J S Ploem's research while affiliated with Leiden University and other places
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Publications (133)
Die Weiterentwicklung vollautomatischer Bildanalysesysteme für die vaginalzytologische Diagnostik haben einen Stand erreicht, der es sinnvoll erscheinen ließ, den Schritt von einem Forschungslabor in ein zytologisches Routinelabor zu tun. An der Universität von Leiden wurden mit dem Vorläufer des automatischen Zellanalysesystems Autoplan MIAC (Modu...
To investigate whether breast cancer cells with unusually high nuclear DNA content are associated with an adverse outcome, Eastern Cooperative Oncology Group investigators selected breast cancer trial patients who suffered an early death (ED) within two years after diagnosis to compare with other trial patients who had a survival of at least 7.5 ye...
An automated cell analysis system (Autoplan-MIAC) for the early detection of precancerous lesions of the cervix was tested under semi-routine conditions in a clinical cytology laboratory. A set of 1500 specimens, highly enriched with abnormal cases, was analysed. Cervical scrapings were collected in suspension and processed by cytocentrifugation fo...
Nuclear DNA content in soft tissue sarcoma was determined by image cytometry using archival, paraffin embedded material. In a retrospective study 138 specimens of 81 patients have been analysed. The ploidy level was correlated to clinical outcome regarding tumor volume and histological grading, the most important prognostic parameters. Ploidy has a...
The preparation of charge-stabilized suspensions of small phosphor particles (0.1-0.3 micron) and their coupling with antibodies to immunoreactive conjugates is described. Phosphor particles consisting of yttriumoxisulfide activated with europium served as a model system in the evaluation of the stabilizing properties of several polycarboxylic acid...
Thirty-eight renal transplant recipients were followed during the first 3 months after transplantation. Once weekly, cultures of urine and buffy coat for cytomegalovirus (CMV) were taken and an immunocytochemical assay for immediate early antigens of CMV (IEA assay) was performed. Thirty patients had evidence of a CMV infection and 11 had a symptom...
Traditionally, cellular constituents labelled with fluorescent markers have been interpreted visually using a normal fluorescence microscope. During the past decades however, developments such as fluorescently labelled monoclonal antibodies and hybridocytochemistry have increased the demands on microscope methods and thereby stimulated instrumentat...
This minireview presents the state of the art with respect to automated detection of micronuclei (MN) in binucleated lymphocytes. Emphasis is on an image analysis technique, based on the principles of mathematical morphology (pattern recognition), which combines a personal computer with an image processing board and a board for microscope control....
In reflection contrast microscopy (RCM) special optics such as a quarter-wave plate and a central stop are used, the latter being unique for Leitz microscope stands and contributing much to contrast enhancement of the image. In this paper we demonstrate that a cap or prism, linked by oil immersion to the glass slide, can be used instead of this cen...
Cervical specimens from 1500 patients were prepared by means of a centrifugation procedure to obtain monolayer specimens suitable for automated screening using a machine. After staining according to Papanicolaou, each specimen was diagnosed by four independent cytologists from two different institutes. Within each institute, noncorresponding screen...
Image cytometry is used more and more for the study of clinical cytology, notably for the determination of morphometrical and densitometrical values, the quantification of monoclonal antibody labelling and the detection of DNA probes after in situ hybridisation. Aspects of automated and interactive image cytometry are discussed, including a brief e...
A method for multiple fluorescence in situ hybridization is described allowing the simultaneous detection of more than three target sequences with only three fluorescent dyes (FITC, TRITC, AMCA), respectively emitting in the green, red, and blue. This procedure is based on the labeling of (DNA) probes with more than one hapten and visualisation in...
This chapter discusses the instruments needed for fluorescence scanning. Scanning microfluorometry is a special form of scanning microphotometry. It has been introduced for the measurement of fluorescence intensities in multiple small spots in the microscope preparation. The method provides information about the distribution of fluorescence intensi...
Image cytometry by means of LEYTAS features analysis of both fresh and archival cellular material. Although not as accurate in ploidy determination as flow cytometry, LEYTAS cytometry incorporates extensive artefact rejection algorithms, thereby allowing detection of low frequency cells. This feature is very useful for the search of rare cells, as...
