Reflection Contrast Microscopy Research Articles

Reflection contrast microscopy. Visualization of (peroxidase-generated) diaminobenzidine polymer products and its underlying optical phenomena.

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 diaminobenzidine (DABox) in immunocytochemically stained specimens, can be made visible. RCM has been successfully applied in demonstrating single copy nucleic acid sequences using in situ hybridization procedures (Landegent et al. 1984). We have systematically studied the aspects of image formation of DABox by RCM by using a model system consisting of glass slides coated with peroxidase containing protein layers to determine the conditions for optimal sensitivity of this detection method. Moreover, investigations were performed to study the relationship between the amount of reflected light and DABox depending on the thickness of the object. Both theoretical and practical evidence is obtained to show that DABox detection by RCM is based on interference phenomena occurring in the layer of DABox, and less on selective reflection. This restricts the type of specimen which can be used for sensitive detection of DABox by RCM. Consequently, in ultrathin (40 nm) sections osmificated DABox was visualized in peroxidatic positive cell organelles with high contrast and resolution. Similar results were obtained with immunoperoxidase stained material embedded in Lowicryl under conditions that did not allow visualization of the staining product by bright field microscopy.

Direct visualization of single copy genes on banded metaphase chromosomes by nonisotopic in situ hybridization.

A rapid method is described for non isotopic in situ mapping of single copy genes directly on G-banded chromosomes by "one-step" regular light microscopy. It is based on hybridizing biotinylated probes to metaphase chromosomes. Biotin residues are detected by rabbit antibiotin antibody and anti-rabbit Ig labelled with peroxidase or colloidal gold. The peroxidase reaction product or colloidal gold signals are amplified by silver precipitation. The final product is a black silver dot at the gene locus on a purple G-banded chromosome. N-ras and alpha-1-antitrypsin genes have been mapped using plasmids with inserts of 1.5 and 1.3kb to 1p13.1 and the junction of 14q31/32 respectively. The signal to noise ratio in these experiments ranged from 32:1-46:1. This technology is at least as sensitive as radioisotopic in situ hybridization and gives results within 1 day of hybridization and has much better resolution. Additionally, genes are visualized by regular light microscopy without specialized techniques such as reflection contrast, fluorescence or phase microscopy. This methodology should facilitate more precise chromosomal gene localization.

Chromosomal insertion of human papillomavirus 18 sequences in HeLa cells detected by nonisotopic in situ hybridization and reflection contrast microscopy.

Genomic insertion of human papillomavirus (HPV) sequences is associated with the genesis of cervical carcinoma, and HPV-induced incipient cellular alterations may also present a requisite for the establishment of cell lines such as HeLa. Considering the theoretical importance of specific viral integration sites, we attempted to detect in HeLa cells the chromosomal location of DNA sequences homologous to HPV-16 and HPV-18 sequences by a nonisotopic high resolution in situ hybridization technique. Chromosome identification following in situ hybridization was possible by counterstaining of the same preparation with Chromomycin A3, Distamycin A, and DAPI. Using this approach, we have assigned HPV-18 integration in HeLa cells to band 8q24 (a site including the locus of the myc-protooncogene), to an abnormal chromosome 22, and to a not yet identified marker chromosome possibly neighboring other oncogenic or activating sites. The sensitive detection technique described in this study presents a new approach involving in situ chromosome hybridization with biotinylated DNA probes in combination with reflection contrast microscopy and subsequent fluorescent R- and C-banding. The method allowed the assignment of a 7-kb HPV-18 DNA probe to human chromosomal sites important in growth regulation and cancerogenesis. It should prove useful in a number of similar studies using other viral and oncogenic DNA probes.

