Negative dielectrophoretic patterning with different cell types

In this paper, a novel method for patterning different cell types based on negative dielectrophoresis (n-DEP), without any special pretreatment of a culture slide, has been described. An interdigitated array (IDA) electrode with four independent microelectrode subunits was fabricated with indium-tin-oxide (ITO) and used as a template to form cellular micropatterns. A suspension of C2C12 cells was introduced into the patterning device between the upper slide and the bottom IDA. In the present system, the n-DEP force is induced by applying an ac voltage (typically 12V(pp), 1MHz) to direct cells toward a weaker region of electric field strength. The cells aligned above one of the bands of IDA within 1min since the aligned areas on the slide were regions with the lower electric field. The application of an ac voltage for 5min allows the cells to adsorb onto the cell culture slide. After removing excess cells, the second cell type was patterned in lines using the same method as with the first set of cells. Periodic and alternate cell lines incorporating two cell types were also fabricated by changing the ac voltage mode. A second cell type was introduced into the device and guided to other areas to form a different pattern. The described system enables two cell types to be patterned in 15min. The patterning method provides a novel tool for use in fundamentals studies of cell biology based on cell-cell interactions between different cell types.     

A simple method for enhancing hybridization efficiency in chromosome and array comparative genomic hybridization

Abstract

The accuracy of comparative genomic hybridization (CGH) analysis is affected by hybridization efficiency. We describe here a simple method for enhancing hybridization efficiency. The hybridization procedure is essentially the same as that of conventional methods. Hybridization solution containing denatured DNA probe mixture was applied to a metaphase chromosome slide or DNA chip slide and covered with a coverslip. In the new method, however, the slide was inverted by turning the coverslip downward prior to hybridization. We termed this method the inverted slide method. To estimate the efficiency of the new method, metaphase chromosome slides and DNA chip slides were treated by both the conventional and inverted slide methods and incubated in a moist chamber at 37°C for 12, 24, 48, and 72 h. Hybridization signals were approximately 1.5 to 2 times brighter on the slides using the inverted slide method than those using the conventional method after 48 and 72 h of incubation. Furthermore, topographical differences in fluorescence intensity were smaller in slides using the inverted-slide method than in those prepared by the conventional method. The inverted slide method is methodologically very simple and improves the resolution of CGH.     

Posters. P3 North west non‐gynaecological cytology EQA pilot CD ROM scheme 2002–2003

The 8th circulation of this scheme was preceded by a CD ROM circulation of selected digital images and presented as individual JPEG file images in a single folder on the CD ROM. The number of images per case ranged from three to eight. The delegates will have an opportunity to assess these themselves prior to uncovering the consensus diagnosis provided by the participants. Of the 76, 29 participants of the scheme provided a completed answer sheet for the image‐based circulation. To date, 19 have now also supplied answers on the actual slide circulation. The same diagnosis on image and slide circulation varied from 5/19 to 19/19 with a mode of 14/19. The case that provided the poorest correlation was because of a generalized undercall of suspicious in a case which achieved almost complete consensus on the slide circulation. It had been the most difficult to photograph. The case that provided the next poorest correlation 11/19 was the case that had the poorest consensus diagnosis on the slide circulation. The two cases that provided the best correlation 18/19 and 19/19 were two cases that provided 100% consensus diagnosis on the slide‐based circulation. Comments were received from 20 participants about the image circulation ranging from ‘great’ to ‘awful’ with the majority of 12 participants not happy for their diagnostic capability to be assessed on such images alone. Two stated that the CD was easier to use on their home computer than their NHS one. In conclusion an image circulation overcomes many of the inherent problems with a slide circulation for EQA purposes and can provide an overall 70% correlation with a slide circulation but a significant number of pathologists do not find this an acceptable method for EQA.     

Freezing by Liquid Carbon Dioxide in Making Slides Permanent

The expansion of liquid CO₂ may be employed in a quick-freeze method for making aqueous slide preparations permanent. An apparatus is described for this purpose which could be duplicated satisfactorily by cutting a 22mm square hole in the top of a standard freezing microtome specimen holder. The edges should be filed smooth to provide a flat surface for the slide to rest on, and clamps added to keep the slide in place while freezing. Once the slide is frozen, the cover slip may be readily removed, leaving practically all of the tissue on the slide. Following simultaneous thawing and dehydration of the slide in 95% alcohol, covering is done with Diaphane or Euparal and a clean, dry cover slip.     

