Single-Molecule Microscopy Reveals Membrane Microdomain Organization of Cells in a Living Vertebrate

It has been possible for several years to study the dynamics of fluorescently labeled proteins by single-molecule microscopy, but until now this technology has been applied only to individual cells in culture. In this study, it was extended to stem cells and living vertebrate organisms. As a molecule of interest we used yellow fluorescent protein fused to the human H-Ras membrane anchor, which has been shown to serve as a model for proteins anchored in the plasma membrane. We used a wide-field fluorescence microscopy setup to visualize individual molecules in a zebrafish cell line (ZF4) and in primary embryonic stem cells. A total-internal-reflection microscopy setup was used for imaging in living organisms, in particular in epidermal cells in the skin of 2-day-old zebrafish embryos. Our results demonstrate the occurrence of membrane microdomains in which the diffusion of membrane proteins in a living organism is confined. This membrane organization differed significantly from that observed in cultured cells, illustrating the relevance of performing single-molecule microscopy in living organisms.     

A model for the study of epithelial migration in wound healing.

A model is described which enables the detailed study of epithelial regeneration in experimentally produced lesions in the common bile duct of the rabbit. The circular on slightly oval defect of 1 mm diameter produced by a specially developed apparatus has a perfectly smooth base. Epithelial migration in this model has been investigated using light microscopy of transverse sections and scanning electron microscopy of whole preparations. Typical changes in the border cells, characterised by the formation of tapered protusions, can be observed as early as two hours after the lesion has been made. Later the cells in the flattened edge of the moving border also show various types of protrusion which rest on the substratum. Mitotic activity in the surface epithelium and crypts in the surrounding region only increases after closure of the lesion, which usually takes place within 16--24 h.     

The use of markers for correlative light electron microscopy

Bioimaging: the visualisation, localisation and tracking of movement of specific molecules in cells using microscopy has become an increasing field of interest within life science research. For this, the availability of fluorescent and electron-dense markers for light and electron microscopy, respectively, is an essential tool to attach to the molecules of interest. In recent years, there has been an increasing effort to combine light and electron microscopy in a single experiment. Such correlative light electron microscopy (CLEM) experiments thus rely on using markers that are both fluorescent and electron dense. Unfortunately, there are very few markers that possess both these properties. Markers for light microscopy such as green fluorescent protein are generally not directly visible in the electron microscopy and vice versa for gold particles. Hence, there has been an intensive search for markers that are directly visible both in the light microscope and in the electron microscope. Here we discuss some of the strategies and pitfalls that are associated with the use of CLEM markers, which might serve as a “warning” that new probes should be extensively tested before use. We focus on the use of CLEM markers for the study of intracellular transport and specifically endocytosis.     

Visualisation studies and glomerular filtration in early diabetic rats

The purpose of this mini-review is to show that more modern multi-photon microscopy approaches allow quantitative glomerular filtration experiments. Modern science has now entered a transition period from light microscopy to multi-photon confocal microscopy. Since the late 20th century, multi-photon microscopy has been applied in the study of organ function. In keeping with observations made in renal physiology and other representative studies throughout this transition period, and in the context of advancing microscopy techniques, this review has been presented as a comment on the glomerular filtration barrier, with a focus on the early aetiopathogenesis of diabetes.     

Sequential scanning electron microscopy of a growing plant meristem

Summary A two-step replica technique has been developed for sequential study of the epidermal cell pattern of a living plant by scanning electron microscopy. This method is nondestructive, allows periodic high resolution observation of the same developing tissue, and can precede use of any destructive technique, such as transmission electron microscopy. The replicas can be trimmed allowing observation of occluded surfaces, such as the areas between leaves, which are inaccessible in continuousin vivo studies. Here we study the developing leaf primordium ofGraptopetalum and discuss potential uses of the technique.     