Detection of a 17 kb unique sequence (T-DNA) in plant chromosomes by in situ hybridization

An approach is described for the detection of a unique sequence, the T-DNA region of the Agrobacterium rhizogenes root-inducing (Ri) plasmid, in plant chromosomes by in situ hybridization. This sequence was introduced into the Crepis capillaris genome (2n=6) by infecting Crepis stem segments with A. rhizogenes. Roots growing from the infection site contain T-DNA and synthesize mannopine, which can be used as a convenient biochemical marker for T-DNA transformation. Southern analysis of DNA isolated from one transformed Crepis root line verified the presence of a single copy of T-DNA (approximate size 17 kb) per diploid Crepis genome. To localize T-DNA, both DNA and RNA probes, labelled with either tritium or biotin, were hybridized to Crepis chromosomes prepared from transformed root tips by a novel spreading method. Biotinylated probes were visualized using reflection-contrast microscopy. In the hybridization experiments described, T-DNA was detected in one homologue of chromosome 3, where it could be assigned to a paracentromeric position in the neighbourhood of the nucleolar organizing region. These results demonstrate that it is possible to localize unique sequences in plant chromosomes by in situ hybridization.

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.

Chromosomal localization of a unique gene by non-autoradiographic in situ hybridization.

During the past few years, several methods have been developed for the detection of specific nucleic acid sequences by in situ hybridization using non-radioactive labels such as fluorochromes, cytochemically detectable enzymes and electron-dense markers. These methods are preferable to autoradiography in terms of speed of performance and topological resolution. Their limited sensitivity, however, has so far restricted their use to the detection of repeated sequences. Here we report single gene detection with a procedure using 2-acetylaminofluorene (AAF)-modified probes, immunoperoxidase cytochemistry and reflection-contrast microscopy. We confirmed the autoradiographic data on the localization of the human thyroglobulin (Tg) gene to the distal end of the long arm of chromosome 8. A mixture of cosmid cHT2-derived subclones of the 3' part of the Tg gene, 22.3 kilobase pairs (kbp) in total, was used as a hybridization probe. This procedure can be used to map other unique sequences, if genomic clones are available from which clones with an appropriate amount of inserts can be isolated.

Lymph node metastasis and cell movement: ultrastructural studies on the rat 13762 mammary carcinoma and Walker carcinoma

The aim of this study was to determine whether tumor cells move actively through the linings of lymph node sinuses. Using 13762 carcinoma in F344 rats, and Walker carcinoma in Wistar rats, 20 X 10(6) tumor cells were injected into the footpad, and the ipsilateral popliteal lymph node examined by transmission electron microscopy. The same tumors were examined by making standard cell spots on plastic or glass surfaces, and examining these by phase and reflection contrast microscopy, fluorescent microscopy after anti-actin and phallacidin staining and transmission electron microscopy. The 13762 cells do not migrate through the lining of the lymph node sinusoid, nor move actively in vitro. Ultrastructural appearances of the Walker rat carcinoma cells suggest that they move actively through the sinus lining. After 24 h in vitro the Walker rat carcinoma cells in the centre of the spot are adherent to the surface. There is some movement of the edge of the sheet, and individual tumor cells at the edge of the sheet move actively and independently. We conclude that the Walker rat carcinoma invades the lining of the lymph node sinusoid by active cell movement, and the 13762 carcinoma does not.

Disruption of microfilament organization after injection of F-actin capping proteins into living tissue culture cells.

Capping proteins are F-actin binding proteins which interfere with the in vitro growth of an actin filament by blocking one of its ends (for recent reviews see refs 1-3). The majority of such proteins described so far "cap' the fast-growing (positive) end of the polar filament, thus reducing the velocity of filament growth while increasing the number of filaments being formed de novo from a monomer pool. We have studied the effects of capping proteins on the organization of actin filaments in living tissue culture cells by microinjection in conjunction with fluorescence, reflection contrast and electron microscopy. Our results, reported here, indicate that capping proteins from different sources disrupt microfilament bundles in a variety of cell types causing their disintegration from the distal end towards the centre of the cell.

Use of reflection contrast microscopy in the analysis of cellular motility.

