Simple method for quantification of fast plasma membrane movements.

We present a method for the quantification of the fast plasma membrane movements that are involved in ruffling, blebbing, fast shape change, and fast translocation. The method is based on the Kontron Vidas image analysis computer program. Video images from cells viewed through an inverted microscope were transmitted to the computer. The procedure was as follows: 4 consecutive video images were averaged (image 1); 28 s later a second set of 4 video images was averaged (image 2); image 2 was subtracted from image 1 and the grey level of each pixel of the resulting image was increased with 128 grey level units, resulting in the subtraction image, showing a uniform grey background speckled with brighter and darker spots corresponding to areas of movement. These spots were discriminated and turned into white objects against a black background. Interactive editing was used to delete artefacts that resulted from floating debris. The total area of the discriminated objects was measured, and the parameter motile area in micron2 per cell was calculated. We have applied our method to the study of motility induced in epithelial cell lines by the tumor promoter 12-O-tetradecanoyl-phorbol-13-acetate and by epidermal growth factor.     

A selective mapping algorithm for computer analysis of voided urine cell images

One of the fundamental targets of the automated image analysis of cytologic preparations is the reduction of computer classification errors due to cells or other objects that do not lend themselves to image segmentation or that have morphologic features that may mislead the cell classification schemes. In prior work from this laboratory, the achievement of this goal was attempted by hierarchical analysis of sequential microscopic objects at high resolution. This paper reports on the successful development and implementation of an automated "selective mapping algorithm" that selects cells at low power for further analysis and eliminates a large proportion of unwanted "objects." The algorithm classifies the objects and extracts appropriate features from a 256 X 240 digital image obtained via a 10 X planachromatic objective. The five-node binary tree classifier used in this triage is described. The algorithm was trained and tested initially on 501 visually classified microscopic "objects," resulting in a correct acceptance rate of 61.3% and correct rejection rate of 81.3%. The selective mapping algorithm was subsequently integrated into the video-based image analysis system constructed at the Montefiore Medical Center for the diagnostic evaluation of sediments of voided urine. The algorithm was then tested on ten cytocentrifuge preparations for a preliminary evaluation of its performance. Up to 100 "objects" per case were selected by the algorithm for further classification by the computer at high power. Of the 810 "objects" selected by the selective mapping algorithm, 344 (42.5%) were classified by the computer at high resolution as cells of diagnostic value ("WELL" cells) and 466 were rejected.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of focus on cell detection and recognition by the Cell Analyzer

The effect of defocusing on the quality of signals from live cells detected by an automated image cytometry device, the Cell Analyzer, was examined. The influence of these effects on the ability of this device to automatically locate cells plated into a tissue culture flask was then determined by measuring the performance of cell detection and recognition procedures as a function of focus setting. Acceptable limits for deviation from the optimal focus setting (as determined by microscope objective position) were found to be similar for both these procedures, ranging from 40 microns below to 25 microns above the optimal focus position. These limits were asymmetrical about ideal focus due to a pronounced asymmetry in the effects of positive and negative defocusing on the cell signal.     

Metabolism of palmitate in cultured rat Sertoli cells

Isolated rat Sertoli cells were incubated in the presence of [1-14C]palmitate at a cell concentration of 1.54 +/- 0.31 mg protein/flask (n = 7). The oxidation of palmitate was concentration dependent and maximal oxidation was obtained at 0.35 mM-palmitate. At a saturating concentration of palmitate the oxidation was linear for at least 6 h. About 65% of the total amount of palmitate oxidized during 5 h at 0.52 mM-palmitate (109 +/- 44 nmol/flask, n = 5) was recovered as CO2 and the rest as acid-soluble compounds. Almost all radioactive acid-soluble compounds which were secreted by the Sertoli cells were shown to be 3-hydroxybutyrate and acetoacetate. The palmitate recovery in cellular lipids and triacylglycerols was 9.4 +/- 5.1 nmol/flask (n = 5) and 3.5 +/- 2.8 nmol/flask (n = 5) respectively. Addition of glucose had no significant effect on palmitate oxidation but caused a 9-fold increase in esterification of palmitate into triacylglycerols. We conclude that cultured rat Sertoli cells can oxidize palmitate to CO2 and ketone bodies and that fatty acids appear to be a major energy substrate for these cells.     