Structural Evidence for α-Synuclein Fibrils Using in Situ Atomic Force Microscopy

Human α-synuclein is a presynaptic terminal protein and can form insoluble fibrils that are believed to play an important role in the pathogenesis of several neurodegenerative diseases such as Parkinson‘s disease, dementia with Lewy bodies and Lewy body variant of Alzheimer‘s disease. In this paper, in situ atomic force microscopy has been used to study the structural properties of α-synuclein fibrils in solution using two different atomic force microscopy imaging modes: tapping mode and contact mode. In the in situ contact mode atomic force microscopy experiments α-synuclein fibrils quickly broke into fragments, and a similar phenomenon was found using tapping mode atomic force microscopy in which α-synuclein fibrils were incubated with guanidine hydrochloride (0.6 M). The α-synuclein fibrils kept their original filamentous topography for over 1h in the in situ tapping mode atomic force microscopy experiments. The present results provide indirect evidence on how 13-sheets assemble into α-synuclein fibrils on a nanometer scale.     

Further Studies on the Replication of Rabies and Rabies-Like Viruses in Organized Cultures of Mammalian Neural Tissues

Organized cultures of mammalian spinal and dorsal root ganglions were used for a comparative study of the neurocytopathology caused by rabies and so-called rabies-like viruses. Electron microscopy and titration of infectivity revised earlier data in which no difference could be demonstrated between street and fixed-virus infection. In the present study, fixed virus produced inclusion bodies without apparent virus assembly. Sequential electron microscopy revealed that the main sites of virus assembly were the membranes of the Golgi complex. In contrast, rabies-like viruses freshly isolated from wild rodents produced inclusion bodies all of which were associated with virus replication. Electron microscopic evidence has led us to classify these strains as street virus. Nonneural cell elements from cultivated ganglions were susceptible to fixed virus and the cultures yielded higher titers of infectivity as compared to those of rabies-like viruses. Virus budding was shown to occur at the cell surface as well as at intracytoplasmic membranes.     

Microsculpture of the wing surface in Odonata: evidence for cuticular wax covering

The insect wing membrane is usually covered by scales, hairs, and acanthae, which serve diverse functions, such as species-specific coloration pattern, decrease of wind resistance during flight or decrease of wing wettability. Representatives of Palaeoptera (Odonata and Ephemeroptera) have no hairy structures on the wing membrane, but both its sides are fine-sculptured. In this study, the nature of the wing covering was studied using acoustic microscopy, scanning- and transmission electron microscopy followed by a variety of chemical treatments. It was shown that wing microsculptures are not cuticular outgrowths, but a wax covering, which is similar to pruinosity, which has been previously described in several odonate taxa. Data from scanning acoustic microscopy revealed that scratches on the wax covering have material density different from the surrounding material. Various functions of the wax covering are discussed.     

Smooth muscle cells in the rat testicular capsule: a developmental study.

This study of the postnatal development (from 1 to 60 days) of smooth muscle elements in the rat testicular capsule has demonstrated that while such elements are identifiable by light microscopy at 30 days, myocytes are present at birth as seen by electron microscopy. The differentiation of smooth muscle from birth to 30 days has been described, by which time it is of adult morphology and content. Perhaps significantly, it is at 30 days that the testis achieves a scrotal position, although sexual maturity does not occur until about 60 days. Presumably, at 30 days the testicular capsule of the rat is capable of the spontaneous contractions which are known to occur in the adult and which are assumed to aid the transport of non-motile spermatozoa from the testis to the spididymis. The presence of occasional striated muscle fibers in the rat testicular capsule as reported previously has not been confirmed by this investigation, although their possible origin is discussed.     

High-Speed Imaging of Amoeboid Movements Using Light-Sheet Microscopy

Light-sheet microscopy has been developed as a powerful tool for live imaging in biological studies. The efficient illumination of specimens using light-sheet microscopy makes it highly amenable to high-speed imaging. We therefore applied this technology to the observation of amoeboid movements, which are too rapid to capture with conventional microscopy. To simplify the setup of the optical system, we utilized the illumination optics from a conventional confocal laser scanning microscope. Using this set-up we achieved high-speed imaging of amoeboid movements. Three-dimensional images were captured at the recording rate of 40 frames/s and clearly outlined the fine structures of fluorescent-labeled amoeboid cellular membranes. The quality of images obtained by our system was sufficient for subsequent quantitative analysis for dynamics of amoeboid movements. This study demonstrates the application of light-sheet microscopy for high-speed imaging of biological specimens.     

Nature and localization of avian lens glycogen by electron microscopy and Raman spectroscopy.