介绍一种半薄切片定位样品的方法

介绍了一种半薄切片定位样品的方法。此法与前人的方法不同之处在于位于载玻片之上被重新包埋于塑料环内的切片与载玻片的分离真人处步骤是“切片被包埋聚合好后,立即从６０￣６５℃温箱内被转入冰箱冷冻室中（－１８℃）放置５￣１０分钟。然后,将载玻征从冷冻室中取出,轻推塑料环,即可使包埋在塑料环内的切片与载玻片分离。这一方法成功地解决了样品中靶细胞的发育时期确定和样品丢失问题。而且还有简单、易操作和成功率高等优     

介绍一种半薄切片定位样品的办法

介绍了一种半薄切片定位样品的方法。此法与前人的方法不同之处在于位于载玻片之上被重新包埋于塑料环内的切片与载玻片的分离方法。具体操作步骤是：切片被包埋聚合好后,立即从60～65 ℃温箱内被转入冰箱冷冻室中(-18 ℃)放置5～10分钟。然后,将载玻片从冷冻室中取出,轻推塑料环,即可使包埋在塑料环内的切片与载玻片分离。这一方法成功地解决了样品中靶细胞的发育时期确定和样品丢失问题,而且还有简单、易操作和成功率高等优点。     

A method for fast safety screening of explosives in terms of crystal packing and molecular stability

Safety prediction is crucial to the molecular design or the material design of explosives, and the predictions based on any single factor alone will cause much inaccuracy, leading to a desire for a method on multi-bases. The presented proposes an improved method for fast screening explosive safety by combining a crystal packing factor and a molecular one, that is, steric hindrance against shear slide in crystal and molecular stability, denoted by intermolecular friction symbol (IFS) and bond dissociation energy (BDE) of trigger linkage respectively. Employing this BDE-IFS combined method, we understand the impact sensitivities of 24 existing explosives, and predict those of two energetic-energetic cocrystals of the observed CL-20/BTF and the supposed HMX/TATB. As a result, a better understanding is implemented by the combined method relative to molecular stability alone, verifying its improvement of more accurate predictions and the feasibility of IFS to graphically reflect molecular stacking in crystals. Also, this work verifies that the explosive safety is strongly related with its crystal stacking, which determines steric hindrance and influences shear slide.     

Color standardization method and system for whole slide imaging based on spectral sensing

In the field of whole slide imaging, the imaging device or staining process cause color variations for each slide that affect the result of image analysis made by pathologist. In order to stabilize the analysis, we developed a color standardization method and system as described below. (1) Color standardization method based on RGB imaging and multi spectral sensing, which utilize less band (16 bands) than conventional method (60 bands). (2) High speed spectral sensing module. As a result, we confirmed the following effect. (1) We confirmed the performance improvement of nucleus detection by the color standardization. And we can conduct without training data set which is needed in conventional method. (2) We can get detection performance of H&E component equivalent to conventional method (60 bands). And measurement process is more than 255 times faster.     

Protein microarrays on hybrid polymeric thin films prepared by self-assembly of polyelectrolytes for multiple-protein immunoassays

We report here the development and characterization of protein microarrays fabricated on nanoengineered 3-D polyelectrolyte thin films (PET) deposited on glass slide by consecutive adsorption of polyelectrolytes via self-assembly technique. Antibodies or antigens were immobilized in the PET-coated glass slides by electrostatic adsorption and entrapment of porous structure of the 3-D polymer film and thus establishing a platform for parallel analysis. Both antigen and antibody microarrays were fabricated on the PET-coated slides, and direct and indirect immunoassays on protein microarrays for multiple-analyte detection were demonstrated. Microarrays produced on these PET-coated slides have consistent spot morphology and provide performance features needed for proteomic analysis. The protein microarrays on the PET films provide LOD as low as 6 pg/mL and dynamic ranges up to three orders of magnitude, which are wider than the protein microarrays fabricated on aldehyde and poly-L-lysine functionalized slides. The PET films constructed by self-assembly technique in aqueous solution is green chemistry based, cost-effective method to generate 3-D thin film coatings on glass surface, and the coated slide is well suited for immobilizing many types of biological molecules so that a wide variety of microarray formats can be developed on this type of slide.     

Image Recognition of Breast Tumor Proliferation Level Based on Convolution Neural Network

Pathological slide is increasingly applied in the diagnosis of breast tumors despite the issues of large amount of data, slow viewing and high subjectivity. To overcome these problems, a micrograph recognition method based on convolutional neural network is proposed for pathological slide of breast tumor. Combined with multi-channel threshold and watershed segmentation, a sample database including single cell, adhesive cell and invalid cell was established. Then, the convolution neural network with six layers is constructed, which has ability to classify the stained breast tumor cells with accuracy of more than 90%, and evaluate the proliferation level with relative error of less than 5%. The experimental result indicates the effectiveness of this approach, and is useful for providing an objective basis for evaluating the malignancy of breast tumors.     

