scholarly journals New Concept and Apparatus for Cytocentrifugation and Cell Processing for Microscopy Analysis

2021 ◽  
Vol 22 (13) ◽  
pp. 7098
Author(s):  
Anna Ligasová ◽  
Karel Koberna
Keyword(s):  
Image Cytometry ◽  
Staining Procedure ◽  
Sample Holder ◽  
Cell Nuclei ◽  
Sample Processing ◽  
Efficient Procedure ◽  
Threaded Connection ◽  
Common Technique ◽  
Microscopy Analysis ◽  
Paper Filter

Cytocentrifugation is a common technique for the capture of cells on microscopic slides. It usually requires a special cytocentrifuge or cytorotor and cassettes. In the study presented here, we tested the new concept of cytocentrifugation based on the threaded connection of the lid and the sample holder to ensure an adjustable flow of solutions through the filters and the collection of the filtered solutions in the reservoir during centrifugation. To test this concept, we developed a device for the preparation of cell samples on circular coverslips. The device was tested for the capture and sample processing of both eukaryotic and prokaryotic cells, cell nuclei, and mitochondria for microscopy analysis including image cytometry. Moreover, an efficient procedure was developed for capturing formaldehyde-fixed cells on non-treated coverslips without cell drying. The results showed that the tested arrangement enables the effective capture and processing of all of the tested samples and the developed device represents an inexpensive alternative to common cytocentrifuges, as only the paper filter is consumed during sample processing, and no special centrifuge, cytorotor, or cassette is necessary. As no additional system of solution removal is required during sample staining, the tested concept also facilitates the eventual automation of the staining procedure.

Generation of digital phantoms of cell nuclei and simulation of image formation in 3D image cytometry

2009 ◽  
Vol 75A (6) ◽  
pp. 494-509 ◽  
Author(s):  
David Svoboda ◽  
Michal Kozubek ◽  
Stanislav Stejskal
Keyword(s):  
Image Formation ◽  
Image Cytometry ◽  
3D Image ◽  
Cell Nuclei ◽  
Digital Phantoms

scholarly journals Detection of Cervical Cancer and High Grade Neoplastic Lesions by a Combination of Liquid‐Based Sampling Preparation and DNA Measurements Using Automated Image Cytometry

2005 ◽  
Vol 27 (1) ◽  
pp. 33-41
Author(s):  
Xiao Rong Sun ◽  
Jian Wang ◽  
David Garner ◽  
Branko Palcic
Keyword(s):  
Cervical Cancer ◽  
Clinical Intervention ◽  
Image Cytometry ◽  
Population Based ◽  
High Grade ◽  
Cell Nuclei ◽  
Dna Amount ◽  
Screening Programs ◽  
Neoplastic Lesions ◽  
Dna Measurements

Objective: To establish if measurements of DNA ploidy could be used to assist cytopathologists and cytotechnologists in population based cervical cancer screening programs in countries where manually reading the slides is impossible due to the lack of sufficient skilled cytotechnologists. The goal of such program is to identify only clinically significant lesions, i.e. those where a clinical intervention to remove the lesion is required immediately. Study Design: A total of 9905 women were enrolled in the study. Cervical samples were taken with a cervix brush that was then placed into a fixative solution. The cells were separated from mucus by mechanical and chemical treatment and then deposited onto microscope slides by a cytocentrifuge. Two slides were prepared from each case; one slide was stained by Papanicolaou stain for manual cytology examination, while the other slide was stained by a DNA specific stain. The latter slide was used to determine the relative amount of DNA in the cell nuclei. Results: A total of 876 women were followed by colposcopy examination where biopsies were taken from the visible lesions or from suspicious areas and histopathology diagnosed 459 as normal or benign cases, 325 as CIN1, 36 as CIN2, 25 as CIN3/CIS, and 31 as invasive cancer. Of these 876 cases, manual cytology called 655 normal or ASCUS, 197 as LSIL, 16 cases as HSIL, and 8 as cancer. DNA measurements found 704 cases having no cells with DNA greater than 5c, 98 cases where there were 1 or 2 cells having DNA amount greater than 5c, and 74 cases where there were 3 or more cells having DNA amount greater than 5c. If manual cytology were to be used to refer all cases of HSIL and cancer to colposcopy and biopsy, 23 lesions that had to be removed would have been discovered (2 CIN2, 11 CIN3/CIS, and 10 cancers), for a sensitivity of 25.0±5.2% at specificity of 99.9±0.1%. If DNA assisted cytology were to be used instead, and all cases having 3 or more cells with DNA amount greater than 5c were to be referred to colposcopy and biopsy, then 50 lesions that had to be removed would have been discovered (10 CIN2, 15 CIN3/CIS and 25 cancers) for the sensitivity of 54.3±6.2% at specificity of 96.9±0.6%. Conclusions: The study suggests that screening for high grade cervical neoplastic lesions and cervical cancer by DNA assisted cytology could be implemented with minimal use of skilled cytotechnologists, at least in those countries where it would be difficult to introduce population based screening for cervical cancer due to the lack of availability of such skilled cytotechnologists.

