Suitable transfection methods for single particle tracing in plant suspension cells.

BACKGROUND: A multitude of different imaging systems are already available to image genetically altered RNA species; however, only a few of these techniques are actually suitable to visualize endogenous RNA. One possibility is to use fluorescently-labelled and hybridization-sensitive probes. In order to yield more information about the exact localization and movement of a single RNA molecule, it is necessary to image such probes with highly sensitive microscope setups. More challenges arise if such experiments are conducted in plant cells due to their high autofluorescence and demanding transfection procedures. RESULTS: Here, we report in planta imaging of single RNA molecules using fluorescently labeled molecular beacons. We tested three different transfection protocols in order to identify optimal conditions for transfection of fluorescent DNA probes and their subsequent detection at the single molecule level. CONCLUSIONS: We found that an optimized heat shock protocol provided a vastly improved transfection method for small DNA molecules which were used for subsequent single RNA molecule detection in living plant suspension cells.

Spatial cluster analysis of nanoscopically mapped serotonin receptors for classification of fixed brain tissue.

We present a cluster spatial analysis method using nanoscopic dSTORM images to determine changes in protein cluster distributions within brain tissue. Such methods are suitable to investigate human brain tissue and will help to achieve a deeper understanding of brain disease along with aiding drug development. Human brain tissue samples are usually treated postmortem via standard fixation protocols, which are established in clinical laboratories. Therefore, our localization microscopy-based method was adapted to characterize protein density and protein cluster localization in samples fixed using different protocols followed by common fluorescent immunohistochemistry techniques. The localization microscopy allows nanoscopic mapping of serotonin 5-HT1A receptor groups within a two-dimensional image of a brain tissue slice. These nanoscopically mapped proteins can be confined to clusters by applying the proposed statistical spatial analysis. Selected features of such clusters were subsequently used to characterize and classify the tissue. Samples were obtained from different types of patients, fixed with different preparation methods, and finally stored in a human tissue bank. To verify the proposed method, samples of a cryopreserved healthy brain have been compared with epitope-retrieved and paraffin-fixed tissues. Furthermore, samples of healthy brain tissues were compared with data obtained from patients suffering from mental illnesses (e.g., major depressive disorder). Our work demonstrates the applicability of localization microscopy and image analysis methods for comparison and classification of human brain tissues at a nanoscopic level. Furthermore, the presented workflow marks a unique technological advance in the characterization of protein distributions in brain tissue sections.

Nano-anchors with single protein capacity produced with STED lithography.

Acrylate nanoanchors of subdiffraction-limited diameter are written with optical stimulated emission depletion (STED) lithography. After incubation, 98% of all nanoanchors are loaded quickly with fluorescently labeled antibodies. Controlling the size of the nanoanchors allows for limiting the number of the antibodies. Direct stochastic optical reconstruction microscopy (dSTORM) imaging, statistical distribution of fluorescence, quantitative fluorescence readout, and single molecule blinking consistently prove that 80% of the nanoanchors with a 65 nm diameter are carrying only one antibody each, which are functional as confirmed with live erythrocytes.

Brain tumor classification using AFM in combination with data mining techniques.

Although classification of astrocytic tumors is standardized by the WHO grading system, which is mainly based on microscopy-derived, histomorphological features, there is great interobserver variability. The main causes are thought to be the complexity of morphological details varying from tumor to tumor and from patient to patient, variations in the technical histopathological procedures like staining protocols, and finally the individual experience of the diagnosing pathologist. Thus, to raise astrocytoma grading to a more objective standard, this paper proposes a methodology based on atomic force microscopy (AFM) derived images made from histopathological samples in combination with data mining techniques. By comparing AFM images with corresponding light microscopy images of the same area, the progressive formation of cavities due to cell necrosis was identified as a typical morphological marker for a computer-assisted analysis. Using genetic programming as a tool for feature analysis, a best model was created that achieved 94.74% classification accuracy in distinguishing grade II tumors from grade IV ones. While utilizing modern image analysis techniques, AFM may become an important tool in astrocytic tumor diagnosis. By this way patients suffering from grade II tumors are identified unambiguously, having a less risk for malignant transformation. They would benefit from early adjuvant therapies.

Ultrastructural characterization of cystic fibrosis sputum using atomic force and scanning electron microscopy.