A rapid method of determining growth characteristics of plant cell populations in batch suspension culture

A non-destructive, simple and accurate method of determining the relative growth rate (RGR) of the packed cell volume (PCV) of plant suspension cells in one Erlenmeyer flask at any time during the incubation period is described. The Erlenmeyer flask was tilted and the length of the chord formed by the surface of the packed cells across the bottom of the flask was measured. The chord length and the log PCV were correlated in a calibration line. The method enables the RGR during the exponential growth phase to be calculated by multiplying the slope of the linear part of the curve of the chord length in time with the slope of the calibration line. In order to investigate other growth parameters and to analyse the accuracy of the method statistically, a four-parameter function for the chord length and a computer program were used. The RGR during the exponential growth phase of cell suspensions of Solanum tuberosum and Haplopappus gracilis appeared to be independent of the PCV of the inoculum. The method appeared to be sufficiently accurate.     

Quantitative precision of an automated, fluorescence-based image cytometer.

Digital image-based cytometry of clinical specimens labeled with fluorescent, disease-specific markers holds promise for becoming an important diagnostic and prognostic technique because the technique can make a diverse range of quantitative biochemical, morphologic, densitometric and contextual measurements on intact specimens. It has been previously shown by us, using an image cytometer (IC) consisting entirely of commercially available components, that the nuclei of individual cells in slide-supported specimens can be detected automatically using a fluorescent DNA stain and image analysis software. The purpose of this study was to determine the precision of the IC for quantifying the integrated fluorescence intensity and area of fluorescent standard beads and nuclei. Integrated intensities could be quantified to between 2.3% and 3.5% precision using a 40x objective lens and between 1.6% and 2.3% using a 20x objective. The main contribution to this uncertainty was 2% inaccuracy in determining the variations in sensitivity over the imaging area. Areas could be quantified to between 0.91% and 2.1% using a 40x objective and between 2.8% and 3.2% using a 20x objective. Significant quantification errors were introduced if the objects were not in focus or were touching each other. Overall, however, these results demonstrated that image cytometry of fluorescence-stained specimens can yield quantitative results with sufficient precision for determining DNA ploidy distributions and for making other measurements on clinical specimens.     

Automated fluorescence image cytometry. DNA quantification and detection of chlamydial infections

Digitized fluorescence microscopy in conjunction with automated image segmentation is a promising approach for screening clinical specimens quickly and reliably. This paper describes the hardware and software of a prototype image-based cytometer that can identify fluorescent objects, discriminate true objects from artifacts and divide overlapping pairs of objects. The use of this image cytometer is discussed for: (1) the measurement of the DNA ploidy distribution of isolated mature rat liver nuclei labeled with 4',6-diamidine-2-phenylindole; (2) the comparison of the DNA ploidy distributions of the same samples measured by image cytometry (ICM) and flow cytometry (FCM); and (3) the quantification of chlamydial infection by double labeling cells with antichlamydiae antibody and Hoechst 33258 for nuclear DNA analysis. Ploidy distributions measured by the automated image cytometer compared favorably to those obtained by FCM. All pairs of overlapping nuclei were automatically detected by an additional computer algorithm, and those pairs that were clearly more than one nucleus by visual inspection were correctly divided. The irregular morphology of the chlamydiae-infected cells meant that 26% of them were not correctly identified in the fluorescein-stained images (as judged by manual inspection), but all cells were nevertheless detected correctly from the images of the Hoechst-stained samples. Automated fluorescence ICM yielded results similar to those obtained with FCM and had the additional benefit of maintaining cell and tissue architecture while preserving the opportunity for subsequent manual inspection of the specimen.     

