High-resolution color video cytophotometry

Comparison was made between cytophotometric measurements obtained using two data acquisition systems, one a microphotometer and the other a rapid video camera system, to ascertain whether the degradation of data with the faster video acquisition system still results in recorded images of sufficient quality to permit computer discrimination between cells of very similar appearance. Normal-appearing intermediate cells from cases with normal cytology and those from patients with dysplasia or malignant disease, as well as the subvisual markers within these cells that have rendered them capable of cytophotometric discrimination, were used for the study. Comparison of the data recorded by the two systems indicates that the diagnostic information is preserved in the change-over to a full-field, video-rate scanning system, with differences in the data caused primarily by differences in the spectral response of the two systems. This was reflected in the substantial differences observed in the color-related features and the lesser differences seen in the textural features, while the morphometric features (outline and shape) were virtually unaffected. The differences were primarily expressed on a cell-to-cell basis; in sets of about 300 cells, which would be used in patient-to-patient comparisons, the feature values showed remarkable consistency between the two systems.     

Cytophotometric analysis of corresponding cytological and histological cervical intraepithelial neoplasia grade III specimens

Cytophotometric analysis of cervical intraepithelial neoplasia grade III (CIN III) was performed in 22 cytological smears (CS) and in 22 corresponding cytospin specimens retrieved from selected areas of paraffin-embedded tissues (PEC). The average time interval between cytological and histological diagnosis was 6 weeks. CIN III nuclei in CS and PEC specimen were Thionin-Feulgen stained and digitized. Beside the visual classification of DNA ploidy patterns, the 2.5c and 5c exceeding rates and the specimen mean and standard deviation values of 21 photometric features were also analyzed. It was shown that, although there was a significant correlation between DNA ploidy patterns in corresponding PEC and CS specimen, the DNA patterns were dissimilar in eight of 22 cases. The DNA index, as represented by 2.5c and 5c exceeding rates, was significantly higher in the CS specimen. High-resolution cytophotometric analysis of cell nuclei in CS and PEC specimens showed significant differences for a large number of nuclear photometric features. These findings can possibly be explained by differences in selection of CIN III cells from CS and PEC specimens and by differences between fixation procedures as used for the two techniques. It was concluded that cytophotometric data of CS and PEC specimens representing CIN III lesions should not be regarded as interchangeable.     

Leukemia-related morphological features in blast cells

This paper investigates the use of image-processing methods to detect leukemia-related morphological differences in mononuclear blast cells. Routinely prepared Pappenheim-stained blood smears were scanned in a high-resolution color TV-microscope system. Eleven blast-cell classes (OMSBC, T-ALL, OMS, ALL, LBL, IBL, AUL, AML, AMOL, AMMOL, and CML) were analyzed with the nonparametric statistical software program "Classification and Regression Trees" (CART). This paper documents the initial statistical evaluation of 62 leukemia-related morphological features that directly measure and analyze the cell-related quantifiable differences occurring in the various blast cells. The 62 cell image features include both common cytophotometric features, and new texture and color features developed for this project. This study found that each leukemia specimen contains a dominant class of blasts that correlates with the specific leukemia, plus a distribution of blasts from related diseases. The present data suggest the existence of a distribution fingerprint pattern for each leukemia.     

DNA ploidy and cytophotometric analysis of cervical intraepithelial neoplasia grade III and invasive squamous cell carcinoma

Cervical intraepithelial neoplasia grade III (CIN III) and squamous cell carcinoma (INV) were examined using DNA ploidy and cytophotometric analysis. Based on hysterectomy, exconisation, and biopsy material from 69 patients in two age categories, analysis was performed in nuclei isolated from selected areas of paraffin-embedded tissue. High percentages of DNA-diploidy in INV lesions were found mainly in the group of patients age 45 years or younger. CIN III lesions in women age 46 or older demonstrated high percentages of DNA-aneuploidy. DNA-polyploidy was most frequent in CIN III lesions in the younger age category. The results of cytophotometric analysis indicated that the overall mean values of 16 nuclear photometric features discriminated significantly between the whole groups of CIN III (n = 37) and INV (n = 32). On an individual patient level, however, the mean feature values showed a large overlap. Based on the results of a stepwise linear discriminant analysis of patient mean values, a combination of geometrical and run-length texture features was used to discriminate between CIN III and INV lesions. The correct classification rate was highest in the category of patients in the older age category. The results of this study indicate age related differences in CIN III and invasive squamous cell carcinoma, and they may be of help in assessing cytophotometric features in the study of progressive and non-progressive CIN lesions.     

