Reflection Contrast Microscopy Research Articles

Multi-scale alignment of respiratory cilia and its relation to mucociliary function

The tracheobronchial tree is lined by a mucociliary epithelium containing millions of multiciliated cells. Their integrated oscillatory activity continuously propels an overlying pollution-protecting mucus layer in cranial direction, leading to mucociliary clearance – the primary defence mechanism of the airways. Mucociliary transport is commonly thought to co-emerge with the collective ciliary motion pattern under appropriate geometrical and rheological conditions. Proper ciliary alignment is therefore considered essential to establish mucociliary clearance in the respiratory system. Here, we used volume electron microscopy in combination with high-speed reflection contrast microscopy in order to examine ciliary orientation and its spatial organization, as well as to measure the propagation direction of metachronal waves and the direction of mucociliary transport on bovine tracheal epithelia with reference to the tracheal long axis (TLA). Ciliary orientation is measured in terms of the basal body orientation (BBO) and the axonemal orientation (AO), which are commonly considered to coincide, both equivalently indicating the effective stroke as well as the mucociliary transport direction. Our results, however, reveal that only the AO is in line with the mucociliary transport, which was found to run along a left-handed helical trajectory, whereas the BBO was found to be aligned with the TLA. Furthermore, we show that even if ciliary orientation remains consistent between adjacent cells, ciliary orientation exhibits a gradual shift within individual cells. Together with the symplectic beating geometry, this intracellular orientational pattern could provide for the propulsion of highly viscous mucus and likely constitutes a compromise between efficiency and robustness.

Applications of reflection-contrast microscopy, including the sensitive detection of the results of in situ hybridisation a review.

The observation with RCM of the reflection from reaction products produced by nonisotopic in situ hybridisation and a peroxidase staining, has recently facilitated the identification of single copy genes. RCM also reveals light microscope structures in stained ultrathin (0.1 μm) epon and lowicryl sections. In such preparations no out-of-focus blur can be observed. Confocal optics are thus not needed to obtain high-quality images of ultrathin sections at the highest conventional light microscope optical resolution, and with higher image-contrast than can be obtained by classical absorption microscopy. Such RCM images provide the same type of information as confocal scanning microscope images. A sequential ultrathin section of the same microscopic specimen can be examined with electron microscopy for a simple and accurate (CLEM) procedure. For some applications RCM can replace (CLSM). LAY DESCRIPTION: With reflection contrast microscopy (RCM), a microscope image can be observed if micromirror-like reflecting substances are present in the reaction product of cytochemical stains. Using an antiflex objective for RCM, the reflected incident light from the stained microscope specimen can be observed with only a small amount of unwanted stray-light in the image background. Stray-light is caused by reflection of incident light from the surface of the lenses in the microscope objective and in the microscope tube (using a 50% beam splitter for epi-illumination). Several authors who compared RCM with bright-field and fluorescence microscopy, found RCM to be a sensitive microscope method in many applications. It has been reported that RCM, using in situ hybridisation methods, can detect DNA sequences in single copy genes. In the literature is mentioned that single copy genes have an estimated mass of less than a trillionth of a gram of DNA. From stained ultrathin microscope sections with less than 0.1 μm thickness, multicoloured images with a high image contrast, optimal resolution and no out-of-focus blur can be observed with RCM. To achieve this, RCM uses an antiflex microscope objective, with a quarter-wave plate mounted on the front lens in combination with a polarising filter cube inserted in the epi-illuminator. RCM can also make use of oblique illumination, for a further increase of the image-contrast and the resolution of the microscope image. A central stop has then to be inserted in the aperture plane of an epi-illuminator.

