomas (grade 1 oncocytic tumors) rarely, if ever, metastasize and are relatively noninvasive locally, their markedly abnormal flow cytometry patterns are of considerable interest. Moreover, DNA polyploidy has not been identified previously in renal tumors. The biological significance and mechanism of DNA polyploidy, and the relationship of DNA polyploidy and DNA aneuploidy to the pathogenesis of oncocytic renal tumors require further laboratory investigation. The clinical use of flow cytometry to classify and to predict the behavior of renal tumors will be complicated, since renal oncocytomas commonly have polyploid and aneuploid DNA histograms

Simple cells in cat striate cortex: responses to stationary flashing and to moving light bars

Cells in the simple family respond to a moving light bar with an average response histogram that is most commonly unimodal (single peak: encounter frequency, 64%) and less commonly bimodal (33%) or trimodal (3%). The mean width of the principal response peak given by hypercomplex I cells is narrower than that of simple cells and they have a lower mean optimal stimulus velocity. In a series of 74 cells (simple, 47; hypercomplex I, 27), detailed comparison of the spatial relations between the response peaks to the moving bar and the subregions to the stationary flashing bar led to the concept of a boundary response. The term "boundary response" refers to an isolated response peak occurring as a moving light bar leaves an OFF subregion that is the last in the sequence of subregions traversed by the bar. The presence of a boundary response leads to an apparent discrepancy between the number of response peaks to a moving light bar and the number of ON subregions in the static-field plot. The boundary response is necessarily completely direction selective. A detailed comparison of the properties of cells as revealed by hand and quantitative methods showed a very good agreement between the two methods in respect to the assignment of cells to the simple, B- and complex cell families. There were, however, serious discrepancies in respect to the receptive field organization of cells in the simple family.(ABSTRACT TRUNCATED AT 250 WORDS)

Very small fat cell populations determined by a modified osmium tetroxide-urea method.

To facilitate investigations on very small fat cell (VSFC) populations in adipose tissue, an alternate method of preparing fat tissue samples was explored. The osmium tetroxide-8M urea method, modified by addition of a 95% ethanol step in tissue processing, centrifugation between steps, and final resuspension in 55% glycerol in 0.01% Triton-saline, was compared with the collagenase method for determination of VSFC populations in Fischer 344 epididymal and Sprague-Dawley retroperitoneal adipose depots. For each method and in both depots, the average histogram of 300 adipocyte diameters, measured by microscopy, was bimodal with the nadir between 30 and 40 micron diameter. The average histogram of fat cells less than 35 micron in diameter showed a separate population of VSFC existed in each depot. The modified osmium-urea method gave better results and was easier to perform than the collagenase method. It has confirmed our earlier results and raises anew questions concerning a role for the natural existence of a VSFC population in the adipose depot.

Early diagnosis of cutaneous T-cell lymphoma by DNA flow cytometry on skin biopsies.

Seven patients with mycosis fungoides early plaque stage with nondiagnostic histology had single-cell DNA content measured by flow cytometry for estimation of clonal ploidy. A total of 63 skin specimens were examined by histology and DNA measurements concurrently during the course of the disease. In addition, six patients had blood samples studied. All seven patients demonstrated aneuploid DNA histograms when the specimens were obtained from skin lesions. In 36 specimens the aneuploid peaks were hyperdiploid. By sequential studies one patient demonstrated two different aneuploid cell clones, one located in the hyperdiploid region and one located in the hypotetraploid region. All patients developed mycosis fungoides which were histologically confirmed, and the time from first aneuploid DNA histogram until diagnostic histology varied from 5 to 21 months (median, 12 months). In six of the patients a normal diploid DNA histogram was found of peripheral blood lymphocytes. The finding of aneuploidy in patients with early mycosis fungoides who still have a nondiagnostic histology emphasizes the value of flow cytometry as a complementary diagnostic aid which facilitates an early diagnosis.

Steady state condition--what does it mean during investigation of oxygen supply?

Judging from the presented data it was possible to maintain steady state conditions during high dose piritramide anesthesia for a period of three and a half hours. Under these carefully defined experimental conditions it will be possible to actually correlate any changes observed in the microcirculatory bed with medication or other interventions. So-called "normal Po2 histograms" taken from different organs of dogs under the above-defined experimental conditions (basic anesthesia with high-dose piritramide, controlled artificial ventilation with 21% O2) are presented in Fig. 12. We feel that any comparison of results from different working groups must be based on exactly defined and standardized experimental conditions, because otherwise the methodological differences would prevent adequate comparability.

