J. S. Ploem

Sensitive detection of hybridocytochemical results by means of reflection-contrast microscopy.

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 of reflection-contrast microscopy. The applicability of the procedure is demonstrated with nucleic acid probes modified with 2-acetylaminofluorene groups. These in turn are localized in situ by an indirect immunoperoxidase reaction. Besides its sensitivity, this simple visualization technique possesses the additional advantages, over absorption and fluorescence microscopy, that it provides a total DNA counterstain and a chromosomal banding pattern.

An immunofluorescence reaction for Schistosoma mansoni using the defined antigen substrate spheres (DASS) system

Abstract Schistosoma mansoni antigens covalently bound to agarose beads were found suitable as a matrix for an immunofluorescent reaction on S. mansoni with sera of infected animals and men. This system proved to be at least as sensitive as the indirect fluorescent antibody reaction with frozen sections of the adult parasite; furthermore, storage of the antigen-bound beads in freeze-dried state proved to be possible. Automated microfluorometry permitted the rapid measurement of a sufficient number of beads for routine applications. The DASS-system is a promising quantitative method for the identification of a variety of antigens using the indirect immunofluorescence method.

High-resolution scanning-densitometry of photographic negatives of human metaphase chromosomes

SummaryA computer-controlled cytophotometrical system is described to determine local absorbance or fluorescence values of chromosomes.The absorbance or fluorescence values within the chromosome are obtained in digital form by interactive scanning of 35 mm photomicrographic negatives. The scanning system consists of a mechanical stage scanner (Scanning Microscope Photometer SMP, Zeiss) connected to a PDP-12 computer (Digital Equipment).A computer program (CHROSCAN) enables the scanning of single chromosome images in a direction perpendicular to the length axis with 0.16 μ increments at the specimen level, and a transmission resolution of 500 linearly spaced intensity levels. Optical density values corrected for the local background of the individual chromosome, and total integrated optical density as well as the ratio of optical density in the long arm over the total optical density are calculated. Photography of a grey wedge together with the metaphase, allows determination of the gamma of each individual photomicrograph and conversion of optical density values into actual absorbance values.The program has a papertape output of the corrected integrated optical density data per lateral scanline to be used as an input for a curve analysis program. It also allows plotting of the integrated optical density distribution over the chromosome length by a Calcomp plotter.In this paper the instrumentation and experimental set up are presented, together with data concerning the reproducibility and accuracy of the photographic and the scanning procedures.

Light microscopical detection of single 5 and 20 nm gold particles used for immunolabelling of plasma membrane antigens with silver enhancement and reflection contrast

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 particles, however, were clearly demonstrable with silver contrast enhancement, both in absorption contrast and in reflection contrast. The method proved to be compatible with cytochemical methods such as acid phosphatase.

Laser scanning fluorescence microscopy

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 and in the immersion oil in LSM provide images of great contrast, even with weakly fluorescent specimens. Confocal LSM permits the visualization of multiple focal layers of thespecimen and 3-D image reconstructions. Combination of images stored in computer memory allow the comparison of phase contrast and fluorescence images of the same area of the specimen enabling multiparameter analysis of cells.

Enzyme assays at the single cell level using a new type of microfluorimeter

SummaryA direct method for the microfluorimetric determination of NADPH2 and 4-methylumbelliferon in the sub-picomole range is described. By means of an inverted fluorescence microscope with epi-illumination it has been possible to measure low concentrations of these fluorophores in volumes down to 17 nl. The aqueous microvolumes to be measured are suspended in oil to prevent evaporation. Since the droplets are sunk on thin teflon foil, to attain a spherical form, they can be measured in a reproducible way. The excitation of the microdroplet takes place from below through the teflon foil, while the fluorescence emission follows the same route back to a photomultiplier. The possibilities of this method are illustrated with the biochemical measurements of glucose-6-phosphate dehydrogenase (G6PD) activity in single fibroblasts. It was possible to distinguish between normal and G6PD deficient cells on a single cell level. The microfluorimetric measurement of lysosomal enzymes with 4-methylumbelliferyl substrates is discussed.

Image analysis combined with quantitative cytochemistry

SummaryThis 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 and interaction facilities. A much wider range of image analysis programs is however, possible with LEYTAS-2, enabling many applications. LEYTAS-1, the earlier version, consists of a Leitz microscope with automated functions, a TV camera, the Texture Analysis System (TAS, Leitz), a four-bit grey value memory and a minicomputer (PDP 11/23). Using this instrumentation 1,500 cervical smears prepared from cell suspensions and stained with acriflavin-Feulgen-Sits have been analysed in a completely automated procedure. Image transformations working in parallel on entire fields, have been used for cell selection and artefact rejection. Resulting alarms, consisting of selected single cells and non-rejected artefacts are stored in the grey value memory, which is displayed on a TV monitor. This option allows visual interaction after the machine diagnosis has been made. The machine diagnosis was correct in 320 out 321 specimens with a severe dysplasia or more serious lesion. The false positive rate in 561 morphologically negative specimens (normal and inflammation) was 16% (machine diagnosis). Visual interaction by subtracting the visually recognized false alarms from the total number of alarms reduces the false positive rate to 11%. In LEYTAS-2, which is based on LEYTAS-1 studies, the microscope is equipped with a new type of objective, enabling the analysis of microscope fields, which are four times as large as in LEYTAS-1. The image analysis part consists of the Modular Image Analysis Computer (MIAC, Leitz) and for alarm storage an eight-bit grey value processor is used. Comparison with LEYTAS-1 shows that cell selection capacities are similar and that the speed is four times higher.

Automated screening for micrometastases in bone marrow smears

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 reproducible slide it has been possible to automate the technique. A 100% concordance was found between the machine and the light microscope in the identification of slides containing moderate to high numbers of tumour cells in bone marrow, and in those containing no tumour cells. However, in those slides containing low numbers of tumour cells (1-10 tumour cells/10(6) normal bone marrow cells) the sensitivity was decreased to 91%. In the presence of non-specific staining the false positive rate was increased from 0% to 22%. This method represents a potential improvement in the assessment of an important clinical staging procedure.

The Use of LEYTAS in Analytical and Quantitative Cytology

This paper describes the application of a television-based system (LEYTAS) in machine analysis of cytological specimens.

Reflection contrast microscopy performed on epi‐illumination microscope stands: Comparison of reflection contrast‐ and epi‐polarization microscopy

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 central stop. Thus, microscope stands with incident illumination may be used for RCM.