Electron microscopy confirms the presence of a high concentration of glycogen particles in the lens nuclear region of birds of flying habit such as the ring-neck dove and pigeon. This observation is consistent with Raman spectroscopy. The glycogen particles in the dove lens, which are approximately 35 nm in diameter, are classified as beta type particles. Although this type has been previously characterized by high rates of glycogen turnover in other tissues, its localization in the lens nucleus indicates that it may serve a structural function rather than as a storage depot of carbohydrate in the lens. In a comparative electron microscopy study, glycogen particles were not observed in the chicken lens.     

New technologies in scanning probe microscopy for studying molecular interactions in cells

Atomic force microscopy (AFM) is a specialised form of scanning probe microscopy, which was invented by Binnig and colleagues in 1986. Since then, AFM has been increasingly used to study biomedical problems. Because of its high resolution, AFM has been used to examine the topography or shape of surfaces, such as during the molecular imaging of proteins. This, combined with the ability to operate under known force regimes, makes AFM technology particularly useful for measuring intermolecular bond forces and assessing the mechanical properties of biological materials. Many of the constraints (e.g. complex instrumentation, slow acquisition speeds and poor vertical range) that previously limited the use of AFM in cell biology are now beginning to be resolved. Technological advances will enable AFM to challenge both confocal laser scanning microscopy and scanning electron microscopy as a method for carrying out three-dimensional imaging. Its use as both a precise micro-manipulator and a measurement tool will probably result in many novel and exciting applications in the future. In this article, we have reviewed some of the current biological applications of AFM, and illustrated these applications using studies of the cell biology of bone and integrin-mediated adhesion.     

Secretory Vesicle-Specific Antibodies in the Confocal Study of Exo–Endocytosis Dynamics

The study of secretory vesicle dynamics is a continuing challenge. Classically it was studied using biochemical techniques, such as subcellular fractionation and immunoprecipitation, combined with time-consuming electron microscopy studies. The recent development of confocal microscopy, giving in-focus optical section images throughout the thickness of a fluorescently labeled sample, allows scientists to study the key events in the secretory cycle at the level of light microscopy. This study demonstrates the use of specific antibodies against marker proteins of two different secretory vesicles (synaptic vesicles and large dense-cored vesicles) to follow their exo–endocytosis dynamics in peripheral adrenergic neurons. Only in recent years has insight grown regarding the presence of both exocytosis pathways in the same neuron. Confocal microscopy is a suitable technique to study aspects of exocytosis, endocytosis, and intracellular sorting and as such improves our knowledge on the interaction between both secretory pathways.     

Secretory vesicle-specific antibodies in the confocal study of exo-endocytosis dynamics.

The study of secretory vesicle dynamics is a continuing challenge. Classically it was studied using biochemical techniques, such as subcellular fractionation and immunoprecipitation, combined with time-consuming electron microscopy studies. The recent development of confocal microscopy, giving in-focus optical section images throughout the thickness of a fluorescently labeled sample, allows scientists to study the key events in the secretory cycle at the level of light microscopy. This study demonstrates the use of specific antibodies against marker proteins of two different secretory vesicles (synaptic vesicles and large dense-cored vesicles) to follow their exo-endocytosis dynamics in peripheral adrenergic neurons. Only in recent years has insight grown regarding the presence of both exocytosis pathways in the same neuron. Confocal microscopy is a suitable technique to study aspects of exocytosis, endocytosis, and intracellular sorting and as such improves our knowledge on the interaction between both secretory pathways.     

The lipid raft hypothesis revisited - New insights on raft composition and function from super-resolution fluorescence microscopy

Recently developed super‐resolution microscopy techniques are changing our understanding of lipid rafts and membrane organisation in general. The lipid raft hypothesis postulates that cholesterol can drive the formation of ordered domains within the plasma membrane of cells, which may serve as platforms for cell signalling and membrane trafficking. There is now a wealth of evidence for these domains. However, their study has hitherto been hampered by the resolution limit of optical microscopy, making the definition of their properties problematic and contentious. New microscopy techniques circumvent the resolution limit and, for the first time, allow the fluorescence imaging of structures on length scales below 200 nm. This review describes such techniques, particularly as applied to the study of membrane organisation, synthesising newly emerging facets of lipid raft biology into a state‐of‐the art model. Editor's suggested further reading in BioEssays: Super‐resolution imaging prompts re‐thinking of cell biology mechanisms Abstract and Quantitative analysis of photoactivated localization microscopy (PALM) datasets using pair‐correlation analysis Abstract     