In this paper, results obtained with a new detection method which combines use of a polyclonal monospecific fluorescent antibody against sickle cell hemoglobin (HbS) (due to a transversion mutation, codon GAG in the normal globin gene is replaced by GTG in the sickle-cell gene, which results in replacement of glutamic acid by valine at position 6 o...
Two techniques are described to enhance the detection of low frequency aneuploid cells in automated cell analysis. One method concerns a cell preparation technique; the other is focused on specific cell selection at the measurement level. The cell preparation method has been designed to select and process the tumour areas in paraffin blocks and can...
Reflection contrast microscopy (RCM) is a sensitive tool to detect minor amounts of precipitated diaminobenzidine (DABox) in immunoperoxidase stained specimens. One of the main issues in immunocytochemistry is the ongoing need for more sensitive and quantitative techniques. Therefore we applied RCM, using a new simple model system, to methods previ...
Modification of cellular DNA has been linked with diverging biological effects, such as cell death, cytostatic effects, mutagenesis and carcinogenesis. Important parameters are the pattern and extent of DNA modification, the proliferative activity of the exposed cell, and its capacity to repair relevant DNA damage. The present paper reviews a speci...
Chemical substances involved in the various aspects of neurotransmission are heterogeneously distributed. Besides specific intraneuronal fluorescence one has to reckon with extraneuronal fluorescence and non-specific fluorescent signals in the background. Consequently the application of quantitative immunocytochemical methods to nervous tissues suc...
It is possible to detect micrometastases in primary breast cancer using immunocytochemical staining of bone marrow smears. However, using the light microscope the procedure is time-consuming and laborious because such cells occur rarely (less than 1 in 10,000). Using an image analysis system, the Leytas machine, and a specially prepared reproducibl...
Image analysis instrumentation presently includes multiparameter microscopy and fast processing. One type of image analysis microscope uses only 1 objective and 2 TV cameras, thereby obtaining 2 magnifications simultaneously: a low magnification for cell selection and a high magnification for further cell analysis. Image analysis is very capable of...
In a retrospective study, a DNA histogram was established from specimens obtained by cystectomy in 65 carcinomas of the bladder (stages pT1 to pT4, pN0, pN1 and pN2). Flow cytometry and automated photocytometry were systematically compared. Automated photocytometry makes it possible to differentiate between diploid, polyploid and aneuploid tumors....
Reflection contrast microscopy (RCM) has proven to be a useful tool for the study of living cells (Ploem 1975). Due to the effective suppression of aspecific reflected light by polarization optics combined with a quarter lambda plate at the front lens of the objective, low intensity reflection signals originating from minor amounts of precipitated...
Forty-five patients with advanced ovarian cancer were studied with both DNA flow cytometry (FCM) and automatic DNA image cytometry carried out with the Leiden Television Analysis System (Leytas). There was a significant difference in survival between the diploid and nondiploid cases as determined by FCM. Furthermore, the presence of nuclei with a h...
Fluorescence laser scanning microscopy (LSM) offers many advantages over conventional fluorescence microscopy. Very strong excitation light can be concentrated on small spots (0.5 microm) of the specimen, enabling the detection of low concentrations of fluorescent substances. The low levels of autofluorescence generated in the microscope objective...
A method is described for the preparation of monolayer smears from paraffin-embedded tissue. The smears are suitable for automated image analysis and DNA measurements while still allowing interpretation of nuclear morphology. The proposed technique uses enzyme treatment and syringing for cell dispersal. The preparation of cell monolayers is perform...
In a retrospective study, paraffin-embedded cystectomy specimens obtained from 46 patients with bladder cancer (stage pT1 — pT4a, pN0, pN2) were analysed for tumor DNA ploidy and proliferation using automated image cytometry (LEYTAS). In 41 cases, DNA ploidy could be measured. Estimation of proliferation was possible in 26 tumors. The number of cel...
An instrument combining scanning electron microscopy (SEM) and light microscopy (LM) was used to study the cell surface characteristics and DNA content of macrophages in murine bone marrow cultures. After a quantitative Feulgen DNA staining, the DNA content of the individual macrophages was measured and their cell surface morphology was studied imm...