Refractive Index of Cells Measured through a Microscope with Variable-Angle TIR

The refractive index in the interior of single cells is interesting because it both indirectly senses the concentration of solutes in the cytoplasm, and also directly affects the evanescent field depth in quantitative studies using total internal reflection (TIR) fluorescence. However, only a few studies have directly measured the intracellular refractive index, notably that of Bereiter-Hahn et al (1979) using quantitative interference reflection contrast (IRC) microscopy. IRC microscopy produces sharp images but does contain some inherent (but partly surmountable) ambiguities, particularly in distinguishing refractive index effects from distance -to-substrate effects. We present an alternative: direct microscopic imaging of a collimated light beam reflected from the substrate/buffer/cell interference at variable angles of incidence. Above the TIR critical angle (which is a strong function of refractive index), the reflection is 100%, but in the immediate sub-critical angle zone, the reflection intensity is a very strong ascending function of incidence angle. By analyzing the angular position of that edge at each location in the field of view, the local refractive index can be estimated. In addition, by analyzing the steepness of the edge, the distance -to-substrate can be separately determined. We show how this non-fluorescence technique can be implemented in a modified objective-based TIR setup and we present results on both calibration samples and on chromaffin cells in primary culture. The optical technique suffers from rather poor lateral resolution, so subcellular organelles cannot be seen easily. However, the technique as combined with appropriate theory and calibration provides reasonable results for both refractive index (∼1.375) and for distance-to-substrate (∼100 nm) for the cells. Supported by NIH RO1-GM110289 to RWH and DA.

Growth plate regulation and osteochondroma formation: insights from tracing proteoglycans in zebrafish models and human cartilage

Proteoglycans are secreted into the extracellular matrix of virtually all cell types and function in several cellular processes. They consist of a core protein onto which glycosaminoglycans (e.g., heparan or chondroitin sulphates), are attached. Proteoglycans are important modulators of gradient formation and signal transduction. Impaired biosynthesis of heparan sulphate glycosaminoglycans causes osteochondroma, the most common bone tumour to occur during adolescence. Cytochemical staining with positively charged dyes (e.g., polyethyleneimine-PEI) allows, visualisation of proteoglycans and provides a detailed description of how proteoglycans are distributed throughout the cartilage matrix. PEI staining was studied by electron and reflection contrast microscopy in human growth plates, osteochondromas and five different proteoglycan-deficient zebrafish mutants displaying one of the following skeletal phenotypes: dackel (dak/ext2), lacking heparan sulphate and identified as a model for human multiple osteochondromas; hi307 (β3gat3), deficient for most glycosaminoglycans; pinscher (pic/slc35b2), presenting with defective sulphation of glycosaminoglycans; hi954 (uxs1), lacking most glycosaminoglycans; and knypek (kny/gpc4), missing the protein core of the glypican-4 proteoglycan. The panel of genetically well-characterized proteoglycan-deficient zebrafish mutants serves as a convincing and comprehensive study model to investigate proteoglycan distribution and the relation of this distribution to the model mutation status. They also provide insight into the distributions and gradients that can be expected in the human homologue. Human growth plate, wild-type zebrafish and fish mutants with mild proteoglycan defects (hi307 and kny) displayed proteoglycans distributed in a gradient throughout the matrix. Although the mutants pic and hi954, which had severely impaired proteoglycan biosynthesis, showed no PEI staining, dak mutants demonstrated reduced PEI staining and no gradient formation. Most chondrocytes from human osteochondromas showed normal PEI staining. However, approximately 10% of tumour chondrocytes were similar to those found in the dak mutant (e.g., lack of PEI gradients). The cells in the reduced PEI-stained areas are likely associated with loss-of-function mutations in the EXT genes, and they might contribute to tumour initiation by disrupting the gradients.

Cell adhesion property affected by cyclooxygenase and lipoxygenase: Opto-electric approach

Expression of cyclooxygenases (COX) and lipoxygenases (LOX) has been linked to many pathophysiological phenotypes, including cell adhesion. However, many current approaches to measure cellular changes are performed only in a fixed-time point. Since cells dynamically move in conjunction with the cell matrix, there is a pressing need for dynamic or time-dependent methods for the investigation of cell properties. In the presented study, we used stable human colorectal cancer cell lines ectopically expressing COX-1, COX-2, and 15LOX-1, to investigate whether expression of COX-1, COX-2, or 15LOX-1 would affect cell adhesion using our opto-electric methodology. In a fixed-time point experiment, only COX-1- and COX-2-expressing cells enhanced phosphorylation of focal adhesion kinase, but all the transfected cells showed invasion activity. However, in a real-time experiment using opto-electric approaches, transmitted cellular morphology was much different with tight adhesion being shown in COX-2 expressing cells, as imaged by differential interference contrast microscopy (DICM) and interference reflection contrast microscopy (IRCM). Furthermore, micro-impedance measurements showed a continued increase in both resistance and reactance of COX- and LOX-transfected cells, consistent with the imaging data. Our data indicate that both COX- and LOX-expressing cells have strong cell-to-cell and cell-to-substrate adhesions, and that cell imaging analysis with cell impedance data generates fully reliable results on cell adhesion measurement.