Analyses of DNA Image Cytometry Uncertainty Caused by Diffractive Blurring

2017 ◽  
Vol 870 ◽  
pp. 369-374
Author(s):  
Irina G. Palchikova ◽  
Evgenii S. Smirnov ◽  
Alexander A. Konev
Keyword(s):  
Quantitative Methods ◽  
Glass Plate ◽  
Image Cytometry ◽  
Local Algorithm ◽  
Cell Nuclei ◽  
Image Study ◽  
Brightness Gradient ◽  
Quantitative Results ◽  
Chromium Films ◽  
Test Objects

Comparison of quantitative methods for the segmentation of nuclei images is performed. A sizeable variability is typical for biological specimens and it induces the elemental uncertainty in experimental data. To remove the variability from comparison we have proposed and built the special test objects of two types simulating the nuclei images. I-type objects are test patterns as sets of circles and squares of specified dimensions in chromium films on a glass plate. II-type objects are images with brightness differentials that simulate diffractive blurring and are built up with MathCad programming environment. Test objects enable objective comparison of characteristics obtained in the course of their quantitative optical-and-structural analysis using various algorithms and programs. We found that the efficiency of the segmentation algorithm depends on diffractive blurring of the image. Specifics of Otsu’s algorithm and local algorithm of brightness gradient are analyzed for finding the segmentation threshold of digital images modeling transmission Feulgen-stained cell nuclei specimens with diffractive blurring. The performed calculations revealed that the border of geometrooptical image practically coincide with the points of inflection on the intensity distribution graph in a test-object image space. Computational experiments show that quantitative results of the morphometric image study defined by the various segmentation algorithms vary within 5%. It is established that the threshold-identifying algorithm based on the brightness gradient is preferable in the image cytometry.

scholarly journals Large-Scale Isolation of Cajal Bodies from HeLa Cells

2002 ◽  
Vol 13 (7) ◽  
pp. 2461-2473 ◽  
Author(s):  
Yun Wah Lam ◽  
Carol E. Lyon ◽  
Angus I. Lamond
Keyword(s):  
Large Scale ◽  
Cultured Cells ◽  
Cajal Body ◽  
Cell Nuclei ◽  
Cellular Processes ◽  
Electron Microscopy Analysis ◽  
Microscopy Analysis ◽  
Composition Structure ◽  
Transcription Complexes

The Cajal body (CB) is a conserved, dynamic nuclear structure that is implicated in various cellular processes, such as the maturation of splicing small nuclear ribonucleoproteins and the assembly of transcription complexes. Here, we report the first procedure for the large-scale purification of CBs from HeLa cell nuclei, resulting in an ∼750-fold enrichment of the CB marker protein p80-coilin. Immunofluorescence, immunoblotting, and mass spectrometric analyses showed that the composition of the isolated CBs was similar to that of CBs in situ. The morphology and structure of the isolated CBs, as judged by transmission and scanning electron microscopy analysis, are also similar to those of CBs in situ. This protocol demonstrates the feasibility of isolating intact distinct classes of subnuclear bodies from cultured cells in sufficient yield and purity to allow detailed characterization of their molecular composition, structure, and properties.