An instrument combining scanning electron microscopy (SEM) and light microscopy (LM) was used to study the cell surface characteristics and DNA content of macrophages in murine bone marrow cultures. After a quantitative Feulgen DNA staining, the DNA content of the individual macrophages was measured and their cell surface morphology was studied imm...
A simple method is described for the selection of tumor areas in paraffin blocks for cytometry. The surface of a paraffin block is stained with the fluorescent dye DAPI. By means of incident fluorescence microscopy on the cut surface of a total block, the tissue can be visualized. Location of the tumor area with the aid of conventional histopatholo...
To detect plasma membrane antigens, cytocentrifuge preparations of the macrophage-like cell line P388D1 were incubated with monoclonal antibodies and labelled with 5 and 20 nm gold particles conjugated to immunoglobulins or protein A. The 20 nm, but not the 5 nm, particles could be observed by reflection-contrast light microscopy. Single 5 nm parti...
This paper describes the application of image analysis combined with a quantitative staining method for the analysis of cervical specimens. The image analysis is carried out with the Leyden Television Analysis System, LEYTAS, of which two versions are described. LEYTAS-1 as well as LEYTAS-2 have both been designed with a high degree of flexibility...
The response of peripheral blood mononuclear cells to pokeweed mitogen (PWM) stimulation was investigated with combined light (LM) and scanning electron microscopy (SEM). The DNA content of the nucleus of a cell (measured by fluorescence) was compared directly with the diameter and morphological features (by SEM). It was found that upon PWM stimula...
A method is described in which the surface morphology of benign and malignant cervical cells is investigated with a combined light microscope-scanning electron microscope, after the measurement of the DNA content of each individual cell in the same instrument. The suspect cells can thus be identified by an increased aneuploid DNA content (greater t...
An instrument for combined scanning electron microscopy (SEM) and light microscopy (LM) to which a photometer unit is attached is described. A special stage in the vacuum chamber of a scanning electron microscope incorporates light microscope optics (objective and condenser) designed for transmission and epi-illumination fluorescence LM. An optical...
This paper describes the application of image analysis combined with a quantitative staining method for the analysis of cervical specimens. The image analysis is carried out with the Leyden Television Analysis System, LEYTAS, of which two versions are described. LEYTAS-1 as well as LEYTAS-2 have both been designed with a high degree of flexibility...
A new sensitive method for visualization of nonautoradiographic hybridization results in microscopic preparations is described. The method is based on the reflection of the incident light by diaminobenzidine precipitates deposited at the site of hybridization during an indirect hybridocytochemical procedure. The reflected light is detected by means...
The aperture-defined microvolume (ADM) method is based on the relatively constant absorbance or fluorescence of a microvolume of homogeneously coloured material, which is defined by the numerical aperture of the objective.
This paper describes the princile of the method and discusses the equipment needed. The main applications reported so far for t...
Incubation of skin explant culture with HLA-A, -B and -C alloantibodies results in detachment of cells at the periphery of the outgrowth. This detachment can be observed within 1 1/2 h, using the reflection contrast microscope. During the detachment of the epithelial cell, characteristic changes occur, such as disorganization of the filopodia and l...
A method is described for the preparation of monolayer smears from paraffin-embedded tissue suitable for automated image analysis and DNA measurements. The proposed technique uses enzyme treatment and syringing for cell dispersal. Slide preparation is performed by centrifugal cytology. After Feulgen staining the quality of the monolayer smears is s...
In order to develop an objective grading system for nuclear atypia in breast cancer, an image analysis technique has been applied for the automated recognition of enlarged and hyperchromatic nuclei in cytology specimens. The image segmentation algorithm, based on the 'top hat' image transformation developed in mathematical morphology, is implemente...
A centrifugation method for depositing cells on cover-glasses for scanning electron microscopy (SEM) is described. This centrifugation procedure provides a defined and standardized morphology of mononuclear cells from human blood. The method circumvents the highly variable flattening of some blood cells such as monocytes, observed with some methods...
A comparative flow-cytometric and image-cytometric study was performed on 166 human breast cancers. Parallel measurements of 67 cases showed a good correlation between the DNA indices measured with each of the techniques. However, minor ploidy abnormalities were detected with flow cytometry. Only with this technique about 70% of the tumors appeared...