BACKGROUND: Cystic fibrosis (CF) lung disease is characterized by perpetuated neutrophilic inflammation with progressive tissue destruction. Neutrophils represent the major cellular fraction in CF airway fluids and are known to form neutrophil extracellular traps (NETs) upon stimulation. Large amounts of extracellular DNA-NETs are present in CF airway fluids. However, the structural contribution of NETs to the matrix composition of CF airway fluid remains poorly understood. We hypothesized that CF airway fluids consist of distinct DNA-NETs that are associated to subcellular structures. METHODOLOGY/PRINCIPAL FINDINGS: We employed atomic force microcopy (AFM) and scanning electron microcopy to ultrastructurally characterize the nature of CF sputum and the role of NETs within the extracellular CF sputum matrix. These studies demonstrate that CF sputum is predominantly composed of a high-density meshwork of NETs and NETosis-derived material. Treatment of CF sputum with different DNases degraded CF NETs and efficiently liquefied the mucous-like structure of CF sputum. Quantitative analysis of AFM results showed the presence of three globular fractions within CF sputum and the larger two ones featured characteristics of neutrophil ectosomes. CONCLUSIONS/SIGNIFICANCE: These studies suggest that excessive NET formation represents the major factor underlying the gel-like structure of CF sputum and provide evidence that CF-NETs contain ectosome-like structures that could represent targets for future therapeutic approaches.

Changes in intraocular lens surface roughness during cataract surgery assessed by atomic force microscopy.

PURPOSE: To analyze the changes in optic surface roughness before and after injection of various intraocular lens (IOL) models using atomic force microscopy (AFM). SETTINGS: Departments of Ophthalmology, Medical University of Graz, General Hospital Linz and University Hospital Basel; Upper Austria University, School of Applied Health and Social Sciences, Linz, Austria. DESIGN: Experimental study. METHODS: The morphology and surface roughness of 3 hydrophobic acrylic IOLs from different manufacturers were analyzed by AFM in liquid using the tapping mode. First, AFM was performed on IOLs taken from the original package without further manipulation. In a second step, under sterile conditions, an experienced cataract surgeon loaded the IOLs into the appropriate injection system and pushed them through a system resembling an IOL implantation in cataract surgery; this was followed by AFM evaluation. Finally, 3 samples of a preloaded hydrophilic acrylic IOL taken from the original cartridge were compared with 3 samples that were pushed through the implantation system. RESULTS: Comparison of the arithmetic mean, standard deviation, root mean square, and surface skewness of the IOLs before and after injection showed a significant increase in surface roughness (P<.05). CONCLUSIONS: Standard application procedures of IOLs may alter the IOL surface. Increases in the surface roughness of IOLs may influence postoperative posterior capsule opacification. Further studies are necessary to evaluate the interfacial properties of IOLs.

Examination of native and carbamide peroxide-bleached human tooth enamel by atomic force microscopy.

Atomic force microscopy (AFM) was used to study the effects of bleaching on the morphology of the enamel surface with nanoscale resolution. Samples of human tooth enamel with native (pumiced) or fine-polished surfaces were examined before and after bleaching with 30% carbamide peroxide. The obtained profilometric AFM data revealed significant morphological surface alterations. After 1 h of bleaching, the surface roughness increased significantly from 19 +/- 4nm to 33 +/- 5 nm. Six-hour bleaching did not produce any significant further increase in enamel surface roughness. The interrod junction depth raised more than twice after 1 h of bleaching. After 6 h of bleaching, a further and significant increase in interrod junction depth was recorded. This alteration might be a consequence of oxidation and a subsequent partial lysis of the tooth enamel matrix proteins.

Correlations between AFM and SEM imaging of acid-etched tooth enamel.

Enamel bond strength is an important factor in restorative dentistry and crucially depends on the enamel roughness. To increase roughness, different etching procedures are employed and profilometric estimations, with probe profilometers, including atomic force microscopy (AFM), have been made. However, no correlation between roughness and bond strength has been found. To search for a possible error source leading to the underestimation of enamel roughness when utilizing probe profilometers, the authors compared scanning electron microscopy and AFM images of acid-etched tooth enamel. The results showed that AFM imaging cannot correctly depict the acid-etched enamel surface, because of the high steepness of the enamel crystallites and the generation of convolute images. This leads to a large underestimation of the profilometric parameters measured with AFM, or other profilometers, on acid-etched tooth enamel surfaces.