DNA analysis by flow cytometry in lymphomatoid papulosis

Summary DNA analysis by flow cytometry revealed normal DNA histograms in three patients (seven skin specimens) with ‘classical’ lymphomatoid papulosis, A fourth patient, also with a‘classical lynphomatoid papulosis, had an increased number of tetraploid nuclei (14%, and 28%). The fifth patient with recurrent tumours, no papules and a more malignant histology showed ancuploidy by sequential examinations (hyperdiploidy and hypertetraploidy) of three skin specimens.

Aneuploidy in cutaneous T-cell lymphoma

A new method is described which makes it possible using skin specimens to perform flow cytometric analysis of DNA content. DNA content analysis was performed on 28 skin specimens and 9 blood samples from 18 patients with mycosis fungoides and Sézary syndrome. The reproducibility was fair, with almost identical results in 6 cases (mycosis fungoides and Sézary syndrome) where two samples (skin specimens or blood samples) were taken 6 hours to 10 days apart. Hyperdiploidy was found in 7 of 11 skin specimens from patients with mycosis fungoides stage I with negative histology. In 13 skin specimens and 3 blood samples from patients with mycosis fungoides stages II and IV, abnormalities including hyperdiploidy, near-tetraploidy and near-hexaploidy were found in 8 of 13 skin specimens and in 2 of 3 blood samples. Four patients with Sézary syndrome were studied: 2 patients in remission showed normal DNA histograms (2 skin specimens, 3 blood samples) and 2 patients with active disease showed aneuploidy in the 2 skin specimens examined and in 1 of 3 blood samples. These studies demonstrate: 1) the importance of flow cytometry as a diagnostic tool for use on skin specimens in the early stages of mycosis fungoides where routine histology is non-diagnostic; 2) the diagnostic and prognostic aid of flow cytometry during the course of mycosis fungoides and Sézary syndrome in addition to the probability of measuring the effect of treatment.

Techniques for minimizing radiometric striping effects in Landsat data

Histogram normalization and statistical methods which are implemented at National Remote Sensing Agency, for minimizing radiometric distortions due to striping in the Landsat data are presented in this paper. Also the paper discusses the method of implementation of the above two methods on Multispectral Data Analysis System (M-DAS).

Effect of increased blood oxygen affinity on skeletal muscle surface oxygen pressure fields.

A chronic left displacement of the blood O2 dissociation curve (ODC) was achieved in rats by administering cyanate over a period of 14 days. Control rats received NaCl. An acute left displacement of the ODC was achieved by exchange transfusion with bisulfite-treated erythrocytes. Control rats for this series received an exchange transfusion with fresh heparinized blood. In both series, skeletal muscle surface O2 pressure fields (expressed as PO2 histograms) were measured with the rats anesthetized, curarized, and artificially ventilated. The animals with chronically left-shifted ODC had normal PO2 histograms (for definition see DISCUSSION) when breathing air; during hypoxia (FIO2 0.12) four of the eight experimental and three of the seven control animals developed abnormal histograms, and all animals had normal histograms on return to air breathing. The majority, but not all, of the animals that were to receive exchange transfusions of left-shifted ODC blood had normal histograms before the transfusion, which caused some to become abnormal and others to become normal. Similar results were obtained in the control animals that received normal blood. The results do not provide evidence for an adverse effect of a left-shifted ODC on muscle tissue oxygenation.

Discrimination of a chestnut‐oak forest unit for geologic mapping by means of a principal component enhancement of Landsat multispectral scanner data

A principal component image enhancement has been effective in applying Landsat data to geologic mapping in a heavily forested area of eastern Virginia. A chestnut‐oak forest unit, which occurs on metavolcanic rocks and some metaclastic rocks in the western Piedmont and on highly weathered upland gravel deposits in the eastern Piedmont, can be discerned on a digitally enhanced Landsat winter image. The image enhancement procedure consists of a principal component transformation, a histogram normalization, and the inverse principal component transformation. The enhancement preserves the independence of the principal components, yet produces a more readily interpretable image than does a single principal component transformation.To determine how the chestnut‐oak forest unit was being enhanced, average Landsat multispectral scanner (MSS) values were extracted for four sample forest types and were calculated through the inverted principal component transformation. Slope and intercept values for the linear histogram normalization were chosen to keep the scale between the raw MSS bands and the inverted principal component (PC) bands constant. Plots of the inverted principal component data show that the most separation between forest types is in inverted PC band 5. The chestnut‐oak forest unit is characterized by a high value for inverted PC band 5 as opposed to a low value for inverted PC band 4. In contrast, raw MSS band 4 is greater than MSS band 5 for the four forest types sampled in the winter image. Such observations cannot be readily deduced from analysis of only the principal component rotation matrix. They may ultimately provide a means to map the distribution of chestnut‐oak forest from Landsat.