Analysis of machine-selected cells with an image analysis system in normal and abnormal cervical specimens

An analysis has been performed of visual diagnostic criteria used in cervical cytology applied to machine selected cells in relation to automated classification based on variables, which can be recorded in an image system with automated cell search and segmentation, feature extraction and classification. A 98% accuracy could be obtained with the choice of the most ideal statistical methods for discrimination and the use of the most powerful variables recorded in the image system when compared with consensus of the visual diagnoses based on established cytological criteria for diagnosis of cancer and precancer of the cervix uteri. The most powerful discriminatory variables in the image system (of 17 recorded) for discrimination between normal and abnormal epithelial cells were, in addition to nuclear extinction, cytoplasmic extinction and cytoplasmic shape. It is concluded that the visual classification of cervical cells is highly accurate with experienced observers and that imaging microscopes can be trained to nearly equal this accuracy with appropriate statistical methods of discrimination. The problem of creating fully automated systems, however, also requires the inclusion of even more effective discriminatory variables and also the solution of such problems as automatic cell search, segmentation, artifact rejection, feature extraction, classification and electronic stability in order to become cost-effective.     

Blood velocity measurement in human conjunctival vessels

The bulbar conjunctiva is one of the few areas in which blood flow in the peripheral vasculature can be directly and noninvasively observed in the human. Although extensive literature exists describing morphological changes which correlate with a variety of systemic diseases in this vasculature, little quantitative data is available on hemodynamics in either normal or abnormal states. The hemodynamic data available are primarily subjective assessments of "low flow." Approaches to place the subjective assessment on more quantitative grounds have usually been based on photographic techniques that have intrinsic inadequacies. The objective of the work reported here was to develop a system capable of providing sequential blood velocity data potentially useful for providing quantitative information on blood flow and its change in the microvessels of the human conjunctiva. The method that has evolved uses a standard Zeiss slit-lamp to image a subject's conjunctival vessels by using a 1-inch Newvicon TV camera with an electronic magnification of 2x. The video image is simultaneously recorded on a video tape recorder (VTR) to an overall system magnification of approximately 4 microm/raster line. The data acquisition phase requires approximately 5 minutes of patient time, whereas the actual determination of blood velocity in individual vessels is done offline through a modification of the dual-slit videodensimetric method. Two independently controllable video cursors are placed axially over the vessel image with the VTR in the still-frame mode. For each consecutive video field, the position of two reference points on the vessel and the position of each cursor relative to these and to each other are encoded into a computer to track the moving image caused by normal eye movement. The computer then determines new cursor coordinates to ensure a constant position within the vessel. The electrical signals obtained for each cursor site and for each video field are cross-correlated to yield the average blood velocity over the sampled time interval. The system has been calibrated in vitro from 0.2 to 2.5 mm/sec, evaluated in experimental animals, and used to measure blood velocity (0.3 to 1.5 mm/sec) in human conjunctival venules with diameters ranging from 20 to 50 microm. At this writing, blood velocity has been recorded during a period of about 3 months in the same vessel of several postmyocardial infarction patients. Thus, the method appears suitable for determining sequential changes in small vessel blood flow in patients over extended periods of time.     