Multicontrast microscopy technique to dynamically fingerprint live-cell focal contacts during exposure and replacement of a cytotoxic medium

Multicontrast microscopy techniques were used to comprehensively and dynamically map the cellular contact area adhering to a substrate. The natural fringe patterns observed with interference reflection contrast microscopy were used to map the dynamic fingerprint of a porcine pulmonary artery endothelial cell's ventral surface and to examine the focal and/or close contacts to the substrate when exposed to a toxic agent Cytochalasin D. In addition, differential interference contrast microscopy sequentially imaged the overall cellular morphological responses to the agent. It was observed that focal contacts, which are tightly attached to the substrate, are strongly resistant to even high doses of the cytotoxic agent and that they also form the basis of cellular recovery after replacement of the cytotoxic medium with fresh medium.

Opto-Electric Cellular Biosensor Using Optically Transparent Indium Tin Oxide (ITO) Electrodes.

Indium tin oxide (ITO) biosensors are used to perform simultaneous optical and electrical measurements in order to examine the dynamic cellular attachment, spreading, and proliferation of endothelial cells (ECs) as well as cytotoxic effects when exposed to cytochalasin D. A detailed description of the fabrication of these sensors is provided and their superior optical characteristics are qualitatively shown using four different microscopic images. Differential interference contrast microscopy (DICM) images were acquired simultaneously with micro-impedance measurements as a function of frequency and time. A digital image processing algorithm quantified the cell-covered electrode area as a function of time. In addition, cytotoxicity effects, produced by the toxic agent cytochalasin D, were examined using micro-impedance measurements, confocal microscopy images of stained actin-filaments, and interference reflection contrast microscopy (IRCM) capable of examining the bottom morphology of a cell. The results of this study show (1) the dynamic optical and electrical cellular characteristics using optically thin ITO biosensors; (2) qualitative agreement between cell-covered electrode area and electrical impedance during cellular attachment; (3) in vitro cytotoxicity detection of ECs due to 3 μM cytochalasin D. The present opto-electric biosensor system is unique in that a simultaneous and integrated cellular analysis is possible for a variety of living cells.

Comparison of reflection contrast microscopy and electron microscopy on the histopathological diagnosis of various kidney diseases

Our aim in this study was to compare reflection contrast microscopy (RCM) with transmission electron microscopy (TEM) to understand whether RCM could be used in the histopathological diagnosis of various kidney diseases as a less expensive and an easier alternative to TEM. The diagnoses of kidney pathologic lesions included Alport syndrome, thin membrane disease, Ig A nephropathy. RCM is a form of light microscope that works in the reflected mode, suitable to observe ultrathin (50-100 nm) plastic sections that is also used in TEM. Our findings showed that RCM showed similar results compared with TEM on these lesions described earlier.

Monitoring live cell viability: Comparative study of fluorescence, oblique incidence reflection and phase contrast microscopy imaging techniques

The relative performances of fluorescence, oblique incidence reflection and phase contrast imaging techniques have been studied for the purpose of monitoring long-term cellular activity and cell viability of several types of normal and cancerous cells in cultures. Time-lapse movies of live cell imaging of untagged and green fluorescent protein (GFP) tagged cell lines are presented. Oblique incidence reflection microscopy is the simplest and least expensive method to implement, appears to be the least phototoxic to cells, and is recommended for use in long-term optical monitoring of cell viability.

