Computerized continuous monitoring of cytochemical enzyme reaction product formation by the Vickers M85A microdensitometer

Summary An experimental set-up for the continuous monitoring of cytochemical enzyme reaction rates is described. The design consists of a 32K Commodore PET microcomputer, upgraded to Basic 4 and containing a graphics facility. The microcomputer is interfaced with a Vickers M85A microdensitometer, which has been modified and equipped with a Zeiss X63 water immersion objective. The software allows for the automatic transfer of density readings from the M85A to the microcomputer, and for the continuous monitoring of the enzyme reaction as its proceeds at short time intervals (i.e. down to 3.3 s). The relationship between the absorbance change of the enzyme final reaction product and time is printed both numerically and graphically. Initial velocity rates are calculated by regression analysis, and statistical evaluation of grouped data is possible.     

An interactive microcomputer-based system for the quantitative analysis of stratified tissue sections

This paper describes a microcomputer-based system that allows diagnostically relevant properties of stratified tissue sections to be objectively measured. The results of detailed nuclear image analyses are examined in the broader context of the position of nuclei within the tissue section and relative to histologic structures and each other. Quantitative measures are obtained for important morphometric and densitometric properties of individual nuclei and mitotic figures and especially for their distribution and orientation within the tissue section relative to the stratum germinativum and each other. Recorded karyometric and histometric parameters include measures of nuclear DNA content (based on optical density measurements), size, roundness, texture, shape, distance to the basal layer, angle with the stratum germinativum, epithelial height and proximate nuclear distance. Statistics generated describe normalized mitotic density as a function of depth in the epithelium, and a composite mitotic index is produced based upon weighting of these densities relative to their distance from the stratum germinativum. These properties and derived statistics may be examined as a function of epithelial depth and nuclear type and may be plotted as a function of other diagnostic features in addition to the observed stratum. The system is one part of microTICAS-STRATEX, an expert diagnostic system for the clinical evaluation of stratified tissue sections, now under development as an outgrowth of the microTICAS system. Results of measurements made with this system will be compared with site-specific and diagnosis-specific reference profiles and used in conjunction with clinical data derived from a data base at the University of Chicago of over 1.5 million patients to generate diagnostic and prognostic evaluations.     

Linearity in dehydrogenase reaction rate studies in tissue sections is affected by loss of endogenous substrates during the reaction

We studied the effect of section thickness on the reaction rate of glucose-6-phosphate dehydrogenase (G6PD) activity in unfixed sections of rat liver by use of continuous monitoring by microdensitometry of the reaction product as it formed in the section during incubation. Tetranitro BT or nitro BT was used as final electron acceptor and polyvinyl alcohol as tissue stabilizer. Each test minus control reaction curve deviated from linearity during the first 2 min of incubation. This was mainly due to loss of low molecular weight endogenous dehydrogenase substrates from the surface of the section. For any given reaction, the same absolute amount of endogenous substrate was lost from each section, and hence a much greater proportion was lost from the thinner sections. Such losses lead to a deficit in (nonspecific) formazan production. There was a greater loss from, and hence a greater deficit in, formazan production in sections incubated at 30 degrees C than at 37 degrees C and when nitro BT was used instead of tetranitro BT, but the greatest loss of endogenous substrates occurred in sections incubated in control media. Therefore, greater losses seemed to occur when the reactions were slower because of failure to overcome the critical supersaturation level of the formazan. A consequence of this was a non-linear test minus control response during the first minutes of the incubation.     

The use of interactive image analysis for demonstrating coexistence

A new approach to establish morphological coexistence using computerized image analysis is described. With this technique, the coexisting pattern in two images is revealed by recording the images via a TV camera on a Zeiss/Kontron IBAS interctive image analyzer. Using an arithmetic or a Boolean algebraic operation, the computer then directly compares the respective patterns obtained for different neuroactive substances and shows the resulting coexisting cells (in white) on a TV-monitor. Also non-coexisting system can be showed in various shades of grey. The method allows for a non-biased, rapid and exact scanning of tissue sections where a possible coexistence may be present.     

Kinetic and morphometric measurements of enzyme reactions in tissue sections with a new instrumental setup

Summary An instrumental setup is described for the measurement of enzyme kinetics and morphometry in tissue sections. It consists of a Vickers M85 microdensitometer and computer-assisted Kontron Videoplan system. The Videoplan system consists of a minicomputer with two mini-floppy disks, a keyboard, a graphic tablet, a TV monitor and a printer/plotter. The measuring component of the M85 is linked to the minicomputer via a BCD interface, and the optical system of the M85 is coupled to a TV camera for display on the monitor screen. The enzyme-kinetic data obtained with the M85 in a specified area of the tissue section (density values as a function of reaction time) are stored in the minicomputer. The measurement process is controlled by a corresponding measuring program. Through correlation analysis (a component of the commercial software) between density values and reaction time, the initial and thus maximum enzyme activity is determined. Upon completion of the kinetic measurements, the measured area of tissue is transferred by the TV camera to the monitor, and the reaction area is described and measured with the graphic tablet in video dialogue and related to the initial enzyme activity. With the setup described, it is possible to make microdensitometric measurements of enzyme activities in a specified tissue area while morphometrically analyzing the associated reaction area. To illustrate the use of the system, enzyme-kinetic (succinate dehydrogenase) and morphometric measurements are performed in tissue sections from the proximal tubule of the rat nephron. Additional applications of the system are discussed.Supported by the Deutsche Forschungsgemeinschaft (SFB 105)     

A technique for section thickness evaluation for microphotometry and image analysis of sectioned nuclei.

The exact knowledge of the section thickness is a requisite for making the necessary corrections on DNA measurements in tissue sections. Several methods have been proposed to evaluate section thickness, each of them with advantages and disadvantages depending on the type of specimen and equipment available. We herein report another method based on preparation of standard material whose optical density varies as a function of its thickness and is sectioned and measured alongside the tissue specimen. The standards consist of celloidin cylinders stained with the PAS reaction and embedded in paraffin. For prior characterization of the cylinders, sections of different thickness were obtained and mounted. The optical density of each section was measured by direct microphotometry or image analysis. The actual thickness of each section was evaluated following re-embedding of piled groups of sections in a paraffin block and transversal sectioning. The thickness was then measured with a micrometric eye-piece. Optical density and actual thickness of each section were plotted on a normogram curve. Once a given tissue is sectioned alongside with the reference cylinder, the actual thickness is determined by its optical density on the normogram curve.     

A method to estimate the DNA content of whole nuclei from measurements made on thin tissue sections

A microdensitometry method that allows estimation of the distribution of the DNA content of nuclei in thin tissue sections is described. The method is based on a theoretical model of Feulgen-stained spherical nuclei, of different sizes, in each of which the DNA is present as a homogeneous solution. In thin sections of nuclei of different sizes, the fraction of DNA per section is inversely proportional to the radius of the nucleus. Histograms of the product of DNA content and radius per nuclear section are independent of nuclear size but depend on total DNA content. The distribution of the total DNA content of nuclei in a section can be estimated from such a histogram. The results of the measurements of a Feulgen-stained rat liver section are described.     

Near‐infrared human finger measurements based on self‐calibration point: Simulation and in vivo experiments

Near‐infrared light allows measuring tissue oxygenation. These measurements relay on oxygenation‐dependent absorption spectral changes. However, the tissue scattering, which is also spectral dependent, introduces an intrinsic error. Most methods focus on the volume reflectance from a semi‐infinite sample. We have proposed examining the full scattering profile (FSP), which is the angular intensity distribution. A point was found, that is, the iso‐path length (IPL) point, which is not dependent on the tissue scattering, and can serve for self‐calibration. This point is geometric dependent, hence in cylindrical tissues depends solely on the diameter. In this work, we examine an elliptic tissue cross section via Monte Carlo simulation. We have found that the IPL point of an elliptic tissue cross section is indifferent to the input illumination orientation. Furthermore, the IPL point is the same as in a circular cross section with a radius equal to the effective ellipse radius. This is despite the fact that the FSPs of the circular and elliptical cross sections are different. Hence, changing the orientation of the input illumination reveals the IPL point. In order to demonstrate this experimentally, the FSPs of a few female fingers were measured at 2 perpendicular orientations. The crossing point between these FSPs was found equivalent to the IPL point of a cylindrical phantom with a radius similar to the effective radius. The findings of this work will allow accurate pulse oximetry assessment of blood saturation.      

Colloidal gold immunostaining on deplasticized ultra-thin sections

We localized tissue antigens on ultra-thin sections by deplasticizing the sections while on the grid, incubating in primary antiserum followed by immunoglobulin-conjugated colloidal gold, and ultimately re-embedding in dilute Epon. This procedure permitted ultrastructural localization of tissue antigens that were previously masked by the embedding plastic surrounding tissue components. In addition, replacement of the plastic matrix on the thin section after immunostaining prevented development of the drying artifacts that occur in unsupported tissue sections. Optimal preservation of components in the tissue sections was achieved despite extensive steps involved in plastic removal and immunostaining. This method may be useful in situations where the number of exposed epitopes on the surface of a thin section is low. The procedure also allows the use of antisera at greater dilutions and provides enhanced immunostaining specificity with low background.     

Incorporation of fluorescent enzyme substrates in agarose gel for in situ zymography

The currently available methods for the detection of proteases in tissue sections are characterized by limited substrate specificity and low sensitivity and are also cumbersome. We have developed a novel in situ zymography method that uses a synthetic substrate conjugated to a fluorescent tag for detection of proteases in tissue sections. In the presence of active enzyme, the fluorescent tag is cleaved off from the substrate peptide chain resulting in an approximately 100-fold increase in the fluorescent signal. In order to minimize the diffusion of the fluorescent tag, the substrate is incorporated into 1% agarose prior to overlaying onto the tissue section. This method retains the morphological details of the tissue section, is highly sensitive and specific for the designated peptide sequence, and provides information regarding the functional status of the enzyme. Thus, this method could be used for detection and monitoring of enzymatic activity in tissue sections for a variety of applications.     

Precise cytochemical measurement of neotetrazolium formazan by scanning and integrating microdensitometry

Summary The extinction values of the formazan of neotetrazolium, deposited in tissue sections, have been measured using a Vickers M 85 scanning and integrating microdensitometer with a scanning spot of 0.25 diameter. These values have been correlated with the amount of formazan in the same sections as determined by elution and spectrophotometry. The extinction at 520 nm did not increase linearly with increasing amounts of formazan in sections incubated for different times. This has been shown to be due to the presence of two formazan components which have different absorption characteristics; a red diffuse colour with a molar extinction coefficient of 20580 and a more granular purple product with a coefficient of 7370. However, at 585 nm, the isosbestic wavelength, the extinction was directly proportional to the amount of formazan in the section, irrespective of the nature of the end product; at this wavelength the molar extinction coefficient was 7200.These results have shown that scanning and integrating microdensitometry can be used for the precise measurement of the amount of neotetrazolium formazan, in picograms, in a single cell of group or cells in a tissue section.     

Intraoperative evaluation of sentinel lymph nodes in breast cancer: comparison of frozen sections,imprint cytology and immunocytochemistry

M. Francz, K. Egervari and Z. Szollosi
Intraoperative evaluation of sentinel lymph nodes in breast cancer: comparison of frozen sections, imprint cytology and immunocytochemistry Objective: We analysed the utility of imprint cytology with rapid immunocytochemistry and frozen section analysis for the evaluation of sentinel lymph nodes in breast cancer patients. Methods: The sensitivity, specificity, and positive and negative predictive values have been calculated for each method individually, each pair and all three together. We compared these results with those of routinely processed paraffin sections. Results: The sensitivity and specificity of each of the three methods for detection of metastatic carcinoma were as follows: 69.4% and 97.8% for touch imprint cytology; 58.3% and 100% for frozen sections; 68.5% and 98.9% for rapid immunocytochemistry. When the methods were combined, the highest accuracy was achieved by touch imprint cytology, frozen sections, touch imprint cytology plus rapid immunocytochemistry, or touch imprint cytology frozen section analysis and rapid immunocytochemistry, each of these having identical sensitivity and specificity of 72.2% and 97.8%, respectively. Conclusions: In our study the combined accuracy of the three methods was the same as combining touch imprint cytology and frozen sections or touch imprint cytology plus rapid immunocytochemistry. Rapid immunocytochemistry provides an additional parameter and preserves tissue for permanent sections.     

Continuousin vitro multiplication of shoot buds of Norway spruce (Picea abies L.) by intermittent application of growth regulators

A culture system was developed which allows continuous production of adventitious buds. Segments of seedlings germinated on media supplemented with different growth regulator combinations were used as explants. Cultivation was carried out in two phases, which alternate permanently: (I) without and (II) with growth regulators. Thus it was possible to establish meristematic tissue cultures from 5–10% of the seedlings tested. They have produced buds now for over 3 years with multiplication rates of about 1.5 within 5–6 weeks.     

Laser-assisted microdissection for real-time PCR sample preparation

Laser-assisted microdissection (LMD) has been developed to procure precisely the cells of interest in a tissue specimen, in a rapid and practical manner. Together with real-time PCR and RT-PCR techniques, it is now feasible to study genetic alterations, gene expression features and proteins in defined cell populations from complex normal and diseased tissues. The process that brings from sample collection to the final quantitative results is articulated in several steps, each of which requires optimal choices in order to end up with high-quality nucleic acid or protein that allows successful application of the final quantitative assays. This review will describe shortly the development of LMD technologies and the principles they are based on. Trying to highlight the advantages and disadvantages of LMD, the main problems related to specimens collection and processing, section preparation and extraction of bio-molecules from microdissected tissue samples have been analysed.     

Radioimmunochemical methods for the quantitative autoradiographic determination of antigens in brain and other tissues

1. We have used horseradish peroxidase-conjugated protein A- and 125I-protein A to develop immunohistochemical and radioimmunohistochemical methods for the localization of antigens in brain and other tissues of the rat. 2. We visualized methionine-enkephalin fibers in the rat brain by incubating tissue sections with a specific polyclonal antibody and peroxidase-conjugated protein A. The method is simple, fast, and less expensive and more sensitive than classical immunohistochemical techniques and the principle could be used to visualize many other tissue antigens. 3. Incubation of tissue samples with specific polyclonal antibodies and 125I-protein A, followed by autoradiography, allows the permanent recording of the radioimmunohistochemical localization of brain methionine-enkephalin, tyrosine hydroxylase, and angiotensin-converting enzyme and of pituitary vasopressin and could be applied to the localization of many other tissue antigens. 4. A new quantitative radioimmunohistochemical technique for methionine-enkephalin allows the determination of the endogenous peptide content in discrete brain nuclei from 16-microns-thick sections. The method is based on the quantitative determination of the amount of 125I-protein A bound to specific tissue areas after incubation with a specific polyclonal antibody, followed by autoradiography and computerized microdensitometry. To quantify the endogenous peptide content, the values obtained are interpolated into a methionine-enkephalin internal standard curve. This standard curve was constructed by measuring endogenous concentrations of methionine-enkephalin by radioimmunoassay in specific brain regions and correlating these values with quantitative autoradiographic determinations in homologous areas of adjacent sections. Similar methods can be developed for other tissue antigens. 5. These new methods allow for the localization and quantification of tissue antigens in very discrete areas of the brain and other tissues and have a wide application in neurobiology and pathology.     

Microcomputer control applied to the Vickers M85/86 microdensitometer

Summary An electronic interface for a Research Machines 380Z microcomputer and a Vickers M85/6 integrating microdensitometer is described, together with outlines of the control software. A typical applications programme, which may be written in several high-level languages, is outlined for the determination of a time course plot for enzyme activity in a tissue section. This system facilitates rapid accurate measurement and analysis of experimental data.     

The use of continuous monitoring and computer-assisted image analysis for the histochemical quantification of hexokinase activity

The effect of endogenous 6-phosphogluconate dehydrogenase (6PGDH) on the histochemical quantification of low-Km hexokinase activity in rat submandibular salivary glands has been investigated using a Seescan Solitaire Plus image analysis system and a modified black and white Newvicon TV camera. Absorbance readings of neutral density filters were close to their known absorbance values. A significant correlation was found between the absorbance of Nitro BT reduction products within sections, with and without the use of a 588 nm interference filter. Furthermore, absorbance readings obtained from 8 microns-thick sections were 1.92 and 2.22 times greater than values obtained from 4 microns-thick sections using 'white' and 588 nm light respectively. The level of background illumination was not critical for absorbance measurements provided it was below the level that saturated the Newvicon camera and was not changed between background and image capture. The greatest variations in absorbance readings on tissue sections were associated with changes in zoom and objective combinations. Our studies indicate that this relatively low cost image analysis system can give reproducible absorbance readings from various structures defined in digitized, captured images of tissue sections. Results from continuous monitoring studies indicated that the high levels of 6PGDH activity in excretory ducts caused a 1.67-fold overestimation of hexokinase activity as assessed by absorbance readings performed throughout a 22-minute reaction period. By contrast, overestimation of hexokinase activity in salivary gland acini only became apparent after 8 min incubation with a 1.4-fold overestimation being seen at 22 min. This difference appears to reflect the relatively low hexokinase and 6PGDH activities present in acini compared with excretory ducts.     

3D confocal reconstruction of gene expression in mouse

Three-dimensional computer reconstructions of gene expression data will become a valuable tool in biomedical research in the near future. However, at present the process of converting in situ expression data into 3D models is a highly specialized and time-consuming procedure. Here we present a method which allows rapid reconstruction of whole-mount in situ data from mouse embryos. Mid-gestation embryos were stained with the alkaline phosphotase substrate Fast Red, which can be detected using confocal laser scanning microscopy (CLSM), and cut into 70 microm sections. Each section was then scanned and digitally reconstructed. Using this method it took two days to section, digitize and reconstruct the full expression pattern of Shh in an E9.5 embryo (a 3D model of this embryo can be seen at genex.hgu.mrc.ac.uk). Additionally we demonstrate that this technique allows gene expression to be studied at the single cell level in intact tissue.     

A relationship between slide quality and image quality in whole slide imaging (WSI)

This study examined the effect of tissue section thickness and consistency--parameters outside the direct control of the imaging devices themselves--on WSI capture speed and image quality. Preliminary data indicates that thinner, more consistent tissue sectioning (such as those produced by automated tissue sectioning robots) results in significantly faster WSI capture times and better image quality. A variety of tissue types (including human breast, mouse embryo, mouse brain, etc.) were sectioned using an (AS-200) Automated Tissue Sectioning System (Kurabo Industries, Osaka Japan) at thicknesses from 2 - 9 microm (at one microm intervals) and stained with H&E by a standard method. The resulting slides were imaged with 5 different WSI devices (ScanScope CS, Aperio, CA; iScan, BioImagene, CA; DX40, DMetrix, AZ; NanoZoomer, Hamamatsu Photonics K.K., Japan; Mirax Scan, Carl Zeiss Inc., Germany) with sampling periods of 0.43 - 0.69 microm/pixel. Slides with different tissue thicknesses were compared for image quality, appropriate number of focus points, and overall scanning speed. Thinner sections (i.e. 3 microm sections versus 7 microm) required significantly fewer focus points and had significantly lower (10-15%) capture times. Improvement was seen with all devices and tissues tested. Furthermore, a panel of experienced pathologist judged image quality to be significantly better (for example, with better apparent resolution of nucleoli) with the thinner sections. Automated tissue sectioning is a very new technology; however, the AS-200 seems to be able to produce thinner, more consistent, flatter sections than manual methods at reasonably high throughput. The resulting tissue sections seem to be easier for a WSI system's focusing systems to deal with (compared to manually cut slides). Teaming an automated tissue-sectioning device with a WSI device shows promise in producing faster WSI throughput with better image quality.     

On the preparation of cryosections for immunocytochemistry

The key preparation steps in the Tokuyasu thawed frozen section technique for immunocytochemistry, namely freezing, sectioning, thawing, and drying, were studied. A spherical tissue culture cell was used as a model system. The frozen hydrated section technique indicated that glutaraldehyde-fixed, 2.1 M sucrose-infused pellets of cells were routinely vitrified by immersion in liquid nitrogen but water was crystallized when lower sucrose concentrations (0.6-1 M) were used. Quantitative mass measurements showed that the fixed cells are freely permeable to sucrose. The frozen hydrated sections were severely compressed but cell profiles regained their circular appearance upon thawing. The average section thickness of our frozen-hydrated sections was 110 nm: this was reduced to 30-50 nm upon thawing, washing, and air-drying. This change was accompanied by severe drying artifacts. By using the methyl cellulose drying technique, this collapse upon air-drying could be significantly reduced, but not completely prevented, giving an average thickness of 70 nm.