Standardized thionin-eosin stain in bronchial cytology. A substitute for hematoxylin-eosin Y staining

A standardized thionin-eosinic acid stain was developed as a quick and highly reproducible staining method for bronchial cytology. Bronchial smears and paraffin-embedded sputum samples were stained with thionin-eosin and with the conventional hematoxylin-eosin Y. Spectral absorption characteristics and staining intensity of thionin-eosin-stained cells were investigated by means of cytophotometry. The staining pattern of thionin-eosin is very close to that of the hematoxylin-eosin stain; the contrast between nucleus and cytoplasm is significantly higher for thionin-eosin. Thionin-eosin can be used for "dye-fixation" of cytologic smears and tissue imprints. Blueing and differentiation (as for hematoxylin-eosin) is not required for thionin-eosin; thus, fixation and staining can be performed within two minutes. The spectral absorption characteristics of thionin-eosin allow reliable automated cytophotometric discrimination of cell nuclei and cytoplasm. The standardized thionin-eosin stain is recommended as a substitute for the hematoxylin and eosin stain in bronchial cytology.     

Variations in the echolocation calls of the European free-tailed bat

The echolocation calls of Tadarida teniotis were studied in an outdoor flight enclosure (captive individuals) and in the wild using single microphones or an array of four microphones. Calls were characterized by measures of 10 call variables. Comparison of individual calls recorded on four microphones arrayed in a tetrahedron with 1 m between each microphone revealed that all calls were not equally detectable by all microphones but that there were no significant differences in call features obtained from calls recorded on all four microphones. A comparison of 47 calls recorded by all four microphones showed no significant differences in the features of the four recordings of each call. Analysis of calls of five individuals flying individually in an outdoor flight cage revealed significant individual differences in call features. In the field, T. teniotis used long, narrowband search-phase calls, usually without harmonics. Analysis of 1876 search-phase echolocation calls of T. teniotis recorded in the field in Israel and Greece in 2002, 2005 and 2006 showed significant year-to-year and site-to-site differences in some call features. When flying in the presence of conspecifics, T. teniotis changed their echolocation calls. We found a range of different buzzes in the wild, and based on their structure we attempted to classify them as feeding and social buzzes. The features of individual calls comprising buzzes differed significantly among buzzes, and yet there were no consistent differences between what we classified as feeding and social buzzes.     

Cytophotometric and electron microscopic study of chronic human skin lymphomas. I. A study of skin manifestations

Electron microscopic and cytophotometric studies of low-grade malignant cutaneous lymphomas and non-malignant skin diseases revealed substantial differences between these groups of diseases. In the latter case, the DNA content per nucleus is diploid but in the former an atypical distribution of DNA content per nucleus (more than 5% aneuploid and polyploid nuclei) is observed in addition to a significant excess of the mean DNA content per nucleus above the diploid standard level. In lymphomas electron microscopy reveals clusters of atypical lymphoid cells with irregularly shaped nuclei, nuclear pockets, nuclear extrusions, network of cytoplasmic microfilaments. These features never occur with skin diseases. The data obtained can be helpful in the diagnostics of the low-grade malignant cutaneous lymphomas.     

Continuous image and electrophysiological recording with real-time processing and control.

Collecting continuous video together with multichannel electrophysiological data and other experimental modalities requires high bandwidth and storage capacities, as well as accurate synchronization to detect correlations between different recorded events. Often, experiments are highly complex, with many variables requiring immediate analysis and feedback during the course of the experiment. In addition, output channels require real-time control with high time resolution. We have explored several approaches to a system that can perform the above functions. The design of our system considered a number of issues, including time intervals between control and acquisition events, longest continuous recording period, data transfer bottleneck considerations, file archiving and format, and real-time display and processing. To demonstrate the system, we describe an experiment for characterizing rapid evoked scattered light changes in neural tissue, in vivo, using simultaneous electronic image acquisition and electrophysiological recording.     

Extraction of cells from paraffin-embedded tissue sections for single-cell DNA cytophotometry

A simple technique is presented for the isolation of cells from paraffin-embedded tissues for Feulgen DNA cytophotometric investigations. Tissue fragments from paraffin blocks were deparaffinized in xylene, rehydrated and refixed in a formalin solution and incubated in a solution of 0.5 pepsin in 0.25% hydrochloric acid. After filtration through a 70 micron mesh and centrifugation, the cells were smeared upon a glass slide. Comparison between the results obtained with freshly prepared imprints and with pepsin-extracted cells of the same tumor showed that the extraction technique does not influence the Feulgen reaction or the DNA distribution pattern. Investigations carried out on bladder and embryonal carcinomas have demonstrated that the method permits an analysis of histologically or histochemically identified tumor cells within individual tissue areas.     