A Method for Staining Certain Bacteria and Antherozoids

A modification of Loeffler's method for staining the flagella of bacteria was employed in staining large forms of bacteria and antherozoids. The bacteria or the antherozoids are killed and fixed in a drop of water on a slide and set aside to dry, before the next step is undertaken. The slide is treated for a period of time, varying from about ten minutes to several hours, in a practically saturated solution of tannic acid. After the slide is thoroly rinsed in water, it is stained with either a single stain or a combination of stains. The slide is then dehydrated with absolute alcohol, cleared, with clove oil, and completed in the usual manner.

The body of the bacterium and that of the antherozoid are well differentiated and the cilia are distinctly brought out by means of the method herein described.

The technic is of especial value in staining the antherozoids of mosses, liverworts, and ferns.     

Detection of suspicious cells and rejection of artefacts in cervical cytology using the Leyden Television Analysis System.

In slide based automation of cervical cytology the first stage of analysis involves finding possibly suspicious cells, or areas on the slide with these types of cells. By using a television based system such as the Leyden Television Analysis System (LEYTAS), a number of detection methods can be applied to rapidly screen a large number of fields automatically for suspicious cells. In this paper, results using a parameter based on increased nuclear DNA content of cells are given and a second detection method based on a chromatin pattern feature, called chromatin contrast, is discussed. Two blind trials on 41 positive and 22 negative cervical slides, using the Leyden Television Analysis System to detect suspicious cells with an increased nuclear DNA content, were promising. In 1 of the 41 positive cases no suspicious cells were found. In the negative specimens, suspicious cells were detected in 1 of 9 cases and 1 of 13 cases, with the two detection parameters investigated. These findings are discussed and some automatic artefact rejection procedures with preliminary results are given.     

玉米和花生根际生物的原位观察初探*

利用改进根箱法对玉米和花生根际生物进行了原位采样和观察。采用载玻片贴片方法直接采集到作物根系分泌物中的生物,并用荧光显微镜进行观察。花生根系分泌物中分布有微生物,但是根系表面未发现生物;玉米根系分泌物中分布有线虫和生物体,根系表面分布有生物体,其形态与根系分泌物中生物体形态相似。表明此方法是一种原位研究根际生物的较为有效的方法。     

Darkfield Adapter for Whole Slide Imaging: Adapting a Darkfield Internal Reflection Illumination System to Extend WSI Applications

We present a new method for whole slide darkfield imaging. Whole Slide Imaging (WSI), also sometimes called virtual slide or virtual microscopy technology, produces images that simultaneously provide high resolution and a wide field of observation that can encompass the entire section, extending far beyond any single field of view. For example, a brain slice can be imaged so that both overall morphology and individual neuronal detail can be seen. We extended the capabilities of traditional whole slide systems and developed a prototype system for darkfield internal reflection illumination (DIRI). Our darkfield system uses an ultra-thin light-emitting diode (LED) light source to illuminate slide specimens from the edge of the slide. We used a new type of side illumination, a variation on the internal reflection method, to illuminate the specimen and create a darkfield image. This system has four main advantages over traditional darkfield: (1) no oil condenser is required for high resolution imaging (2) there is less scatter from dust and dirt on the slide specimen (3) there is less halo, providing a more natural darkfield contrast image, and (4) the motorized system produces darkfield, brightfield and fluorescence images. The WSI method sometimes allows us to image using fewer stains. For instance, diaminobenzidine (DAB) and fluorescent staining are helpful tools for observing protein localization and volume in tissues. However, these methods usually require counter-staining in order to visualize tissue structure, limiting the accuracy of localization of labeled cells within the complex multiple regions of typical neurohistological preparations. Darkfield imaging works on the basis of light scattering from refractive index mismatches in the sample. It is a label-free method of producing contrast in a sample. We propose that adapting darkfield imaging to WSI is very useful, particularly when researchers require additional structural information without the use of further staining.     

Elimination of Material that Obscures Stained Chromosomes in Squashes of Vicia faba Root Tips

A procedure for elimination of cytoplasmic debris from Vicia faba root tip cells is: (1) a root tip previously fixed in 3:1 absolute alcohol-acetic acid and stained by the Feulgen method is placed on a slide and squashed in a small drop of water, (2) a cover slip is applied and the cells are flattened with a hand-operated lever device supplying 35 pounds pressure onto a 22 × 22 mm cover glass, (3) the slide is quick-frozen, the cover slip is removed, and the slide is dropped immediately into water, (4) the slide is cleared through an alcohol-xylene dehydration series and permanently mounted. The significant result of this procedure is the consistent presence of clear, flat cells showing excellent definition of chromosomes.     