Part of membrane-bound Aβ exists in rafts within senile plaques in Tg2576 mouse brain

To clarify whether rafts are the site of abnormal amyloid β protein (Aβ) deposition, we examined the ultrastructural localization of both flotillin-1 (pre-embedding) and Aβ (post-embedding) in Tg2576 mouse brains. After observing the exact areas of senile plaques by reflection contrast microscopy, we observed these same plaques under an electron microscope. Membrane-bound Aβ was predominantly observed on plasma membranes of small processes in diffuse plaques. Non-fibrillar and fibrillar Aβ was increased in primitive plaques, and the fibrillar form was predominant in mature plaques. The number of flotillin-1-positive rafts per field in mature plaques was prominently less than those outside of the plaques, in diffuse plaques and in primitive plaques. The colocalization of flotillin-1 with Aβ42 appeared approximately 10% of flotillin-1-positive rafts within senile plaques, while there was no colocalization found outside of the plaques. This study ultrastructurally demonstrated that part of membrane-bound Aβ exists in lipid rafts within senile plaques, and suggests that rafts could be one of the sites for initial Aβ deposition.

2] Reflection interference contrast microscopy

This chapter describes reflection interference contrast microscopy (RICM). The term “RICM” is used in the chapter to cover all optical configurations and practical applications of the method. The method by which the light reflected from a semitransparent, epi-illuminated specimen contributes to the image formation in a microscope has been given different names, depending on details of the optical configuration of the microscope and on the particular application. It was introduced into cell biology by Curtis using the name “interference reflection microscopy” (IRM), and this name, or its variant, “reflection interference microscopy” (RIM), is often used in studies of adhesion of living cells. Ploem used the term “reflection contrast microscopy” (RCM) on the introduction of the antiflex method to increase image contrast. In RICM of living cells, reflections from the cell plasma membrane and intracellular membranes contribute to the image formation by interfering with each other and with the light reflected from the interface between the underlying glass surface and the liquid medium.

Neurohistological examination of the inferior glenohumeral ligament of the shoulder

The neural histology of the anterior band of the inferior glenohumeral ligament (IGHL) was studied in 11 fresh shoulder specimen using a special silver impregnation technique. Between the collagen fibers small myelinated and unmyelinated dendrites could be detected. The appearance of neurovascular structures in the adjacent synovial layer clearly exceeded the typical supply to soft tissues. Analysing about 11,000 sections Ruffini mechanoreceptors that are known to be slow adapting were found on the humeral insertion of the band. The sections containing these neural end organs were identified by means of transillumination and reflection-contrast microscopy and reconstructed using three-dimensional image processing. The presence of neural structures including Ruffini corpuscles in these most important passive stabilizers of the shoulder joint shows that these ligaments function also as an active safety device. There slow adaption is a prerequisite for muscular reflexes counteracting the tensile stresses to which the passive stabilizing structures of the shoulder are exposed. A disruption of the continuity of these structures by mechanical forces or surgery can reduce the biofeedback and proprioceptive quality and thus lead to a decrease of shoulder function and/or stability. These observations should be taken into account when planning surgical interventions involving the IGHL. Procedures like capsule shifts or plications may affect mechanoreceptor orientation and concentrations, thereby affecting the interaction between these structures and the synergistic muscles. When possible, these intervention should avoid receptor-dense regions while attempting to restore normal anatomical orientation and tissue tension.

Local cell membrane deformations due to receptor-ligand bonding as seen by reflection microscopy.

Understanding surface receptor clustering and redistribution processes at the cell-matrix contact zone requires detailed knowledge of the spatial integration of these molecules in the architecture of this complex interface. Here we present and discuss critically a procedure to extract such information combining reflection contrast microscopy (RCM) and reflection interference microscopy (RIM). As model system, we used living human umbilical vein endothelial cells (HUVEC) adhering to laminin-coated surfaces and investigated the distribution of the alpha2beta1 (CD29/CD49b) integrin at the contact zone of these cells. First, we applied freeze-fracture electron microscopy to gain information on microscopic details of the alpha2beta1 distribution at the contact zone. Next, we visualized and analyzed the overall lateral distribution of the integrins applying RCM using immunogold-labeling with 10 nm labels and a special silver enhancement technique. We found that RCM can be used to determine the lateral position of the marked receptor molecules to an accuracy of about 100-200 nm, instead of large morphological changes at the contact zone during silver enhancement. Finally, we combined RCM with RIM and analyzed the interference pattern of the contact zone around the label positions. Thus, we were able to detect changes of the average shape of the cell membrane due to receptor-ligand bonding of a size down to the resolution of the techniques.