Lavaged and in situ rat alveolar macrophages were compared with respect to lysozyme content and size in order to assess the extent to which macrophages from pulmonary lavages reflect the in situ cell population. This relationship was studied in normal rats and in rats with pulmonary granulomas induced by glucan stimulation (10 mg/kg given intraveno...
The expression of HLA-DR by T lymphocyte subpopulations recognized by monoclonal antibodies and flow cytometry was monitored in 10 normal controls, 15 patients on hemodialysis, 25 recipients of a renal allograft with stable graft function, 16 transplant recipients suffering rejection episodes, and 4 transplant recipients with cytomegalovirus (CMV)4...
Die zytopathologische Untersuchung zu Früherkennung und Diagnose von Tumoren des Blasenepithels ist ein wertvolles und empfindliches Verfahren (de Voogt et al. 1977; Koss 1979). Zellen des Blasenepithels in Spontanurin oder Spüllösung ermöglichen solche zytologischen Untersuchungen. Die Fragestellung ist dabei die Früherkennung von Tumoren vor Mani...
Sixteen proteins were demonstrated by crossed immunoelectrophoresis (CIE) of Triton X-100 solubilized megakaryoblasts against a rabbit antiserum. One of these reacted with a recently developed monoclonal antibody (SA-1) prepared by immunization with cells from a patient with megakaryoblastic leukemia. Leukemic cells from 8/15 patients with AML stai...
Reticulocytes in fixed human blood samples were stained for RNA with the fluorescent dye pyronin Y and measured by flow cytometry. The resulting relative frequency distributions of the RNA fluorescence intensities concurred with the different stages in maturation from early reticulocytes to mature red cells. A computer program was written to calcul...
A computer-controlled image-analysis system (LEYTAS) was used to classify 328 cytological urinary specimens automatically into positive and negative. Classification was based on the presence of cells with increased DNA content or high chromatin contrast. These cells are automatically detected by the LEYTAS system and stored in image memories. Fully...
Reticulocytes in fixed human blood samples were stained for RNA with the fluorescent dye pyronin Y and measured by flow cytometry. The resulting relative frequency distributions of the RNA fluorescence intensities concurred with the different stages in maturation from early reticulocytes to mature red cells. A computer program was written to calcul...
Peripheral blood T lymphocyte subpopulations were monitored in 45 consecutive adult recipients of cadaveric renal allografts by using monoclonal antibodies and flow cytometrie. All patients were treated with low dose corticosteroids and azathioprine. In 37 patients pre-transplant OKT4/OKT8 ratios were available. Six of 26 patients (23%) with pre-tr...
Flow cytometry and image cytometry, two measuring techniques in the field of analytical cytology, can be used sequentially on the same cytological sample. Cells stained with a fluorochrome for the determination of for example, DNA or RNA content are first analysed in suspension by flow cytometry. The results of the fluorescence analysis of the indi...
This paper describes the application of a television-based system (LEYTAS) in machine analysis of cervical specimens. LEYTAS basically consists of a Leitz microscope, the texture analysis system (TAS, Leitz), a TV camera, a 4-bit grey value memory and a minicomputer (PDP 11/23). A series of 1176 Feulgen stained cervical smears has been analysed in...
An automated method for measurement of proteinase activities using fluorogenic substrates is described. Enzyme assays were performed in polystyrene microtitration trays as normally used for the enzyme-linked immunosorbent assay technique. The reaction products were measured using an inverted fluorescence microscope equipped with a photometer. Data...
The interaction between histochemistry and microscopy for quantifying the morphology and function of cells in sections of tissue is reviewed.
In principle, the morphological parameters of cells can be measured in suitably-stained sections using image analysers. In practice, however, the individual cells often cannot be distinguished from each other...
The application of arc lamps to flow cytometers is discussed and epiillumination for jet-in-air cell sorters is introduced. An epiilluminator/detector unit equipped with a mercury arc lamp constructed for a commercially available cell sorter is described. Experiments in which laser and mercury arc lamp illumination were compared show that the signa...