Recognition and determination of the following activities of living cancer cells on glass substrates can be greatly facilitated by the use of reflection contrast microscopy: 1. stationary versus translocative motility, 2. migration over/under other cells, 3. actual locomotory activity of cells with a polarized shape usually associated with this type of motility. In addition, reflection contrast is useful for recognizing the presence of fibroblasts in cancer cell populations.

The distribution of fibronectin in attachment sites of chick fibroblasts

Primary chick heart fibroblasts were cultured on glass coverslips and examined by reflection contrast microscopy and then by indirect immunofluorescence microscopy using affinity purified antifibronectin antibody. Fibronectin was found to be primarily localized in areas of ‘close’ contact and in intensely staining fibrous webs that were usually external to the cytoplasmic matrix. Focal contacts, in which there is intimate contact between the cell and the substratum by localized attachment, failed to react with the anti-fibronectin antibody despite a variety of extraction procedures designed to increase the accessibility of antibody to this type of attachment site. It is concluded that in chick fibroblasts, fibronectin participates in cell attachment at areas of close contact and fibrillar attachment sites, but is often excluded from the attachment pads of focal contacts.

Quantitative morphological analysis of erythrocytes by reflection contrast microscopy.

In reflection contrast erythrocytes show characteristic interference lines consisting of alternating maxima and minima. The distance between neighboring interference lines corresponds to a difference in cell thickness of about 113 nm. Some visual and graphic methods for quantitative morphological analysis are described; their application is demonstrated using unstained and stained blood smears of normal individuals and patients with hereditary spherocytosis, sickle cell anemia, and thalassemia.

Cell-to-substrate adhesions during spreading and locomotion of carcinoma cells: A study by microcinematography and reflection contrast microscopy

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 contrast, cells from a human epidermoid carcinoma (LICR-OC-1) first attain a fully spread configuration before some cells slightly round up again for a slow locomotory activity of short range and duration. Reflection contrast showed that during spreading and locomotion, the cells from both carcinomas displayed a predominance of grey, the color associated with close contacts. Fully spread cells, on the other hand, presented a multitude of focal contacts in individually different arrangements of black streaks and dots, randomly distributed over the entire cell area. The functional meaning of this heterogeneity in the arrangement of focal contacts in fully spread cells is not yet understood. The importance of close contacts for spreading and locomotion, however, seems to be established and is in agreement with findings reported for other cell types engaged in the same activities. It is therefore suggested that the formation of substrate contacts depends on cellular activity rather than on the cell type.

Ingestion of latex beads by filopodia of adherent mouse peritoneal macrophages. A scanning electron microscopical and reflection contrast microscopical study

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 surface, the cells were studied with a reflection contrast microscope prior to the preparative procedure. The results indicate that reflection-contrast microscopy and SEM both show the same filopodia for a given cell. The filopodia appear to be functional components of the cytoplasm, as shown by their ability to ingest latex beads.

Corrugated attachment membrane in WI-38 fibroblasts: alternating fibronectin fibers and actin-containing focal contacts.

The distributions of both fibronectin (LETS, CSP) fibers and focal contacts to the substratum, as viewed by fluorescence and reflection contrast microscopy, respectively, have been compared in freshly plated WI-38 human fibroblasts. Most frequently, the actual focal attachment plaques did not contain fibronectin fluorescence and, furthermore, fibronectin spots and fibers often alternated with focal contacts. Overlap, however, was observed between focal contacts and the endings of actin-containing stress fibers [see also Wehland, J., Osborn, M. & Weber, K. (1979) J. Cell Sci. 37, 257-273]. Thus, the fibroblast attachment membrane might best be described as a corrugated sheet that undulates between alternating microfilaments and fibronectin fibers, at the points of closest and farthest distance to the substratum, respectively.

Quantitative reflection contrast microscopy of living cells.