Reflection contrast microscopy (RCM): a forgotten technique?

Reflection contrast microscopy (RCM), which utilizes the optical phenomena caused by oblique epi-illumination in combination with a specific optical apparatus, provides an approach for exploring biological phenomena in greater detail. The lack of stray reflection makes it superior to other microscopes. It bridges light and electron microscopic capabilities by allowing the analysis of ultrathin sections beyond the usual light microscopic magnification. By using consecutive image analysis, quantitation can be achieved. The wide range of applications of RCM can be combined with most microscopical techniques, so extending the spectrum of information that can be gathered. Twenty-five years after the development of RCM, there is still scope for its application in modern cell biology. Copyright © 2000 John Wiley & Sons, Ltd.

Reflection contrast microscopy (RCM): a forgotten technique?

Reflection contrast microscopy (RCM), which utilizes the optical phenomena caused by oblique epi-illumination in combination with a specific optical apparatus, provides an approach for exploring biological phenomena in greater detail. The lack of stray reflection makes it superior to other microscopes. It bridges light and electron microscopic capabilities by allowing the analysis of ultrathin sections beyond the usual light microscopic magnification. By using consecutive image analysis, quantitation can be achieved. The wide range of applications of RCM can be combined with most microscopical techniques, so extending the spectrum of information that can be gathered. Twenty-five years after the development of RCM, there is still scope for its application in modern cell biology.

Ultrastructural evidence of early non-fibrillar Aβ42 in the capillary basement membrane of patients with hereditary cerebral hemorrhage with amyloidosis, Dutch type

The C-terminal profile and ultrastructure of small and presumably early capillary amyloid beta protein (Abeta) deposits were investigated in four patients with hereditary cerebral hemorrhage with amyloidosis, Dutch type. The C terminus of the 40 (Abeta40) or the 42 (Abeta42) amino acid form of Abeta was gold labeled in serial, ultrathin sections on glass slides for reflection contrast microscopy and on grids for electron microscopy. In all studied subjects, reflection contrast microscopy revealed capillaries with focal Abeta42 immunolabeling in the absence of Abeta40 labeling. In the adjacent electron microscopic section, Abeta42 labeling was confined to the capillary basement membrane. The majority of Abeta42(+)40(-) deposits showed no amyloid fibrils. Abeta42(+)40(-) deposits were sometimes observed in an unremarkable basement membrane but usually showed increased electron density and reticular structures. A small subset of Abeta42(+)40(-) deposits with basement membrane changes showed few amyloid fibrils. Abeta42(+)40(+) capillary deposits always showed definite fibrils and were larger than Abeta42(+)40(-) capillary deposits. The present findings suggest that in capillaries the accumulation and subsequent polymerization of Abeta42, possibly in conjunction with basement membrane changes, precedes the definite fibril formation with Abeta40.

Reflection contrast microscopy for high resolution detection of (3)H-estradiol in ultrathin sections of human stratum corneum.

A single autoradiographical method for light and electron microscopy (LM and EM) is presented. Human skin, containing (3)H-estradiol ((3)H-E2) after an in vitro permeation experiment, was processed via a non-extractive tissue preparation protocol, comprising cryo-fixation, freeze-drying, osmium tetroxide vapor fixation, and Spurr resin embedding. Semithin sections were processed for LM autoradiography, while ultrathin sections were processed both for high-resolution LM and EM autoradiography. The autoradiographs were visualized by bright-field microscopy (BFM), reflection contrast microscopy (RCM), and transmission electron microscopy to evaluate the potentials of RCM visualization in high-resolution LM autoradiography. RCM visualization of ultrathin vs. semithin resin sections showed an improved stratum corneum morphology. Histological staining was superfluous. The localization of (3)H-E2 in human stratum corneum using high-resolution LM autoradiography and RCM was as accurate as with high-resolution EM autoradiography.