Ca-Histochemistry in slime mold plasmodia

1978 ◽  
Vol 36 (2) ◽  
pp. 130-131
Author(s):  
Ulrich Dierkes
Keyword(s):  
Physarum Polycephalum ◽  
Carbon Coating ◽  
Freeze Drying ◽  
Freeze Fracture ◽  
Freeze Substitution ◽  
Uranyl Acetate ◽  
Slime Mold ◽  
Staining Procedure ◽  
Protoplasmic Streaming ◽  
Intracellular Ca

Calcium is supposed to play an important role in the control of protoplasmic streaming in slime mold plasmodia. The motive force for protoplasmic streaming is generated by the interaction of actin and myosin. This contraction is supposed to be controlled by intracellular Ca-fluxes similar to the triggering system in skeleton muscle. The histochemical localisation of calcium however is problematic because of the possible diffusion artifacts especially in aquous media.To evaluate this problem calcium localisation was studied in small pieces of shock frozen (liquid propane at -189°C) plasmodial strands of Physarum polycephalum, which were further processed with 3 different methods: 1) freeze substitution in ethanol at -75°C, staining in 100% ethanol with 1% uranyl acetate, and embedding in styrene-methacrylate. For comparison the staining procedure was omitted in some preparations. 2)Freeze drying at about -95°C, followed by immersion with 100% ethanol containing 1% uranyl acetate, and embedding. 3) Freeze fracture, carbon coating and SEM investigation at temperatures below -100° C.

Intranuclear Crystals in Regenerating Canine Kidneys

1973 ◽  
Vol 31 ◽  
pp. 412-413
Author(s):  
D.G. Osborne ◽  
L.J. McCormack ◽  
M.O. Magnusson ◽  
W.S. Kiser
Keyword(s):  
Epithelial Cell ◽  
Epithelial Cells ◽  
Tubular Epithelial Cell ◽  
Tubular Epithelial Cells ◽  
Cell Nuclei ◽  
Crystalline Structures ◽  
Small Crystals ◽  
Membrane Bound

During a project in which regenerative changes were studied in autotransplanted canine kidneys, intranuclear crystals were seen in a small number of tubular epithelial cells. These crystalline structures were seen in the control specimens and also in regenerating specimens; the main differences being in size and number of them. The control specimens showed a few tubular epithelial cell nuclei almost completely occupied by large crystals that were not membrane bound. Subsequent follow-up biopsies of the same kidneys contained similar intranuclear crystals but of a much smaller size. Some of these nuclei contained several small crystals. The small crystals occurred at one week following transplantation and were seen even four weeks following transplantation. As time passed, the small crystals appeared to fuse to form larger crystals.

Preparation of cross-sectional samples for near-surface TEM studies

1987 ◽  
Vol 45 ◽  
pp. 380-381
Author(s):  
R.C. Dickenson ◽  
K.R. Lawless
Keyword(s):  
Standard Sample ◽  
Surface Region ◽  
Specimen Preparation ◽  
Thick Sheet ◽  
Drill Bit ◽  
Sample Holder ◽  
Metal Interface ◽  
Cross Sectional ◽  
Near Surface ◽  
Cold Worked

In thermal oxidation studies, the structure of the oxide-metal interface and the near-surface region is of great importance. A technique has been developed for constructing cross-sectional samples of oxidized aluminum alloys, which reveal these regions. The specimen preparation procedure is as follows: An ultra-sonic drill is used to cut a 3mm diameter disc from a 1.0mm thick sheet of the material. The disc is mounted on a brass block with low-melting wax, and a 1.0mm hole is drilled in the disc using a #60 drill bit. The drill is positioned so that the edge of the hole is tangent to the center of the disc (Fig. 1) . The disc is removed from the mount and cleaned with acetone to remove any traces of wax. To remove the cold-worked layer from the surface of the hole, the disc is placed in a standard sample holder for a Tenupol electropolisher so that the hole is in the center of the area to be polished.