THE VISUAL DISCRIMINATION OF INTENSITY AND THE WEBER-FECHNER LAW

1. A study of the historical development of the Weber-Fechner law shows that it fails to describe intensity perception; first, because it is based on observations which do not record intensity discrimination accurately, and second, because it omits the essentially discontinuous nature of the recognition of intensity differences. 2. There is presented a series of data, assembled from various sources, which proves that in the visual discrimination of intensity the threshold difference ΔI bears no constant relation to the intensity I. The evidence shows unequivocally that as the intensity rises, the ratio See PDF for Equation first decreases and then increases. 3. The data are then subjected to analysis in terms of a photochemical system already proposed for the visual activity of the rods and cones. It is found that for the retinal elements to discriminate between one intensity and the next perceptible one, the transition from one to the other must involve the decomposition of a constant amount of photosensitive material. 4. The magnitude of this unitary increment in the quantity of photochemical action is greater for the rods than for the cones. Therefore, below a certain critical illumination—the cone threshold—intensity discrimination is controlled by the rods alone, but above this point it is determined by the cones alone. 5. The unitary increments in retinal photochemical action may be interpreted as being recorded by each rod and cone; or as conditioning the variability of the retinal cells so that each increment involves a constant increase in the number of active elements; or as a combination of the two interpretations. 6. Comparison with critical data of such diverse nature as dark adaptation, absolute thresholds, and visual acuity shows that the analysis is consistent with well established facts of vision.     

Developing and Integrating Advanced Movement Features Improves Automated Classification of Ciliate Species

Recent advances in tracking technologies such as GPS or video tracking systems describe the movement paths of individuals in unprecedented details and are increasingly used in different fields, including ecology. However, extracting information from raw movement data requires advanced analysis techniques, for instance to infer behaviors expressed during a certain period of the recorded trajectory, or gender or species identity in case data is obtained from remote tracking. In this paper, we address how different movement features affect the ability to automatically classify the species identity, using a dataset of unicellular microbes (i.e., ciliates). Previously, morphological attributes and simple movement metrics, such as speed, were used for classifying ciliate species. Here, we demonstrate that adding advanced movement features, in particular such based on discrete wavelet transform, to morphological features can improve classification. These results may have practical applications in automated monitoring of waste water facilities as well as environmental monitoring of aquatic systems.     

A charge coupled device-based image cytophotometry system for quantitative histochemistry and cytochemistry

A rapid, semiautomated cytophotometry system for quantitative histochemistry and cytochemistry was constructed. The system consists of a Fairchild charge coupled device (CCD) image camera, a Zeiss Universal microscope, a Datacube analog to digital converter, and a digital Equipment Corporation LSI 11/23 computer operating under RT-11. Computer programs were written in FORTRAN and the MACRO assembly language for the acquisition of data from the CCD device. These data were then used for image segmentation, image display, and calculation of total optical density, perimeter, cell area, and several shape features. The reproducibility of measurement made with the CCD-based cytophotometry system was tested by repeated measurements. The coefficient of variation was estimated to be 1.7% for total optical density and 0.9% for cell area. The CCD-based cytophotometry system was further evaluated by comparing results with measurements made on the same cells with a scanning stage cytophotometer using the HIDACSYS computer programs. Correlation coefficients of 0.96 for total optical density and 0.91 for cell area were obtained between the two systems. We conclude that the high-speed, dimensional stability, small size, and linearity of the CCD-based cytophotometry system will make it useful for quantitative histochemistry and cytochemistry.     