Mind the gap: the minimal detectable separation distance between two objects during active electrolocation

In a food‐rewarded two‐alternative forced‐choice procedure, it was determined how well the weakly electric elephantnose fish Gnathonemus petersii can sense gaps between two objects, some of which were placed in front of complex backgrounds. The results show that at close distances, G. petersii is able to detect gaps between two small metal cubes (2 cm × 2 cm × 2 cm) down to a width of c. 1·5 mm. When larger objects (3 cm × 3 cm × 3 cm) were used, gaps with a width of 2–3 mm could still be detected. Discrimination performance was better (c. 1 mm gap size) when the objects were placed in front of a moving background consisting of plastic stripes or plant leaves, indicating that movement in the environment plays an important role for object identification. In addition, the smallest gap size that could be detected at increasing distances was determined. A linear relationship between object distance and gap size existed. Minimal detectable gap sizes increased from c. 1·5 mm at a distance of 1 cm, to 20 mm at a distance of 7 cm. Measurements and simulations of the electric stimuli occurring during gap detection revealed that the electric images of two close objects influence each other and superimpose. A large gap of 20 mm between two objects induced two clearly separated peaks in the electric image, while a 2 mm gap caused just a slight indentation in the image. Therefore, the fusion of electric images limits spatial resolution during active electrolocation. Relative movements either between the fish and the objects or between object and background might improve spatial resolution by accentuating the fine details of the electric images.     

Strategies for rare-event detection: an approach for automated fetal cell detection in maternal blood.

This article explores the feasibility of the use of automated microscopy and image analysis to detect the presence of rare fetal nucleated red blood cells (NRBCs) circulating in maternal blood. The rationales for enrichment and for automated image analysis for "rare-event" detection are reviewed. We also describe the application of automated image analysis to 42 maternal blood samples, using a protocol consisting of one-step enrichment followed by immunocytochemical staining for fetal hemoglobin (HbF) and FISH for X- and Y-chromosomal sequences. Automated image analysis consisted of multimode microscopy and subsequent visual evaluation of image memories containing the selected objects. The FISH results were compared with the results of conventional karyotyping of the chorionic villi. By use of manual screening, 43% of the slides were found to be positive (>=1 NRBC), with a mean number of 11 NRBCs (range 1-40). By automated microscopy, 52% were positive, with on average 17 NRBCs (range 1-111). There was a good correlation between both manual and automated screening, but the NRBC yield from automated image analysis was found to be superior to that from manual screening (P=.0443), particularly when the NRBC count was >15. Seven (64%) of 11 XY fetuses were correctly diagnosed by FISH analysis of automatically detected cells, and all discrepancies were restricted to the lower cell-count range. We believe that automated microscopy and image analysis reduce the screening workload, are more sensitive than manual evaluation, and can be used to detect rare HbF-containing NRBCs in maternal blood.     

Oscillating perfusion of cell suspensions through three-dimensional scaffolds enhances cell seeding efficiency and uniformity

We developed a bioreactor for automated cell seeding of three-dimensional scaffolds by continuous perfusion of a cell suspension through the scaffold pores in oscillating directions. Using quantitative biochemical and image analysis techniques, we then evaluated the efficiency and uniformity of perfusion seeding of Polyactive foams as compared to conventional static and spinner flask methods. Finally, we assessed the efficacy of the perfusion seeding technique for different scaffolds and cell types. Perfusion seeding of chondrocytes into Polyactive foams resulted in "viable cell seeding efficiencies," defined as the percentages of initially loaded cells that were seeded and remained viable, that were significantly higher (75 +/- 6%) than those by static (57% +/- 5%) and spinner flask seeding (55% +/- 8%). In addition, as compared to static and spinner flask methods, cells seeded by perfusion were respectively 2.6-fold and 3.8-fold more uniformly distributed and formed more homogeneously sized cell clusters. Chondrocytes seeded by perfusion into Hyaff-11 nonwoven meshes were 26% and 63%, respectively, more uniformly distributed than following static and spinner flask seeding. Bone marrow stromal cells seeded by perfusion into ChronOS porous ceramics were homogeneously distributed throughout the scaffold volume, while following the static method, cells were found only near the top surface of the ceramic. In summary, we demonstrated that our cell seeding perfusion bioreactor generated constructs with remarkably uniform cell distributions at high efficiencies, and was effective for a variety of scaffolds and different mesenchymal cell types.     

