Automatic Tissue Differentiation Based on Confocal Endomicroscopic Images for Intraoperative Guidance in Neurosurgery.

Diagnosis of tumor and definition of tumor borders intraoperatively using fast histopathology is often not sufficiently informative primarily due to tissue architecture alteration during sample preparation step. Confocal laser microscopy (CLE) provides microscopic information of tissue in real-time on cellular and subcellular levels, where tissue characterization is possible. One major challenge is to categorize these images reliably during the surgery as quickly as possible. To address this, we propose an automated tissue differentiation algorithm based on the machine learning concept. During a training phase, a large number of image frames with known tissue types are analyzed and the most discriminant image-based signatures for various tissue types are identified. During the procedure, the algorithm uses the learnt image features to assign a proper tissue type to the acquired image frame. We have verified this method on the example of two types of brain tumors: glioblastoma and meningioma. The algorithm was trained using 117 image sequences containing over 27 thousand images captured from more than 20 patients. We achieved an average cross validation accuracy of better than 83%. We believe this algorithm could be a useful component to an intraoperative pathology system for guiding the resection procedure based on cellular level information.

Sub-micrometer-scale mapping of magnetite crystals and sulfur globules in magnetotactic bacteria using confocal Raman micro-spectrometry.

The ferrimagnetic mineral magnetite Fe3O4 is biomineralized by magnetotactic microorganisms and a diverse range of animals. Here we demonstrate that confocal Raman microscopy can be used to visualize chains of magnetite crystals in magnetotactic bacteria, even though magnetite is a poor Raman scatterer and in bacteria occurs in typical grain sizes of only 35-120 nm, well below the diffraction-limited optical resolution. When using long integration times together with low laser power (<0.25 mW) to prevent laser induced damage of magnetite, we can identify and map magnetite by its characteristic Raman spectrum (303, 535, 665 cm(-1)) against a large autofluorescence background in our natural magnetotactic bacteria samples. While greigite (cubic Fe3S4; Raman lines of 253 and 351 cm(-1)) is often found in the Deltaproteobacteria class, it is not present in our samples. In intracellular sulfur globules of Candidatus Magnetobacterium bavaricum (Nitrospirae), we identified the sole presence of cyclo-octasulfur (S8: 151, 219, 467 cm(-1)), using green (532 nm), red (638 nm) and near-infrared excitation (785 nm). The Raman-spectra of phosphorous-rich intracellular accumulations point to orthophosphate in magnetic vibrios and to polyphosphate in magnetic cocci. Under green excitation, the cell envelopes are dominated by the resonant Raman lines of the heme cofactor of the b or c-type cytochrome, which can be used as a strong marker for label-free live-cell imaging of bacterial cytoplasmic membranes, as well as an indicator for the redox state.

Repulsive bimodal atomic force microscopy on polymers.

Bimodal atomic force microscopy can provide high-resolution images of polymers. In the bimodal operation mode, two eigenmodes of the cantilever are driven simultaneously. When examining polymers, an effective mechanical contact is often required between the tip and the sample to obtain compositional contrast, so particular emphasis was placed on the repulsive regime of dynamic force microscopy. We thus investigated bimodal imaging on a polystyrene-block-polybutadiene diblock copolymer surface and on polystyrene. The attractive operation regime was only stable when the amplitude of the second eigenmode was kept small compared to the amplitude of the fundamental mode. To clarify the influence of the higher eigenmode oscillation on the image quality, the amplitude ratio of both modes was systematically varied. Fourier analysis of the time series recorded during imaging showed frequency mixing. However, these spurious signals were at least two orders of magnitude smaller than the first two fundamental eigenmodes. Thus, repulsive bimodal imaging of polymer surfaces yields a good signal quality for amplitude ratios smaller than A(01)/A(02) = 10:1 without affecting the topography feedback.

Label-free live-cell imaging with confocal Raman microscopy.

Confocal Raman spectroscopy is a noninvasive alternative to established cell imaging methods because it does not require chemical fixation, the use of fluorescent markers, or genetic engineering. In particular, single live-cell, high-resolution imaging by confocal Raman microscopy is desirable because it allows further experiments concerning the individually investigated cells. However, to derive meaningful images from the spectroscopic data, one must identify cell components within the dataset. Using immunofluorescence images as a reference, we derive Raman spectral signatures by means of information measures to identify cell components such as the nucleus, the endoplasmic reticulum, the Golgi apparatus, and mitochondria. The extracted signatures allow us to generate representations equivalent to conventional (immuno)fluorescence images with more than three cell components at a time, exploiting the Raman spectral information alone.

Synthesis of well-ordered COF monolayers: surface growth of nanocrystalline precursors versus direct on-surface polycondensation.

Two different straightforward synthetic approaches are presented to fabricate long-range-ordered monolayers of a covalent organic framework (COF) on an inert, catalytically inactive graphite surface. Boronic acid condensation (dehydration) is employed as the polymerization reaction. In the first approach, the monomer is prepolymerized by a mere thermal treatment into nanocrystalline precursor COFs. The precursors are then deposited by drop-casting onto a graphite substrate and characterized by scanning tunneling microscopy (STM). While in the precursors monomers are already covalently interlinked into the final COF structure, the resulting domain size is still rather small. We show that a thermal treatment under reversible reaction conditions facilitates on-surface ripening associated with a striking increase of the domain size. Although this first approach allows studying different stages of the polymerization, the direct polymerization, that is, without the necessity of preceding reaction steps, is desirable. We demonstrate that even for a comparatively small diboronic acid monomer a direct thermally activated polymerization into extended COF monolayers is achievable.