Modular domains of focal adhesion-associated proteins.

Attachment of cells to the extracellular matrix is mediated by structures called “focal adhesions.” Focal adhesions can be readily visualized by reflection contrast microscopy in cells grown in culture either on plastic or a defined extracellular matrix (ECM; e.g., fibronectin, collagen), appearing as spear tip-like structures connecting the extracellular matrix with the ventral plasma membrane (Jockusch et al. 1995). The cytoplasmic face of such structures is decorated with the termini of numerous actin stress filaments, indicating a direct linkage with the actin cytoskeletal network. Focal adhesions not only provide the cell with a point of adhesive contact with the extracellular matrix, but also function to mediate signals that regulate cell growth, migration and apoptosis (Burridge and Chrzanowska-Wodnicka 1996; Hynes 1992; Jockusch et al. 1995). The regulated assembly of focal adhesions at the leading edge of the cell is a central step in cell migration and more specialized processes such as axonal growth (Hynes 1992; Lauffenberger and Horwitz 1996). Loss of adhesion to the ECM can lead to cell cycle arrest or in some cells the induction of programmed cell death (Rouslahti and Reed 1994). Thus, adhesive interactions with the ECM play a critical role in regulating a variety of intracellular signaling pathways.

Intracellular localization of the human receptor for the globular domains of C1q.

This study was performed to determine the localization of the recently described receptor for the globular domain of C1q, gC1qR. In contrast to previous reports, we were not able to detect significant surface expression of gC1qR on Raji cells, monocytes, neutrophils, human or rat mesangial cells, the endothelial cell line EA.hy 926, or HUVEC using FACS analysis. Only by using digoxigenin-conjugated Abs could some surface staining of gC1qR be observed on rat mesangial cells and neutrophils. However, after permeabilizing these cells with saponin, a strong positive intracellular staining for gC1qR was observed by FACS, fluorescence microscopy on coverslips, and confocal laser scanning microscopic analysis. By reflection contrast microscopy and electron microscopy on ultrathin sections of permeabilized Raji cells, it was shown that gC1qR is present in double membranous cytoplasmic vesicles located in the proximity of the plasma membrane. To determine whether certain conditions could induce surface expression of gC1qR, Raji cells were either stimulated with T cell growth factor, LPS, or driven to apoptosis by incubation with fenretinide or by serum depletion. None of the conditions resulted in significant surface expression of gC1qR. Our hypothesis that gC1qR is not a surface molecule but a soluble molecule that is secreted by cells is supported by the observation that gC1qR is found in significant concentrations in supernatants of several cultured cells and in normal human and rat sera. Our results suggest that the recently described gC1qR is not a cell surface receptor, but a soluble binding protein with affinity for the globular heads of C1q. Excreted gC1qR might act as a potential fluid phase regulator of complement activation.

A new procedure for high resolution light microscopy on peripheral nervous tissue

▼Basic research into clinical and therapeutical problems of nervous pathways requires increasingly differentiated information about detailed structures. Light microscopical devices commonly in use do not allow good simultanous discrimination between myelin sheaths, intracellular space of Schwann cells, or interstitial structures and especially between different fibre types (Ref. 1). However, automatic detection by image analysis is often necessary for statistical validation of results (Ref. 2). Discrimination procedures applied to images obtained by conventional light microscopical techniques often fail. In particular, small nerve fibres cannot be detected reliably (Ref. 3). These features can be detected by evaluation of extensive areas by transmission electron microscopy but this is very expensive. Reflection contrast microscopy (RCM) achieves better resolution than conventional light microscopy (Ref. 4, 5, 6). Furthermore, images obtained by RCM have an intense contrast and are thus very suitable for automatic image analysis (Ref. 7). Therefore, we sought to adapt this procedure to histology of neural tissues. This tip describes a method to investigate fixed peripheral nervous tissue by means of RCM. We found that RCM applied on highly osmificated (by the OTOTO method) (Ref. 8, 9, 10) and subsequently counter-stained specimens: