Serial section method for cytofluorometric determinations of the DNA content of component cells of the human cochlea

Summary We describe a method for measuring the DNA content of the component cells of the organ of Corti using serial sections of human cochleae obtained at autopsy. Cochleae were fixed in Carnoy's solution and embedded in Acrytron E, a water-miscible methacrylate resin. A procedure was developed to reduce the background fluorescence in methacrylate-embedded sections; the resin was pretreated with ion-exchange resin (Amberlite IRA-410). Experiments showed that pretreatment reduce the background fluorescence practically to zero. Seventy 3 m-thick serial sections were prepared on fluorescence free glass slides and stained with azocarmin G and acriflavine-Feulgen. After postirradiation using blue excitation light, the amount of Feulgen-DNA present in the target nucleus in each section was determined using a microfluorometer. The amount of DNA in the entire nucleus was determined by adding together the DNA contnet of the segments of the nucleus. The characteristic appearance of the organ of Corti made it easy to detect these cells; under green excitation light the cells of this organ exhibited red cytoplasmic azocarmin-G fluorescence. Due to the relatively wide internuclear spaces, cytofluorometry of individual nuclei could be performed without interference from the neighboring cells. Our technique using serial sections allowed us to measure the DNA contnet of individual cells and obtain histological information about particular cells and their neighboring cells. Several polyploid cells were found among the Hensen's cells in the cochlea, while all other component cells of the organ of Corti were diploid.     

A new whole-mount DNA quantification method and the analysis of nuclear DNA content in the stem-cell niche of Arabidopsis roots

A semi-automated method to quantify fluorescence intensity of objects in intact organs and tissues, composed of several cell layers, has been designed. The method has been developed on whole-mount propidium-iodide stained Arabidopsis thaliana (Arabidopsis) root tips, in which the DNA content of individual nuclei could be quantified. A diameter of less than 150 μm makes this organ most appropriate for whole-mount imaging. Further advantages are the lack of chlorophyll and transparent cell walls, with only a little background fluorescence. The method has a great advantage over flow cytometry, as the information regarding the positions of nuclei is maintained, and nuclei with aberrant DNA content can be re-assessed individually, which facilitates the efficient distinction between technical artefact and aberrant DNA content. Our averaging 3D method calculates the average of the summed fluorescence intensities of all sections of a nucleus and interpolates the missing sections, thereby allowing for the correction of detection problems. Furthermore, this method has the advantage of detecting objects in tissues covering multiple cell layers. The results of our method in Arabidopsis root tips showed that the quiescent centre cells, which rarely divide, are diploid, and are arrested in G1 or G0. Most stem cells, with the exception of those of the vascular tissue, are diploid cells, and their rather low division rate is caused by an elongated G1 phase. In contrast, the majority of the vascular stem cells are tetraploid.     

Standardization of the post-irradiation method to eliminate primary fluorescence in cytofluorometry.

Thorough irradiation of specimen with the strong excitation light after fluorescent Feulgen staining destroys the primary fluorescence in the background with stabilization of specific fluorescence of pararosaniline (post-irradiation method). An apparatus to perform effective post-irradiation was developed and irradiation condition for DNA cytofluorometry on a pararosaniline Feulgen stained smear was standardized. After 10 h irradiation on this condition, the primary fluorescente in the background is almost completely cleared away. Proportionality between amount of fluorescence and DNA content is perfectly preserved and standard deviation in each class of ploidy becomes to be less than 5% of the mean DNA value. The standardized post-irradiation enables us to obtain reproducibly the same values of fluorescence on Feulgen stained nuclei of the same cell population in different smears even if the staining conditions in Schiff's solution might be different to some extent.     

Standardization of the post-irradiation method to eliminate primary fluorescence in cytofluorometry

Summary Thorough irradiation of specimen with the strong excitation light after fluorescent Feulgen staining destroys the primary fluorescence in the background with stabilization of specific fluorescence of pararosaniline (post-irradiation method).An apparatus to perform effective post-irradiation was developed and irradiation condition for DNA cytofluorometry on a pararosaniline Feulgen stained smear was standardized. After 10 h irradiation on this condition, the primary fluorescence in the background is almost completely cleared away. Proportionality between amount of fluorescence and DNA content is perfectly preserved and standard deviation in each class of ploidy becomes to be less than 5% of the mean DNA value.The standardized post-irradiation enables us to obtain reproducibly the same values of fluorescence on Feulgen stained nuclei of the same cell population in different smears even if the staining conditions in Schiff's solution might be different to some extent.Partly supported by Alexander von Humboldt-Stiftung     

Combined morphometrical and syntactic structure analysis as tools for histomorphological insight into human lung carcinoma growth

Histological sections of formalin-fixed, paraffin-embedded tissue comprising 60 surgical specimens of human lung carcinoma were Feulgen stained. The histomorphological images were transferred to an automated image analysing system (VISIAC) and analysed as follows. The geometrical centers of tumor cell nuclei were defined as vertices, and the minimum spanning tree (MST) was calculated based on the two-dimensional distance between the vertices. Segmentation of the images was performed semiautomatically by interactive definition of nuclei of interest and automated detection of nuclear boundaries. Several morphometric features of tumor cell nuclei were measured including size, DNA-content (extinction), and form factor, and were set in relation to parameters of the MST. The following results were obtained: DNA-content and tumor cell nucleus size ('center cell') of different microscopic tumor growth patterns are related to the number of nearest neighboring cells. No relation was found in the neighboring (surrounding) cells. The different cell types of lung carcinoma, i.e., the different microscopic tumor textures expressed the relation of center cell features to the parameters of MST. A high amount of DNA content in branching points of the MST for epidermoid carcinoma may be interpreted as carcinoma growing in epidermoid textures tend to proliferate from tumor cell nuclei related to at least one neighboring cell. The opposite was found for large cell anaplastic carcinoma (no perceptible microscopic textures of the tumors) which showed the highest DNA content in tumor cell nuclei but which was not related to any neighboring cells. This technique allows analysis of growth centers and microenvironment conditions in human lung cancer in relation to tumor texture at the light microscopy level.     

Cytochemical identification of quiescent and growth-activated tumor cells

Cytochemical determinations of the amount of protein in the cell nucleus together with the amount of DNA give a background for judging the growth activity of the individual cell. A series of human breast carcinomas of different malignancy grades was studied, and it was shown that judged by their types of DNA profiles, the tumors of low malignancy grades were characterized by growth-arrested cells while the majority of cells in highly malignant tumors were intensely growth activated. This indicates that determinations of proteins in the nucleus together with DNA determinations can be used to better judge malignancy grades in individual breast carcinomas, which is of considerable clinical importance.     

Brush cells of the mouse gallbladder

Summary The brush cells (BC) of the mouse gallbladder were studied using light and electron microscopy (transmission and scanning) to determine their shape and distribution. Specimens were fixed in glutaraldehyde and postfixed in ferrocyanide-reduced osmium tetroxide. BC selectively stained with toluidine blue could be identified by means of light microscopy and subsequently studied in serial semithin and ultrathin sections. The results revealed that the shape of the BC is flask-like. A slender, occasionally branched cytoplasmic process emerges from the bulk cell body and extends through the basal region of neighboring epithelial elements to the basement membrane. Examination of the entire gallbladder epithelial surface by scanning electron microscopy revealed that the BC are numerous in the neck region of the organ but only scanty or even absent in wide areas of the corpus region. Their number increases again in the fundic region. These results demonstrate a preferential regional distribution of BC in the gallbladder, which is discussed in relation to a possible functional significance of the BC.     

对同一细胞内DNA、RNA和蛋白含量相关测定的显微荧光光度法

用荧光染料DAPI、PyroninY和FITC分别染同一细胞内DNA、RNA和蛋白.在紫外光、绿光和兰光顺序激发后,用MPVⅡ显微荧光光度计测量反映单个细胞内DNA、RNA和蛋白含量的荧光强度.根据荧光发射光谱分析,每种染料荧光之间的干扰是可以忽略的.测量结果与FCM获得的结果是一致的.显微荧光光度术对单个细胞多参数相关测量的优点是简单、便宜,并可用于对活细胞获得形态学和定量细胞化学的组合信息.     

基于原子力显微镜测量内耳螺旋器的弹性特征

应用原子力显微镜分析内耳螺旋器(Corti器)不同部位的弹性特征。采用豚鼠内耳基底膜底回新鲜标本,用原子力显微镜在液相接触式测量,获得不同部位力曲线。经计算,对应Corti器相当于Hensen细胞、外毛细胞、柱细胞、内毛细胞、内指细胞、盖膜的部位及基底膜底面局部,其杨氏模量均值分别为46±1.7、59±0.9、250±31、140±2.8、430±29.9、210±7.2和230±8.8 kPa。结果表明,基底膜径向排列的组织结构不同,杨氏模量存在明显差异,在整块基底膜标本上测量Corti器各结构的杨氏模量能更准确地反映它们在生理状态下的弹性特征。     

A detection method for lactic dehydrogenase activity in the inner ear

A method for the detection of lactic dehydrogenase enzymatic activity in outer hair cells of the rabbit is described. The membranous labyrinth with temporal bone was prefixed in glutaraldehyde. After being placed into the incubation medium, it was postfixed in osmium tetroxide. Specimens of the organ of Corti were removed. Then the specimens were embedded in water-soluble glycol and cut with a cryostat for light microscopy, and also they were embedded in Epon and cut for light and electron microscopy. Sectioning of the membranous labyrinth was very easily made when the specimens were embedded in both the water-soluble glycol and the Epon. The structures of the frozen sections as well as the Epon-embedded ones were well preserved. In the frozen sections the preservation and localization of reaction products were thoroughly kept, but monoformazan of the Epon-embedded sections was soluble.     

Measurement of fluorescence using digital integration of video images

The authors describe a system for the quantitation of fluorescence light output by individual cells using the signal obtained from a silicon intensifier target video camera. The video image is digitized to 4 bits (16 levels), and a 512 X 512 matrix is constructed and stored in 128K of video memory. Areas to be measured are user-specified by means of a light pen entry system. Recorded intensities are integrated under microprocessor control to provide a measure of total fluorescence in the selected areas. The distribution of light intensities per pixel over the 16-level gray scale as well as morphometric data are also obtainable. Linear response to transmission and fluorescence standards was verified. Reproducibility of the system was evaluated using fluorescent beads and glutaraldehyde-fixed chick red blood cells, which gave coefficients of variation comparable to those obtainable from other systems. Measurements of DNA per nucleus of human diploid fibroblasts using Hoechst dye 33258 yielded the two sharp peaks corresponding to the 2C and 4C values of DNA expected from such cells. We conclude that digital video measurement of fluorescence provides meaningful data and has considerable promise as a sensitive tool for the quantitation of fluorescence at the cellular level.     

Cytophotometry of DNA in serial sections]

DNA of leprosy cells in serial paraffine sections stained after Felgen were studied photometrically in scanning microscope SPM with electron computer Wang 720C. Significant differences in extinction values (up to 30--35%) between individual sections within the same series were revealed. Cumulative results of different series were rather uniform. It was concluded that photometric studies of serial sections should be carried out for reliable determination of DNA.     

Simultaneous photometry of the DNA and silver-grain count in liver cells labelled with 3H-thymidine or 3H-leucine

A modified method was proposed for reflected light simultaneous measurement of DNA content and of the silver grain number in the nucleus or cytoplasm of the same cell. Specimens-smears of isolated liver cells incorporated 3H-thymidine and 3H-leucine were prepared on coverslips and after processing were mounted on the slide glasses with smeared side facing downwards to avoid the influence of grains on DNA content measurements. To decrease the background, label measurements were carried out in polarized light. It was shown that the intensity of 3H-leucine incorporation in hepatocytes increases proportionally with cell ploidy degree.     

Measurement of DNA content in single cells morphologically identified on smears

A method was developed for measuring the nuclear DNA content in single cells previously identified on a bone marrow smear stained by the Wright-Giemsa method. The smear was first photographed and the location of individual cells, identified by morphology, was recorded on a cell map. The smear was then bleached with 50% acid ethanol and absolute methanol, and re-stained by the Feulgen method in 0.05% pararosaniline Schiff's reagent (pH 2.3) at 7 degrees C for 10 min. Nuclear red fluorescence was observed and the intensity of this fluorescence was proportional to the amount of DNA after prior irradiation of smears with green light for 9 hr. The method is useful for measuring cell DNA content in heterogeneous cell populations when morphological cell identification is required.     

Three-dimensional motion of the organ of Corti

The vibration of the organ of Corti, a three-dimensional micromechanical structure that incorporates the sensory cells of the hearing organ, was measured in three mutually orthogonal directions. This was achieved by coupling the light of a laser Doppler vibrometer into the side arm of an epifluorescence microscope to measure velocity along the optical axis of the microscope, called the transversal direction. Displacements were measured in the plane orthogonal to the transverse direction with a differential photodiode mounted on the microscope in the focal plane. Vibration responses were measured in the fourth turn of a temporal-bone preparation of the guinea-pig cochlea. Responses were corrected for a "fast" wave component caused by the presence of the hole in the cochlear wall, made to view the structures. The frequency responses of the basilar membrane and the reticular lamina were similar, with little phase differences between the vibration components. Their motion was rectilinear and vertical to the surface of their membranes. The organ of Corti rotated about a point near the edge of the inner limbus. A second vibration mode was detected in the motion of the tectorial membrane. This vibration mode was directed parallel to the reticular lamina and became apparent for frequencies above approximately 0.5 oct below the characteristic frequency. This radial vibration mode presumably controls the shearing action of the hair bundles of the outer hair cells.     

A new version of the Ag?NOR technique. A combination with DAPI staining

Summary The Ag−NOR staining technique is widely used for visualizing nucleolar organizer regions (NORs) in various plant and animal tissues. We describe a simple and time-saving combination of Ag−NOR staining with DNA detection by fluorescence microscopy. This modification was tested on cultured cells and semi-thin sections of plastic-embedded tissues. Of the different fixatives and embedding media used in our studies, the best results (i.e., high selectivity of staining, and lack of or very low background precipitation) were obtained with fixation in methanol-acetone at −20°C for cultured cells, and fixation in 4% formaldehyde followed by embedding in Histocryl resin for tissue sections. The optimal time of Ag−NOR staining was determind experimentally for all materials tested. The specificity of the staining was checked at the electron microscopical level. Especially good results were obtained by mixing epifluorescence with standard bright-field illumination. In such a combination, Ag−NOR-positive nucleoli, or their fibrillar centres and dense fibrillar components, were clearly visible against a bright background of nuclear DNA.     

DNA cytofluorometry on large and small cell nuclei stained with pararosaniline Feulgen

Summary To examine reliability of DNA cytofluorometry with conventional Feulgen staining, measurement was carried out on smears of small and large neurons of human cerebellum, using a Digital-Microfluorometer with incident green light excitation. Nuclear fluorescence of the inner granule neuron and the Purkinje cell came to good agreement with each other when the staining was reduced so that the maximal extinction of the excitation beam nowhere exceeded the value of 0.1. The reduction of the feulgen staining is realized with unimpaired proportionality by treatment with 0.05% Schiff's reagent (pH 2.3) at 7–10° C for 10 min. It is concluded that this staining condition assures accurate determination of DNA content irrespective of size of the nucleus and of pattern of the chromatin distribution.     

Light-Harvesting Function in the Diatom Phaeodactylum tricornutum: II. Distribution of Excitation Energy between the Photosystems

The distribution of excitation energy between photosystems I and II (PSI and PSII) was investigated in the marine diatom Phaeodactylum tricornutum (Bohlin) using light-induced changes in fluorescence yield and rate of modulated O₂ evolution. The intensity dependence of the fast fluorescence rise in dark adapted cells (±DCMU) suggests that light absorbed by the major antenna complex was not delivered preferentially to PSII but is more equally distributed between the photosystems. Reversible, slow fluorescence yield changes measured in the absence of DCMU were correlated with decreased initial fluorescence and rate constants for PSII photochemistry, increased variable fluorescence, alteration of the fluorescence excitation and emission spectra, and could be effected by either 510 nm (PSII) or 704 nm (PSI) light. Slow, reversible fluorescence yield changes were also observed in the presence of DCMU, but were characterized by a loss of both initial and variable fluorescence and could not be induced by PSI light. The absence of slow changes in the yield of fluorescence and rate of modulated O₂ evolution, following addition or removal of PSI background light to modulated PSII excitation, does not support regulation of excitation energy density in PSI at the expense of PSII. The results suggest that adjustments are made at the level of excitation energy transfer to the PSII reaction center which prevent prolonged loss of photosynthetic capacity. Energy distribution is regulated by ionic distributions independently of the plastoquinone pool redox state. These differences in light-harvesting function are probably a response to the aquatic light field and may account for the success of diatoms in low and variable light environments.     

Fluorescence in situ hybridization to interphase cell nuclei in suspension allows flow cytometric analysis of chromosome content and microscopic analysis of nuclear organization

Summary Fluorescence hybridization to interphase nuclei in liquid suspension allows quantification of chromosome-specific DNA sequences using flow cytometry and the analysis of the three-dimensional positions of these sequences in the nucleus using fluorescence microscopy. The three-dimensional structure of nuclei is substantially intact after fluorescence hybridization in suspension, permitting the study of nuclear organization by optical sectioning. Images of the distribution of probe and total DNA fluroescence within a nucleus are collected at several focal planes by quantitative fluorescence microscopy and image processing. These images can be used to reconstruct the three-dimensional organization of the target sequences in the nucleus. We demonstrate here the simultaneous localization of two human chromosomes in an interphase nucleus using two probe labeling schemes (AAF and biotin). Alternatively, dual-beam flow cytometry is used to quantify the amount of bound probe and total DNA content. We demonstrate that the intensity of probe-linked fluorescence following hybridization is proportional to the amount of target DNA over a 100-fold range in target content. This was shown using four human/hamster somatic cell hybrids carrying different numbers of human chromosomes and diploid and tetraploid human cell lines hybridized with human genomic DNA. We also show that populations of male, female, and XYY nuclei can be discriminated by measuring their fluores-cence intensity following hybridization with a Y-chromosome-specific repetitive probe. The delay in the increase in Y-specific fluorescence until the end of S-phase is consistent with the results recorded in previous studies indicating that these sequences are among the last to replicate in the genome. A chromosome-17-specific repetitive probe is used to demonstrate that target sequences as small as one megabase (Mb) can be detected using fluorescence hybridization and flow cytometry.