Das Leytas Bildanalysesystem wurde zur automatisierten Klassifizierung von 157 zytologischen Präparaten des Blasenepithels aus Spontanurin eingesetzt. Während der Analyse mikroskopischer Präparate wurden mehr als 99% normaler Zellen und Artefakte (z.B. überlappende Zellkerne) eliminiert und Objekte mit erhöhtem DNS-Wert oder dunklem Chromatin selek...
This paper describes the application of a television-based system (LEYTAS) in machine analysis of cytological specimens.
An image analysis program for automated screening of entire Papanicolaou stained slides, using a television based analysis system (LEYTAS), is described. A series of 45 specimens has been screened to test this program, that consists principally of nuclear contour finding, size and thresholding operations on the nucleus and artefact rejection proced...
Urinary cytology has significantly contributed to the diagnosis of bladder cancer. More specifically, it has enhanced the diagnostic yield of carcinoma-in-situ (8). It has improved prognostic grading and it has an important place in the follow-up of treated bladder cancer patients. The reliability of urinary cytology has been confirmed in numerous...
Gegenwärtig werden in zunehmendem Maße zytopathologische Untersuchungen zur Diagnose von Tumoren des Blasenepithels durchgeführt. Die Fragestellung ist dabei die Früherkennung von Tumoren vor Manifestation invasiver Stadien [6]. Die zytologische Auswertung von urologischem Material ist ein wertvolles und empfindliches Verfahren [11,5]. Zur optimale...
The immunological specificity of the Amsterdam rabbit antiserum against human prostatic and phosphatase was studied on paraffin sections of 200 prostatic carcinomas and 330 control tissues using an indirect peroxidase technique. Peripheral blood leucocyte smears were also investigated with a fluorescent technique. In a limited number of cases, the...
A fluorescent staining procedure based on pyronin Y is described. The technique has been used to stain RNA in human reticulocytes for subsequent flow analysis and sorting. Histograms of fluorescence values of mature red blood cells and reticulocytes obtained by flow cytometry of stained blood samples, contain more information than offered by conven...
Die Aspirationszytologie mit Hilfe der Franzennadel ist neben der Nadelbiopsie in den letzten Jahren zur Routinediagnostik beim Prostatakarzinom geworden. Eine Vergleichsuntersuchung mit diesen beiden Techniken hat eine 90%ige Übereinstimmung festgestellt. Neue objektive und quantitative Parameter wie die Zytomorpho metrie und die Zytofluorometrie...
Scanning electron microscopy (SEM) of mouse peritoneal macrophages attached to glass shows that these cells have filopodia, i.e., cord-like extensions arising from the cell surface. To confirm that these extensions are not the result of the preparative procedure required for SEM or cell-surface material left behind by cells moving on the substrate...
Motility and patterns of adhesion were determined by time-lapse cinematography and reflection contrast microscopy for two types of carcinoma cells, selected for their different motile behavior and not for their malignancy. Cells from the V2 rabbit carcinoma become locomotory soon after having established the necessary contact to the substratum. In...
The quantitative aspects of formaldehyde induced fluorescence (FIF) of noradrenaline (NA) in sympathetic nerve fibres were studied in whole mount preparations of the iris of the rat. Both "static" and scanning microfluorimetric measurements showed a close linear relationship between the FIF intensity and the NA content after pharmacological treatme...
A high resolution (0.5 micrometer), two-dimensional microfluorimetric scanning procedure was used to quantitate the formaldehyde-induced fluorescence of noradrenaline in the iris of the rat. Scanning of large areas (50 x 50 micrometer) in the sympathetic innervated dilator muscle revealed an overlap between measurements of nerve fibers and of backg...
The Leyden Television Analysis System, LETYAS, consists of a number of hardwired modules for the processing of images at television speed. Incorporating a high degree of flexibility, LEYTAS is well suited to various fields of biomedical research. This paper described the hardware configuration of the system and the software program, TAL, which has...
For automated read-out of immunogalactosidase assays with a fluorogenic substrate, performed in microtitration trays, an inverted fluorescence microscope equipped with a photometer and a scanning stage and operated by a microprocessor has been used. Trays (60 and 96 wells) were scanned in 1 min and corrected measurements and a histogram of the resu...
The application of a computer-assisted morphometry technique for the analysis of muscle biopsies is described. This method involves digitizing the contours of muscle fibrecross-sections using a graphic tablet combined with a specially developed illuminated cursor for tracing directly on the microscopic image via a drawing tube. This approach permit...