Mammalian cells in culture (BHK-21, PtK2, Friend, human flia, and glioma cells) have been observed by reflection contrast microscopy. Images of cells photographed at two different wavelengths (546 and 436 nm) or at two different angles of incidence allowed discrimination between reflected light and light that was both reflected and modulated by interference. Interference is involved when a change in reflected intensity (relative to glass/medium background reflected intensity) occurs on changing either the illumination wavelength or the reflection incidence angle. In cases where interference occurs, refractive indices can be determined at points where the optical path difference is known, by solving the given interference equation. Where cells are at least 50 nm distant from the glass substrate, intensities are also influenced by that distance as well as by the light's angle of incidence and wavelength. The reflected intensity at the glass/medium interface is used as a standard in calculating the refractive index of the cortical cytoplasm. Refractive indices were found to be higher (1.38--1.40) at points of focal contact, where stress fibers terminate, than in areas of close contact (1.354--1.368). In areas of the cortical cytoplasm, between focal contacts, not adherent to the glass substrate, refractive indices between 1.353 and 1.368 were found. This was thought to result from a microfilamentous network within the cortical cytoplasm. Intimate attachment of cells to their substrate is assumed to be characterized by a lack of an intermediate layer of culture medium.

Locomotion and adhesion of neutrophil granulocytes: Effects of albumin, fibrinogen and gamma globulins studied by reflection contrast microscopy

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 have a positive chemokinetic effect on neutrophils suspended in Gey's solution. Standard gamma globulin (SGG) or acid-treated gamma globulin with polymers have marked negative chemokinetic activity. Three different mechanisms are presumably responsible for the low rate of locomotion observed in Gey's solution alone, Gey's solution containing acid-treated gamma globulin with polymers or SGG, respectively: ( a) too firm adhesion to the substratum; ( b) lack of adhesion to the substratum; and ( c) impaired capacity to perform shape changes. The relationship between attachment of cells to the substratum and the rate of neutrophil locomotion has been investigated. It appears that the pattern of adhesion rather than cell attachment as measured by the proportion of neutrophils adhering to the substratum is a meaningful correlate to locomotion. Two different patterns of adhesion can be distinguished by means of reflection-contrast microscopy: ( a) the pattern characterized by uniform grey areas is compatible with efficient locomotion; ( b) a pattern characterized by large black areas at the cell periphery. It is associated with neutrophils in Gey's solution which fail to displace themselves efficiently. This suggests that reflection-contrast microscopy may be helpful in distinguishing contacts allowing locomotion to occur from contacts impeding neutrophil locomotion.

In vivo distribution and turnover of fluorescently labeled actin microinjected into human fibroblasts.

Graessmann's microinjection technique was chosen to introduce fluorescently labeled muscle actin and other proteins into WI-38 human fibroblasts. The injected cells were examined during culture by fluorescence and reflection contrast microscopy. Within 30 min after injection, rhodamine-labeled actin was incorporated into a distinct network of fluorescent filaments, resembling the stress fibers observed by classical immunofluorescence microscopy. Cytochalasin b prevented the formation of these fibers, but colchicine did not. Neighboring fibers often converged into distinct focal points that appeared to be concentrated near the base of the cell. Examination of these fluorescent fibers and focal points by reflection contrast microscopy confirmed their close location to the substratum. After 6 hr of culture, fluorescent actin and the control proteins were segregated into granules located mainly near the cell nucleus. Thus, the injected actin both enters the intrinsic actin pool and participates in an assembly and disassembly of filamentous structures. Segregation into granules traces the natural turnover of this protein within the cell.

Adhesion patterns of cell interactions revealed by reflection contrast microscopy

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 a flattened configuration. Such a change took place when leukemia cells came into contact with other spread cells and started to move under these cells. Reflection contrast further showed that in the pathway of the locomoting L5222 cells the adhesions of the overlying spread cells were momentarily detached from the substrate. It is concluded that the combination of reflection contrast and phase contrast represents a good tool for gaining new information on the interaction of motility and formation of adhesions.