The rejection of noncellular artifacts in Papanicolaou-stained slide specimens by an automated high-resolution system. Identification of important cytometric features

The important cytodiagnostic features that permit discrimination of typical cell types by high-resolution image analysis and pattern recognition techniques have been previously studied in detail. An automated system for the diagnosis of Papanicolaou-stained specimens must also deal, however, with the "real world" of extraneous noncellular artifacts and debris found on every slide. Features that are ideal for the separation of typical normal and abnormal cells may not be adequate by themselves to reject these objects. A new set of discriminatory features must be found. In order to identify those features, a large set of images acquired using the TICAS high-resolution television rapid-scanning system was analyzed and studied. These images, from a variety of slide types, included normal cells, abnormal cells and noncellular artifacts identified by low-resolution preprocessing logic as suspicious enough to warrant high-resolution study. The results indicate that the more important features for such discrimination are not those traditionally important in distinguishing abnormal from normal cells but include color relations, shape measures, boundary properties and texture features.     

Surface immobilization of anchorage-dependent mammalian cells

Anchorage-dependent HeLa cells were successfully cultured on two fibrous materials (A07 and R100) with porosities of 75-125 and 40 mum, void fractions of 92% and 81%, and fiber diameters of 7.6 and 10.2 mum, respectively, in 100-mL spinner flasks and 2-L stirred tank bioreactors. The matrix was formed into a fixed vertical spiral configuration. All cultures displayed rapid (/=95%) to the matrix, uniform coverage of the immobilizing area with viable cells, and no significant amount of cell debris in the medium. Spinner flask cultures indicated that the denser material R100 showed better results in terms of final cell density. The growth of HeLa cells on material R100 in both culture systems was similar to that observed in tissue culture dishes (specific growth rate approximately 0.03-0.04 h(-1), maximum cell density of 8 x 10(6)-9 x 10(6) cells . mL(-1), and yields of 0.4 x 10(8) cells . mM(-1) on glucose and 2 x 10(8)-3 x 10(8) cells . mM(-1) on glutamine). Scale-up of this culture technique in a 2-L bioreactor under perfusion with pH and dissolved oxygen (DO) control yielded cell densities of up to 1.6 x 10(6) cells . mL(-1). Two other anchorage-dependent mammalian cells (ADC) known to be cultured with difficulty in roller bottles or with micro carriers were easily grown on material R100 in spinner flasks. The performance of this culture technique was compared to other ADC culture systems.     

Automated identification of circulating tumor cells by image cytometry

Presence of circulating tumor cells (CTC), as detected by the CellSearch System, in patients with metastatic carcinomas is associated with poor survival prospects. CellTracks TDI, a dedicated image cytometer, was developed to improve the enumeration of these rare CTC. The CellSearch System was used to enumerate CTC in 7.5 mL blood of 68 patients with cancer and 9 healthy controls. Cartridges containing the fluorescently labeled CTC from this system were reanalyzed using the image cytometer, which acquires images with a TDI camera using a 40×/0.6 NA objective and lasers as light source. Automated classification of events was performed by the Random Forest method using Matlab. An automated classifier was developed to classify events into CTC, apoptotic CTC, CTC debris, leukocytes, and debris not related to CTC. A high agreement in classification was obtained between the automated classifier and five expert reviewers. Comparison of images from the same events in CellTracks TDI and CellTracks Analyzer II shows improved resolution in fluorescence images and improved classification by adding bright-field images. Improved detection efficiency for CD45-APC avoids the classification of leukocytes nonspecifically binding to cytokeratin as CTC. The correlation between number of CTC detected in CellTracks TDI and CellTracks Analyzer II is good with a slope of 1.88 and a correlation coefficient of 0.87. Automated classification of events by CellTracks TDI eliminates the operator error in classification of events as CTC and permits quantitative assessment of parameters. The clinical relevance of various CTC definitions can now be investigated.     

基于最小矩形法的柑桔横径实时检测方法研究

农产品的大小是农产品分级的重要特征之一．本文以柑桔为研究对象,对利用计算机图像处理技术检测柑桔横径的方法进行了探索．研究了柑桔图像的平滑和边缘提取方法,比较了利用Sobel算子和跟踪虫（tracking bug)法所得到的柑桔边缘,发现跟踪虫法寻找边界的速度比用Sobel算子快,且不需要进行细化处理．为了适应实际生产中柑桔方向的随机性和外形的不规则性的要求,使柑桔尺寸检测的方法有更好的适应性,设计了一种利用柑桔的最小外接矩形（MER）求最大横径的方法,并在柑桔的外形尺寸检测中进行了验证,实际最大横径Dr与预测最大横径的相关性为0．9982,利用此方法估测果径的最大误差在1．30mm以内,最大相对误差为2.64％,平均相对误差为1.35％,GB／T12947-91鲜柑桔分级标准中均以5mm为分等标准差,故MER法的精度能满足实际生产中柑桔分级精度的要求．本研究结果不仅为进一步研究开发基于图像处理技术的柑桔品质检测系统打下了基础,而且可用于对其他农产品进行检测．     