Skeletal muscle oxygen pressure fields in artificially ventilated, critically ill patients.

The MDO (Mehrdraht Dortmund oberfläche) oxygen electrode was used in a study of skeletal muscle oxygen pressure fields, presented as histograms, in critically ill patients artificially ventilated with gas mixtures of different oxygen concentrations. The histograms were compared with forearm blood flow measurements performed with strain gauge plethysmography. Local blood flow and permeability-surface area product (PS) were also studied by the simultaneous clearances of 133xenon and 51Cr-EDTA. The histogram distribution type was normal, i.e. approximately Gaussian, at arterial oxygen pressure levels between 10 and 18 kPa. At arterial oxygen pressures outside this range the histogram distribution types were abnormal, i.e. they showed a non-symmetrical distribution of oxygen pressure values, but their mean was approximately the same as in the normal histogram. However, there were significantly higher tissue oxygen pressure mean values in the patients (3.43 kPa) than in a group of healthy human volunteers (2.25 kPa). Mean forearm blood flow and the clearances of 133xenon and 51Cr-EDTA showed marked variations during the measurements both intraindividually and interindividually. Mean forearm blood flow and mean clearances of 133xenon showed opposite trends compared with arterial oxygen pressures. Mean clearances of 51Cr--EDTA and means PS showed only minor variations at the different arterial oxygen pressure levels.

Evaluation of automated whole-blood platelet counts and particle sizing.

The Coulter Counter model S-Plus measures the erythrocyte, leukocyte, and platelet counts, and the mean cell volume of erythrocytes and platelets. Two additional features are novel to the instrument: erythrocyte distribution width (RDW), an index of erythrocyte volume heterogeneity, and mean platelet volume. For 677 subjects, we compared these measurements with complete blood counts obtained by a standard instrument, platelet counts from platelet-rich plasma (PRP), and histograms of erythrocyte and platelet volumes generated by free-standing equipment. The two methods correlated well (r = 0.97) for the complete blood count and the mean platelet volume. The whole-blood platelet count correlated well (r = 0.97) with the PRP platelet count, with whole-blood counts on average 5% higher than PRP counts. The whole-blood index of erythrocyte volume heterogeneity, RDW, correlated less well (r = 0.81) with the erythrocyte coefficient of variation measured by histogram. RDW was abnormal for three of 100 normal subjects with normal histograms. RDW was less sensitive (78% vs. 97%) and less specific (94% vs. 97%) than erythrocyte histograms in distinguishing iron deficiency from heterozygous alpha- or beta-thalassemia. We conclude that the Coulter Counter Model S-Plus reliably measures complete blood cell count, platelet count, and mean cell volumes, but analyzes erythrocyte volume distribution less well than does a histogram.

Improved tissue perfusion (Po2 histograms) in extracorporeal circulation using membrane instead of bubble oxygenators