The future is cold: cryo-preparation methods for transmission electron microscopy of cells

Our knowledge of the organization of the cell is linked, to a great extent, to light and electron microscopy. Choosing either photons or electrons for imaging has many consequences on the image obtained, as well as on the experiment required in order to generate the image. One apparent effect on the experimental side is in the sample preparation, which can be quite elaborate for electron microscopy. In recent years, rapid freezing, cryo-preparation and cryo-electron microscopy have been more widely used because they introduce fewer artefacts during preparation when compared with chemical fixation and room temperature processing. In addition, cryo-electron microscopy allows the visualization of the hydrated specimens. In the present review, we give an introduction to the rapid freezing of biological samples and describe the preparation steps. We focus on bulk samples that are too big to be directly viewed under the electron microscope. Furthermore, we discuss the advantages and limitations of freeze substitution and cryo-electron microscopy of vitreous sections and compare their application to the study of bacteria and mammalian cells and to tomography.     

Expansion microscopy: A chemical approach for super-resolution microscopy

Super-resolution microscopy is a series of imaging techniques that bypass the diffraction limit of resolution. Since the 1990s, optical approaches, such as single-molecular localization microscopy, have allowed us to visualize biological samples from the sub-organelle to the molecular level. Recently, a chemical approach called expansion microscopy emerged as a new trend in super-resolution microscopy. It physically enlarges cells and tissues, which leads to an increase in the effective resolution of any microscope by the length expansion factor. Compared with optical approaches, expansion microscopy has a lower cost and higher imaging depth but requires a more complex procedure. The integration of expansion microscopy and advanced microscopes significantly pushed forward the boundary of super-resolution microscopy. This review covers the current state of the art in expansion microscopy, including the latest methods and their applications, as well as challenges and opportunities for future research.     

Use of automated microscopy for the detection of disseminated tumor cells in bone marrow samples

The use of automated microscopy has reached the maturity necessary for its routine use in the clinical pathology laboratory. In the following study we compared the performance of an automated microscope system (MDS) with manual method for the detection and analysis of disseminated tumor cells present in bone marrow preparations from breast carcinoma patients. The MDS System detected rare disseminated tumor cells among bone marrow mononuclear cells with higher sensitivity than standard manual microscopy. Automated microscopy also proved to be a method of high reproducibility and precision, the advantage of which was clearly illustrated by problems of variability in manual screening. Accumulated results from two pathologists who had screened 120 clinical slides from breast cancer patients both by manual microscopy and by use of the MDS System revealed only two (3.8%) missed by the automatic procedure, whereas as many as 20 out of 52 positive samples (38%) were missed by manual screening.     

New possibilities of studying microbial objects by laser interference microscopy

A novel method, laser interference microscopy, has been developed for studying the morphofunctional state of bacterial cells and the structure of bacterial communities. The following potentialities of the method are shown: rapid determination of the cell structure and subcellular structures (nucleus zone, vacuoles, lamellar structures) and the physiological state of the cell, as well as the study of the structure of bacterial communities (biofilm). The method does not require any additional preparation of cells before the investigation (fixation, staining, treatment with contrasting substances), which reduces the possible appearance of artifacts to a minimum and enables one to use laser interference microscopy for in vivo investigations.     

Fatty acyl moieties: improving Pro-rich peptide uptake inside HeLa cells.

In the field of drug delivery there has been a continuous study of powerful delivery systems to aid non permeable drugs in reaching their intracellular target. Among the systems explored are cell penetrating peptides (CPPs), which first garnered interest a decade ago when the interesting translocation properties of the pioneer CPPs Tat and Antp were described. A new family of CPPs has recently been described as non cytotoxic Pro-rich vectors with favorable profiles for internalization in HeLa cells. Fatty acyl moieties that can tune a peptide's interaction with the lipophilic environment of a cell membrane have been incorporated into the Pro-rich sequence. Improvements in cellular uptake of peptides modified with fatty acyl groups, as studied by confocal microscopy and flow cytometry, as well as the results obtained by the interaction of these peptides with a model dioleoylphosphatidylcholine (DOPC) membrane and transmission electron microscopy (TEM), illustrate the importance of the fatty acyl moieties for efficient internalization.