Problems with oxygen analysis in the system MgO-Al2O3-SiO2 with the electron microprobe

1992 ◽  
Vol 50 (2) ◽  
pp. 1624-1625
Author(s):  
Michel Fialin ◽  
Guy Rémond
Keyword(s):  
Transition Metal ◽  
Metal Oxides ◽  
Transition Metal Oxides ◽  
Electrostatic Charge ◽  
Carbon Layer ◽  
Sample Holder ◽  
Rock Matrix ◽  
Electrical Discharges ◽  
Thin Carbon ◽  
Beam Instabilities

Oxygen-bearing minerals are generally strong insulators (e.g. silicates), or if not (e.g. transition metal oxides), they are included within a rock matrix which electrically isolates them from the sample holder contacts. In this respect, a thin carbon layer (150 Å in our laboratory) is evaporated on the sections in order to restore the conductivity. For silicates, overestimated oxygen concentrations are usually noted when transition metal oxides are used as standards. These trends corroborate the results of Bastin and Heijligers on MgO, Al2O3 and SiO2. According to our experiments, these errors are independent of the accelerating voltage used (fig.l).Owing to the low density of preexisting defects within the Al2O3 single-crystal, no significant charge buildup occurs under irradiation at low accelerating voltage (< 10keV). As a consequence, neither beam instabilities, due to electrical discharges within the excited volume, nor losses of energy for beam electrons before striking the sample, due to the presence of the electrostatic charge-induced potential, are noted : measurements from both coated and uncoated samples give comparable results which demonstrates that the carbon coating is not the cause of the observed errors.

Material analysis techniques using the ion mill

1996 ◽  
Vol 54 ◽  
pp. 1034-1035
Author(s):  
J. P. Benedict ◽  
R. M. Anderson ◽  
S. J. Klepeis
Keyword(s):  
Polished Surface ◽  
Surface Material ◽  
Analysis Techniques ◽  
Material Analysis ◽  
Optical Inspection ◽  
Electron Microscopy Analysis ◽  
Microscopy Analysis ◽  
Argon Ions ◽  
The Impact ◽  
Scanning Electron

Ion mills equipped with flood guns can perform two important functions in material analysis; they can either remove material or deposit material. The ion mill holder shown in Fig. 1 is used to remove material from the polished surface of a sample for further optical inspection or SEM ( Scanning Electron Microscopy ) analysis. The sample is attached to a pohshing stud type SEM mount and placed in the ion mill holder with the polished surface of the sample pointing straight up, as shown in Fig 2. As the holder is rotating in the ion mill, Argon ions from the flood gun are directed down at the top of the sample. The impact of Argon ions against the surface of the sample causes some of the surface material to leave the sample at a material dependent, nonuniform rate. As a result, the polished surface will begin to develop topography during milling as fast sputtering materials leave behind depressions in the polished surface.

A pre-embedding double staining procedure used to observe the effect of fixation on the activity of intercellular adhesion molecule on T and B cells

1990 ◽  
Vol 48 (3) ◽  
pp. 378-379
Author(s):  
Jane E. Ramberg ◽  
Shigeto Tohma ◽  
Peter E. Lipsky
Keyword(s):  
B Cells ◽  
Adhesion Molecule ◽  
Colloidal Gold ◽  
Intercellular Adhesion ◽  
Intercellular Adhesion Molecule ◽  
Staining Procedure ◽  
Double Staining ◽  
T And B Cells ◽  
Microtiter Plates ◽  
Streptavidin Peroxidase

Intercellular adhesion molecule (ICAM-1) appears to be a ligand for LFA-1 dependent adhesion in T cell mediated cytotoxcity. It is found on cells of both hematopoietic and non-hematopoietic origin. While observing the activity of ICAM-1 on the surfaces of interacting T and B cells, we found that we could successfully carry out a pre-embedding double staining procedure utilizing both colloidal gold and peroxidase conjugated reagents.On 24-well microtiter plates, mitomycin-treated T4 cells were stimulated with 64.1 (anti-CD3) for one hour before the addition, in some instances, of B cells. Following a 12-48 hour incubation at 38°C, the cells were washed and then immunostained with a colloidal gold conjugated RFB-4 (anti-CD22); biotinylated R6.5 (anti-ICAM-1); followed by streptavidin/peroxidase. This method allowed us to observe two different antigens without concern about possible cross-reaction of reagents. Because we suspected ICAM-1 and R6.5 were sensitive to fixation, we tried varying concentrations of fresh paraformaldehyde before R6.5, after R6.5 and after streptavidin/peroxidase. All immunostaining and washing was done on ice with ice cold reagents.

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