We made an experimental study of dogs undergoing extracorporeal circulation and used bubble oxygenators (BOs) (N = 8) or membrane oxygenators (MOs) (N = 7). Hematologic changes were related with alterations in tissue oxygen supply, which reflect microcirculatory changes. The tissue oxygen supply was measured with an eight-wire P o 2 electrode on the surface of the sartorius muscle. From 104 P o 2 values a frequency distribution curve (P o 2 histogram) was computed. Hematologic studies, in particular platelet numbers and function (adenosine diphosphate-induced aggregation), were performed. The extracorporeal circuit was primed with about 1 L of homologous blood. A momentary fall of platelet numbers and function occurred after the start of partial bypass (400 ml/min). Arterial pressures frequently showed an initial rise followed by a severe but transient drop in pressure, while the continuously monitored muscle surface P o 2 dropped to zero for a longer period. During total bypass, tissue oxygen supply was restored and was sufficient in both the BO and MO groups, as indicated by the improved P o 2 histograms (means between 3.45 and 4.50 kPa). After extracorporeal circulation a clear distinction appeared between the BO and the MO groups. In the BO group impaired P o 2 histograms (mean 1.83 ± 0.53 kPa) were observed, which coincided with a high systemic vascular resistance (mean 0.69 ± 0.14 kPa min/L/kg), low cardiac output (71 ± 9 ml/min/kg), and decreased platelet numbers (33.10 9 /L ± 16) and function (11% ± 6%). In comparison, the MO group had normal histograms (mean 5.45 ± 0.82 kPa, p < 0.05), normal systemic vascular resistance values (0.42 ± 0.07 kPa min/L/kg, N.S.), normal cardiac output (101 ± 12 L/min/kg, p < 0.05), and better maintained platelet numbers (128.10 9 /L ±23, p < 0.0005) and function (30% ± 11, p < 0.01). In this study, advantages of MO over BO perfusion could be quantitated in terms of better tissue oxygen supply after extracorporeal circulation, correlating with a better hemocompatibility. P o 2 muscle surface histograms appeared to be a reliable method to evaluate tissue oxygen supply, which reflects tissue perfusion.

Microfluorimetric scanning of sympathetic nerve fibers: quantification of neuronal and extraneuronal fluorescence with the aid of histogram analysis.

A high resolution (0.5 micrometer), two-dimensional microfluorimetric scanning procedure was used to quantitate the formaldehyde-induced fluorescence of noradrenaline in the iris of the rat. Scanning of large areas (50 x 50 micrometer) in the sympathetic innervated dilator muscle revealed an overlap between measurements of nerve fibers and of background (smooth muscle). In order to discriminate between both populations, the scan data were converted into fluorescence histograms (256 fluorescence classes, and subjected to mathematical analysis. The characteristics of the background histogram were obtained from scans of iris preparations devoid of fluorescent nerve fibers (pretreatment of the donor rat with reserpine or sympathetic denervation). A curve-fitting program was applied on those fluorescence classes in the normal iris histogram that represent measurements on pure background and resulted in a full background histogram. After subtraction of this background histogram from the original histogram, a nerve fiber histogram was obtained. The validity of the algorithm was evaluated by scanning iris preparations with varying background intensities. The results showed that quantification of the nerve fiber fluorescence was independent of variation in the background.

Discrete changes of the fluorescence yield from cells vitally stained with ethidium bromide (EB), as determined by flow cytometry

Cultured L cells were scraped from the glass surface and directly suspended in solutions of ethidium bromide (EB) in Tris buffer. After different staining times the fluorescence distributions were analysed with a flow cytometer. At 5 min after staining the cells yielded a normal DNA-specific histogram, but then a second peak at about 30–50% beyond the normal G1 fluorescence appeared and grew with increasing staining time. This effect of a discrete transition into a state of higher fluorescence was not dependent on the proliferative state of the cultures but was influenced by the temperature of the suspension and by serum and RNase. The effect proved not to be correlated with a G0–G1 transition and shows the possibility of artefacts in the determination of G0 G1 ratios by vital staining.

The influence of stimulus velocity on the responses of single neurones in the striate cortex.

1. Using a multi-histogram technique forty-seven response-velocity curves were prepared for a variety of visual stimuli presented to twenty-one cells in the striate cortex of the anaesthetized, paralysed cat. 2. The character of each velocity-response curve varied according to the measurement used in assessing a response. Reasons are advanced for sampling the response over a single bin of short duration at the peak of the discharge in each average response histogram. 3. The sharpness of tuning varied markedly throughout the population of cells but it was not possible to establish any definitive class differences. 4. For simple and complex cell categories there was considerable overlap in both the range of effective stimulus velocities and the distribution of the optimal velocities. An observation not emphasized in the past was that some simple cells responded to very fast stimuli while a number of complex cells were driven by very slowly moving stimuli. 5. Generally changes in stimulus parameters such as the polarity of contrast of a moving edge, its orientation or direction of movement produced only slight modifications in the profile of the velocity-response curve. 6. The abolition of the response of simple cells that failed to be driven by rapidly moving stimuli was shown to be due to the entry of the stimulus into the inhibitory flank distal to the discharge region. When the movement of the stimulus was confined to the discharge region there was little evidence of velocity dependence in the response. The duration over which the inhibition from the distal flank remained effective was evaluated for representative simple cells.