The Leyden Television Analysis System (LEYTAS) is applied to the automated analysis of five positive specimens (2 cases of severe dysplasia, 2 cases of carcinoma in situ and 1 case of invasive carcinoma) and five negative specimens (4 cases of inflammation and 1 normal specimen). Two detection criteria based on size and contrast (so called 'top hat...
Compares shape parameters most commonly mentioned in biomedical literature; Fourier analysis and Young's concept of bending energy. Two alternatives for the bending energy are given: one based on piecewise linear approximation of the contour leading to the definition of a new class of parameters using the runlength spectrum of the contour, and the...
Serum peptides containing classical anaphylatoxin (CAT) produce marked chemotatic orientation of human neutrophil granulocytes without modifying cell attachment to the substratum. Furthermore gradients of adhesion produced with gammaglobulins fail to induce morphological orientation of neutrophils. The results suggest that chemotaxis is not a speci...
One of the aims of the research of our group is the development of an automated system for prescreening and for quantitative evaluation of cervical cytology preparations. As an approach towards this goal a relatively large number of slides obtained from patients of different clinics has been analyzed by semi-automated cy-tofluorometry for the prese...
Two-dimensional microfluorimetric scanning was used to quantitate the formaldehyde-induced fluorescence within nerve fibers (neuronal) and between nerve fibers (extraneuronal) in the iris of the rat. After treatment with reserpine or surgical sympathectomy, extraneuronal fluorescence of the iris decreased to 60%. This indicates that about 40% of ex...
Proteins as well as materials of low molecular weight have marked effects on the rate of locomotion, adhesion and cell shape of human neutrophil granulocytes in vitro. Plasma protein preparations differ qualitatively with respect to their chemokinetic activity. Human serum albumin (HSA), fibrinogen and acid-treated gamma globulin without polymers h...
Reflection contrast in combination with phase contrast microscopy was utilized for the study of adhesion patterns of locomotive L5222 rat leukemia cells. It was found that for cells moving in a spherical shape on the glass surface, adhesions were very faint. This inconspicuous pattern, however, became very distinct, as soon as the cells changed to...
The specificity and quantitative reliability of the Feulgen-acriflavine-SO2 procedure was tested on polyacrylamide model films containing DNA. Noncovalent binding of acriflavine to DNA was observed when the washing procedure, as used in the classical way, was applied. The noncovalently bound acriflavine could be removed with an extra wash in acid-e...
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 aut...
This paper describes instruments for microfluorometry and microspectrophotometry, an interactive graphic tablet linked to a microprocessor for semi-automated morphometry, and systems using rapid scanning stages or television scanning techniques for automated analysis of biological material. Some applications of these techniques in clinical work are...
Texture parameters of the nuclear chromatin pattern can contribute to the automated classification of specimens on the basis of single cell analysis in cervical cytology. Current texture parameters are abstract and therefore hamper understanding. In this paper texture parameters are described that can be derived from the chromatin pattern after seg...
This paper describes an automated microscope developed for operation in conjunction with the Leyden Television Analysis System. It features automated control of magnification, illumination, movement of scanning stages, and fine focus. These functions are controlled by means of a microcomputer. This enables a flexible design and relieves the supervi...
Citations
... The standard technique for reticulocyte count is sating with fluorescent dyes, a technique which has been later incorporated into flow cytometry. [9][10][11] The present study aims not to replace flow cytometry as the gold standard tool, but to evaluate an image analysis based software as a potential automated cell classifier. ...