Acquisition of circadian bioluminescence data in Gonyaulax and an effect of the measurement procedure on the period of the rhythm

During measurements of the circadian (approximately 24-hr) rhythms of spontaneous bioluminescence in the marine dinoflagellate Gonyaulax polyedra, the individual cultures in vials were shielded from otherwise constant dim light for 1-3 min every 20-60 min by a photomultiplier housing that was moved from vial to vial. The high-frequency dark pulses caused a small but consistent shortening of the free-running circadian period, but there was no indication that the dark pulses caused entrainment. Hardware and software components of the microcomputer-controlled data collection system are described. A microcomputer controlled the movement of the photomultiplier and acquired the data via an analog-to-digital converter. The algorithms distinguished and separately recorded background glow, intermittent flashes, and total light from populations ranging in number from 10(3) to 10(5) cells in volumes from 1 to 10 ml. Fast video display techniques allowed continuous on-line viewing of incoming data, together with a display of the data recorded over the preceding day or two. Detection of mechanical and software errors coupled with recovery systems maintained high reliability of data collection.     

Prognostic significance of DNA image cytophotometry for osteosarcoma

OBJECTIVE: To investigate the prognostic significance of DNA image cytophotometric data. STUDY DESIGN: Twenty-six osteosarcomas in patients without lung metastases were investigated for several cytophotometric data. In 24 cases, these data were correlated with the clinical course of the patients to assess the prognostic value of nuclear DNA content in osteosarcomas. RESULTS: Of all osteosarcomas, 96% showed aneuploid DNA content. Patients with tumors having a 2c deviation index (2cDI) of 12.00, DNA malignancy grade (DNA-MG) of 2.0, a mean DNA content (MDC) of 4.95 c, DNA index (DI) of 1.75 or mean nuclear area (MNA) of 130 microns 2 had a significantly lower overall survival rate as compared to those with lower values (P < .05). CONCLUSION: Image cytophotometric features, such as 2cDI, DNA-MG, MDC, DI and MNA, are of prognostic value in patients with osteosarcoma and free of lung metastases.     

On-line transmission electron microscopic image analysis of chromatin texture for differentiation of thyroid gland tumors

Nuclei of the cells from the thyroid gland were analyzed in a transmission electron microscope by direct TV scanning and on-line image processing. The method uses the advantages of a visual-perception model to detect structures in noisy and low-contrast images. The features analyzed include area, a form factor and texture parameters from the second derivative stage. Three tumor-free thyroid tissues, three follicular adenomas, three follicular carcinomas and three papillary carcinomas were studied. The computer-aided cytophotometric method showed that the most significant differences were the statistics of the chromatin texture features of homogeneity and regularity. These findings document the possibility of an automated differentiation of tumors at the ultrastructural level.     

Computer-based image analysis for the automated counting and morphological description of microalgae in culture

A largely unexplored area is the application of digital image processing to counting and sizing of microalgal cells from culture. Commercial systems are available, but have not been tested, nor necessarily optimized for high speed counting and sizing of phytoplankton. The present work describes the design, construction, specifications and comparative performance of an inexpensive system optimized for counting and sizing microalgal cells. This system has been tested with cells of the picoplankton to nanoplankton size ranges (1–20 μm). The hardware was a widely available standard microcomputer, an inexpensive video camera and monitor, and a video digitization board (frame grabber). A modifiable menu-driven program (PHYCOUNT) was written and provisions made to make this program available to other workers. The program is constructed such that it can be adapted to a variety of hardware setups Video digitization boards). Comparison of growth curves for microagae revealed there were no significant differences in division rate and cell yield as assessed by the image analysis method compared to manual counts with a hemacytometer. Several hundred cells were counted routinely within 10–15 s, far exceeding the counting rate achieved by hand tally. A variable transect feature allowed sampling every nth pixel and provided a substantial increase in execution speed. More than 1000 counts can be done per day. A protocol for the use of 96-well plates of polyvinyl chloride as counting chambers contributed to the processing of large numbers of samples rapidly. Other routines developed provided subtended area, defined the coordinates of cell perimeter, and derived cell length and width. The calculation of the latter two parameters was usually done off-line as data output is in standard numerical form accessible by other programs. Experience with daily use of the PHYCOUNT program and imaging hardware reveal that the system is reliable for cell counting and sizing. The presence of bacteria in the algal cultures does not affect cell counting or sizing.