A TMA de-arraying method for high throughput biomarker discovery in tissue research

Background

Tissue MicroArrays (TMAs) represent a potential high-throughput platform for the analysis and discovery of tissue biomarkers. As TMA slides are produced manually and subject to processing and sectioning artefacts, the layout of TMA cores on the final slide and subsequent digital scan (TMA digital slide) is often disturbed making it difficult to associate cores with their original position in the planned TMA map. Additionally, the individual cores can be greatly altered and contain numerous irregularities such as missing cores, grid rotation and stretching. These factors demand the development of a robust method for de-arraying TMAs which identifies each TMA core, and assigns them to their appropriate coordinates on the constructed TMA slide.

Methodology

This study presents a robust TMA de-arraying method consisting of three functional phases: TMA core segmentation, gridding and mapping. The segmentation of TMA cores uses a set of morphological operations to identify each TMA core. Gridding then utilises a Delaunay Triangulation based method to find the row and column indices of each TMA core. Finally, mapping correlates each TMA core from a high resolution TMA whole slide image with its name within a TMAMap.

Conclusion

This study describes a genuine robust TMA de-arraying algorithm for the rapid identification of TMA cores from digital slides. The result of this de-arraying algorithm allows the easy partition of each TMA core for further processing. Based on a test group of 19 TMA slides (3129 cores), 99.84% of cores were segmented successfully, 99.81% of cores were gridded correctly and 99.96% of cores were mapped with their correct names via TMAMaps. The gridding of TMA cores were also extensively tested using a set of 113 pseudo slide (13,536 cores) with a variety of irregular grid layouts including missing cores, rotation and stretching. 100% of the cores were gridded correctly.     

Zymomonas mobilis cell viability: measurement method comparison

Comparison of three different cell viability methods: slide count, plate count and methylene blue staining techniques, applied onZymomonas mobilis cultures, was performed. The slide technique proved to be faster and more accurate than the plate count method, and both of them far more reliable than the standard methylene blue method which constantly overestimated theZymomonas cell viability. The slide technique is advantageous also because it gives information on the cell morphology changes, notably the abnormal cell elongation, in the ethanol fermentation.     

Comparison between slide and flow cytophotometric DNA measurements in breast tumors

Nuclear DNA analysis was performed in 37 human mammary adenocarcinomas in order to elucidate the difficulties and pitfalls connected with the interpretation of DNA histograms obtained using different methodologic approaches. For each tumor, DNA profiles were obtained by means of slide microspectrophotometry on a fine needle aspirate, slide cytophotometry on a 4-micron histologic section and flow cytometry on a suspension prepared from a cube of fresh tissue. When the DNA histograms were interpreted according to criteria usually applied to discriminate low-grade malignant tumors from high-grade malignant tumors, some tumors classified as euploid by one method were classified as aneuploid by another method. The main reasons for this weak correlation seem to be in specimen preparation and in tumor cell representation within the specimen between the methods. Another reason is that slide and flow techniques exhibit different sensitivities for malignancy-associated nuclear DNA changes: minor alterations of the DNA content of the tumor stemlines seem to be more exactly reported by means of the flow technique whereas structural alterations of the nuclear chromatin seem to be more sensitively recorded by means of the slide technique. It is suggested that thorough control of each step of the various DNA analysis procedures and the use of information obtainable by slide and flow techniques taken together may significantly improve the prognostic value of DNA measurements.     

Technic for Studying Chromosome Structure

The methods described are modifications of various technics for the study of spiral structure in chromosomes. They enable permanent preparations to be made with better fixation and allow the use of stains which give clear and more critical definition. The first method described involves the use of ammonium, hydroxide (880 vols.) fumes for the treatment of pollen mother cells before fixation. Anthers of Tradescantia are smeared on a slide and wet in a 3% cane sugar solution. The preparation is then immediately placed in a dish of fixative where it remains for two hours. The slide can then be washed, bleached and stained with gentian violet or hematoxylin. It was found that fumes of nitric acid, hydrochloric acid and glacial acetic acid gave similar results. For the second method, boiling water is used for pre-treatment. A smear is made on a slide and immersed in boiling water for five to ten seconds. The smear is then fixed and treated in the usual manner.