![[object Object]](https://i1.rgstatic.net/ii/profile.image/277598304260104-1443196001697_Q64/Ger-Koper.jpg)
... Attempts to unravel the complexity of intracellular processes such as multienzyme reactions (Higgins, 1965;Kacser, 1983Kacser, , 1986Burns, 1973, 1979;Rapoport, 1974, 1977;Werner and Heinrich, 1985;Van Dam et al., 1980;Westerhoff, 1983;Westerhoff and Chen, 1984;Westerhoff and Van Dam, 1986;Westerhoff et al., 1981 or multiorganelle interactions (Kohen et al., , 1989a have stimulated the development of microspectrofluorometric methods (Caspersson et al., 1965;Chance and Thorell, 1959;Daudel et al., 1974;Hirschberg et al., 1978;Kohen et al., 1983aKohen et al., ,b, 1984Kohen et al., , 1986aKohen et al., ,b,c, 1989aPloem, 1989;Rousseau, 1957;Rost, 1973;Sloane and Loeser, 1963;West, 1965) which allow the spatiotemporal mapping (Kohen, , 1989a of bioregulatory mechanisms monitored by fluorescence of endogenous or exogenous probes (Kohen, 1989a). Simultaneous progress in three areas has proved particularly helpful in these studies: the development of new fluorescence probes of cell organeUes and constituents, the emergence of highly sensitive low hght-level detectors for microspectrofluorometry and micromanipulatory procedures to obtain rapid shifts of steady or transient metabohc states (Chance, 1952;Chance and Wilhams, 1955). ...
... In addition, the 7-medG antibody used identifies imidazole-ringopened 7-medG (13) and the alkali treatment converts the ring-closed form 7-medG to the ring-opened form. The sensitivities for this method have been calculated to be around one adduct in 10 6 normal nucleotides for O 6 -medG and approximately one adduct in 10 5 normal nucleotides for 7-medG (14). The numbers of nuclei strongly stained with DNA methylation adducts were counted in all cells comprising lesions and 1000 cells of non-lesion sites, determined histopathologically in separate sequential sections stained with H&E. ...
... However, during the seventies it became possible to develop interactive image analysis systems, albeit with very limited capacity, typically with a memory size of a few hundred kB and a monochrome or binary display. These systems were used to explore new image segmentation, feature extraction, and classification designs which led to a new generation of systems in the early 1980-ies such as BioPEPR [15], FAZYTAN [16], Cerviscan [17], LEYTAS [18], and at the authors' laboratory the Diascanner [19]. ...
... The application of a polarizer combined with a lambda quarter wave plate for elliptical polarization and re-polarization of the front lens, as well as a suitably aligned analyser, allows only the re¯ections from the section to reach the eyepiece ( Figure 1). The re¯ections are created with 45u epiillumination; this combination is described by Leica as the Ploemopak epi-illuminator [7]. The conical, annular illumination combines the characteristics of darkground illumination. ...
![[object Object]](https://i1.rgstatic.net/ii/profile.image/272378697613346-1441951550838_Q64/Frans-Prins.jpg)
... The slides were developed at 20 ³C in Kodak D19b (2 min) or Gold-Elon Ascorbic acid developer (GEA, gold latensi®cation 5 min, elon ascorbic acid 5 min) and ®xed in a non-hardening ®xative (20% Na 2 S 2 O 3 e5H 2 O/2.5% K 2 S 2 O 5 /H 2 O). For LM autoradiography the slides were examined with a Leitz Orthoplan microscope (Leitz, Wetzlar, Germany) equipped for re¯ection contrast microscopy (RCM, for details see (15)). For EM autoradiography the ultrathin sections with the accompanying ®lms were¯oated on water, covered with grids, picked up with para®lm and examined with a JEOL JEM-100S transmission electron microscope at an accelerating voltage of 60 kV. ...
... Although diffraction limit is surpassed in fluorescence imaging (Sheppard, 1988;Hell & Wichmann, 1994;Gustafsson, 2000;), resolution improvement in other conventional optical techniques such as reflection and other modalities are constantly attempted (Ploem, 1975;Brakenhoff et al., 1979;Koerten et al., 1979;1980;Cox et al., 1982a;Cox et al., 1982b;Brakenhoff et al., 1984;Cornelesetenvelde et al., 1989;Prins et al., 1993;Brakenhoff & Muller, 1996;Prins et al., 2006;Azeredo et al., 2016;Ploem & Prins, 2017;Sivaguru et al., 2017b;Sivaguru et al., 2018a). Superresolution techniques such as stimulated emission and depletion (Hell & Wichmann, 1994), structured illumination (Gustafsson, 2000), Airyscan (Sheppard, 1988;Huff, 2015) and PALM/STORM (Betzig et al., 2006) have recently been commercialised to go beyond diffraction limit of resolution in biological samples labelled with fluorescent targets. ...