Polymer brushes on graphene.

A critical bottleneck for the widespread use of single layer graphene is the absence of a facile method of chemical modification which does not diminish the outstanding properties of the two-dimensional sp(2) network. Here, we report on the direct chemical modification of graphene by photopolymerization with styrene. We demonstrate that photopolymerization occurs at existing defect sites and that there is no detectable disruption of the basal plane conjugation of graphene. This method thus offers a route to define graphene functionality without degrading its electronic properties. Furthermore, we show that photopolymerization with styrene results in self-organized intercalative growth and delamination of few layer graphene. Under these reaction conditions, we find that a range of other vinyl monomers exhibits no reactivity with graphene. However, we demonstrate an alternative route by which the surface reactivity can be precisely tuned, and these monomers can be locally grafted via electron-beam-induced carbon deposition on the graphene surface.

Nanostructure and mechanics of mummified type I collagen from the 5300-year-old Tyrolean Iceman.

Skin protects the body from pathogens and degradation. Mummified skin in particular is extremely resistant to decomposition. External influences or the action of micro-organisms, however, can degrade the connective tissue and lay the subjacent tissue open. To determine the degree of tissue preservation in mummified human skin and, in particular, the reason for its durability, we investigated the structural integrity of its main protein, type I collagen. We extracted samples from the Neolithic glacier mummy known as 'the Iceman'. Atomic force microscopy (AFM) revealed collagen fibrils that had characteristic banding patterns of 69 +/- 5 nm periodicity. Both the microstructure and the ultrastructure of dermal collagen bundles and fibrils were largely unaltered and extremely well preserved by the natural conservation process. Raman spectra of the ancient collagen indicated that there were no significant modifications in the molecular structure. However, AFM nanoindentation measurements showed slight changes in the mechanical behaviour of the fibrils. Young's modulus of single mummified fibrils was 4.1 +/- 1.1 GPa, whereas the elasticity of recent collagen averages 3.2 +/- 1.0 GPa. The excellent preservation of the collagen indicates that dehydration owing to freeze-drying of the collagen is the main process in mummification and that the influence of the degradation processes can be addressed, even after 5300 years.

Torsional noise of a colloidal probe in contact with surface-grafted PEG layers.

Surface coatings modify the interaction between microparticles and surfaces. To analyze the effect of a PLL-g-PEG surface coating on the sticktion of a microparticle, we analyzed the torsional Brownian fluctuations of a colloidal probe attached to an atomic force microscope (AFM) cantilever. Results obtained with uncoated surfaces indicate that the torsional oscillation due to noise was only weakly affected by hydrodynamic effects. Upon mechanical contact, however, the uncoated probe stuck to the uncoated surface. Coating probe or surface with a PLL-g-PEG brush polymer reduced the lateral interaction (sticktion and friction) of the colloidal probe. For such a combination, the torsional fluctuations persisted during mechanical compression of the brush layer. The results demonstrate that a PLL-g-PEG coating can effectively reduce the lateral interaction of a microparticle with a surface and prevent sticktion.

Amplitude and frequency modulation torsional resonance mode atomic force microscopy of a mineral surface.

Scanning probe imaging in a shear force mode allows for the characterization of in-plane surface properties. In a standard AFM, shear force imaging can be realized by the torsional resonance mode. In order to investigate the imaging conditions on mineral surfaces, a torsional resonance mode atomic force microscope was operated in amplitude (AM) and frequency modulation (FM) feedback. Freshly cleaved chlorite was investigated, which showed brucite-like and talc-like surface areas. In constant amplitude FM mode, a slight variation in energy dissipation was observed between both surfaces. Amplitude and frequency vs. distance curves revealed that the tip was in repulsive contact with the specimen during imaging.

Nanoscale residual stress-field mappingaround nanoindents in SiCby IR s-SNOM and confocal Raman microscopy.

We map a nanoindent in a silicon carbide (SiC) crystal by infrared (IR) scattering-type scanning near-field optical microscopy (s-SNOM) and confocal Raman microscopy and interpret the resulting images in terms of local residual stress-fields. By comparing near-field IR and confocal Raman images, we find that the stress-induced shifts of the longitudinal optical phonon-frequencies (LO) and the related shift of the phonon-polariton near-field resonance give rise to Raman and s-SNOM image contrasts, respectively. We apply single-frequency IR s-SNOM for nanoscale resolved imaging of local stress-fields and confocal Raman microscopy to obtain the complete spectral information about stress-induced shifts of the phonon frequencies at diffraction limited spatial resolution. The spatial extension of the local stress-field around the nanoindent agrees well between both techniques. Our results demonstrate that both methods ideally complement each other, allowing for the detailed analysis of stress-fields at e.g. material and grain boundaries, in Micro-Electro-Mechanical-Systems (MEMS), or in engineered nanostructures.