An Apparatus for Measuring Volumes of Small Objects

An apparatus for measuring volumes of small objects such as tissue blocks is described. The apparatus measures volumes by fluid displacement and consists of a micropipette adapted to fit the mouth of an Erleiuneyer flask, a Luer adaptor fused to the side of the flask, and a glass syringe. When assembled with fluid enclosed, the fluid rises to a low level in the micropipette. Withdrawal of fluid into the syringe lowers the fluid level below the mouth of the flask. The micropipette is raised, the object to be measured is placed in the flask, and the micropipette is joined to the flask again. Fluid returned to the flask from the syringe rises to a higher level in the micropipette. The difference between the two fluid levels equals the volume of the object measured.

This apparatus gives reproducible measurements and can be calibrated for absolute volume determination. It is inexpensive to construct and easy to use.     

Biological treatment of the components of solid oligomeric waste from a nylon-6 production plant

On partial analysis of the solid oligomeric waste of a nylon-6 production plant, it was found to contain ε-caprolactam, 6-aminocaproic acid (6-ACA) and its linear and cyclic oligomers. Out of four bacterial isolates capable of utilizing caprolactam as the sole growth substrate, Alcaligenes faecalis was found to be the most potent and utilized 90% of caprolactam in 24 h. In shake flask experiments, when the solid waste after solubilization was treated with a consortium of bacteria of four different genera, except the cyclic oligomers, all the other constituents were found to be degraded. A reduction of the chemical oxygen demand (COD) of the solid waste to the level of 63–66% was obtained when it was treated with either a consortium of the bacterial isolates or only a single isolate, A. faecalis. Alcaligenes faecalis could bring about a decrease of 95% in the caprolactam content of the solid waste, while 6-ACA and its linear oligomers were almost completely degraded. Alcaligenes faecalis cells adapted on solid waste could degrade the linear oligomers at a faster rate as compared to cells adapted on caprolactam. However, cyclic oligomers could not be degraded in either case. When solid waste, partially hydrolysed with acid to yield 6-ACA as the major constituent, was treated with the consortium of bacterial isolates, a 95% reduction in the COD was achieved. This revised version was published online in November 2006 with corrections to the Cover Date.     

Radiation dosimetry by automatic image analysis of dicentric chromosomes.

A system for scoring dicentric chromosomes by image analysis comprised fully automatic location of mitotic cells, automatic retrieval, focus and digitization at high resolution, automatic rejection of nuclei and debris and detection and segmentation of chromosome clusters, automatic centromere location, and subsequent rapid interactive visual review of potential dicentric chromosomes to confirm positives and reject false positives. A calibration set of about 15,000 cells was used to establish the quadratic dose response for 60Co gamma-irradiation. The dose-response function parameters were established by a maximum likelihood technique, and confidence limits in the dose response and in the corresponding inverse curve, of estimated dose for observed dicentric frequency, were established by Monte Carlo techniques. The system was validated in a blind trial by analysing a test set comprising a total of about 8000 cells irradiated to 1 of 10 dose levels, and estimating the doses from the observed dicentric frequency. There was a close correspondence between the estimated and true doses. The overall sensitivity of the system in terms of the proportion of the total population of dicentrics present in the cells analysed that were detected by the system was measured to be about 40%. This implies that about 2.5 times more cells must be analysed by machine than by visual analysis. Taking this factor into account, the measured review time and false positive rates imply that analysis by the system of sufficient cells to provide the equivalent of a visual analysis of 500 cells would require about 1 h for operator review.     

A method to separate plant roots from soil and analyze root surface area

Analysis of the effects of soil management practices on crop production requires knowledge of these effects on plant roots. Much time is required to wash plant roots from soil and separate the living plant roots from organic debris and previous years’ roots. We developed a root washer that can accommodate relatively large soil samples for washing. The root washer has a rotary design and will accommodate up to 24 samples (100 mm diam. by 240 mm long) at one time. We used a flat-bed scanner to digitize an image of the roots from each sample and used a grid system with commercially-available image analysis software to analyze each sample for root surface area. Sensitivity analysis and subsequent comparisons of ‘dirty’ samples containing the roots and all the organic debris contained in the sample and ‘clean’ samples where the organic debris was manually removed from each sample showed that up to 15% of the projected image could be coveredwith debris without affecting accuracy and precision of root surface area measurements. Samples containing a large amount of debris may need to be partitioned into more than one scanning tray to allow accurate measurements of the root surface area. Sample processing time was reduced from 20 h, when hand separation of roots from debris was used, to about 0.5 h, when analyzing the image from an uncleaned sample. The method minimizes the need for preprocessing steps such as dying the roots to get better image contrast for image analysis. Some information, such as root length, root diameter classes and root weights, is not obtained when using this technique. Root length measurements, if needed, could be made by hand on the digital images. Root weight measurement would require sample cleaning and the advantage of less processing time per sample with this method would be lost. The significance of the tradeoff between information not obtained using this technique and the ability to process a greater number of samples with the time and personnel resources available must be determined by the individual researcher and research objectives.     

Bladder cancer diagnosis by computer image analysis of cells in the sediment of voided urine using a video scanning system

A video-based computerized semiautomated image analysis system was applied to the diagnostic evaluation of 119 sediments of voided urine: 103 from patients with a broad variety of neoplastic and nonneoplastic disorders of the lower urinary tract and 16 normal controls. Each specimen was presented to the machine as a single cytocentrifuge preparation, preserved in 2% Carbowax in 50% ethanol and stained-by the Papanicolaou method. Five hundred sequential "objects" were scanned within an area of 9 sq mm on each slide. "Objects" of no diagnostic value, such as dirt, debris, inflammatory cells, cell clusters, poorly preserved cells, etc., were eliminated from the final diagnostic analysis by a computer-based hierarchic triage system. The final specimen classifier was based on the cell images identified by the computer as well-preserved normal (NEG), atypical (ATY I), suspicious (ATY II) and malignant (POS) cells. For specimen classification by computer, the four categories of "abnormal," "inadequate," "acellular" and "negative" were defined. For high-grade tumors, the performance of the specimen classifier was generally comparable to the visual diagnosis. The specimen classifier unexpectedly identified twice as many low-grade papillary urothelial tumors as abnormal than did the visual analysis. Several false "alarms" were recorded by computer in patients with benign prostatic hypertrophy and prostatic carcinoma, some of whom had atypical urothelium. One of the 16 negative controls was misdiagnosed by the computer as abnormal. The possibility that the video system recognizes nuclear abnormalities not perceived by the human eye is being investigated further. The details of the computer analysis are reported, and the value of the system is discussed. The system appears to be promising as a future laboratory instrument, although it requires further extensive testing.     

A rapid, semi-automated method for scoring micronuclei in mononucleated mouse lymphoma cells

A semi-automated scoring system has been developed to provide rapid, accurate assessment of micronuclei in preparations of mononuclear mouse lymphoma L5178Y cells. Following exposure to a range of test agents, flat, single-cell preparations were produced from exponentially growing cultures by cytocentrifugation. Following staining with 4'-6-diamidino-2-phenylindole (DAPI), cells were scanned by use of the MicroNuc module of Metafer 4 v 3.4.102, after modifying the classifier developed for selecting micronuclei in binucleate cells to increase its sensitivity. The image gallery of all cells was then sorted to bring aberrant cells to the top of the gallery to assess visually the numbers of cells with micronuclei, as distinct from other debris. Slide quality was shown to be paramount in obtaining accurate results from an automated scan and the data obtained compared very well with the incidence of micronuclei scored conventionally by microscopy. Compared with manual scoring the time saving is considerable, as more than 2000 images are captured in approximately 2min, with subsequent visual assessment of aberrant cells in the image gallery taking about 1-2min/slide. By scanning all aberrant cells, the system also captures additional information on necrotic, apoptotic and fragmented cells. Although optimised for mouse lymphoma cells, it should be simple to adapt the method for any cell type growing in suspension.