Microscopic Structures

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1 Microscopic Structures Image Analysis Metal, 3D Image (Red-Green) The microscopic methods range from dark field / bright field microscopy through polarisation- and inverse microscopy to techniques like differential interference contrast (DIC) or circular polarisation (CP). The integrated software quantifies the morphological structures. Thus the computer calculates, for example, distributions or fineness of fibre bundles or areas of corrosion. Digital Optical Microscopy (DOM) Neurons The basic technique is to extract sharp image information from several virtual layers of the object like in confocal microscopy, but with the advantage to use all microscopic standard techniques. The result is visualised as 3 dimensional images. It is also possible to overlay two or more images, e. g. produced by different techniques, like DIC (differential interference contrast) combined with fluorescence. Magnifications over 2000 can be achieved. Pollen

2 Microscopic Structures Confocal Microscopy Only light coming from the focal plane is being detected. Minimal volume element: 350 nm x 350 nm and 500 nm in z-direction (Excitation at 532 nm) Pinhole is realized by optical fibers Confocal microscope, based on a Witec alpha300 system 2 µm Confocal microscopy of a test pattern for lateral resolution measurement Confocal Setup Back Scattering Microscopy Back Scattering Microscopy of a Silicon Chip Back Scattering Microscopy of a Glioblastoma multiforme cell

3 Nanoscopic Analysis Scanning Probe Microscopy (SPM) Atomic Force Microscope by Digital Instruments Scanning probe microscopy measures the distance dependent interaction between the sample surface and a sensor tip. In this way, the topography of conducting and isolating samples on an atomic scale can be analysed. Depending on the sensor tip different measuring modes can be applied. Features: - AFM, MFM - Tapping, Contact and Phase Imaging x 125 µm x 5 µm scanning range - ~7 nm x ~7 nm x ~0.2 nm resolution limit - electrochemical cell - flow cell Test grating CD groves Atomic Force Microscopy (AFM) The AFM is the most common tool for imaging, measuring and manipulating materials on a nanoscale. The topography of a surface can be imaged by scanning mechanically a silicon tip over the surface. Different surface forces lead to a deflection signal of a cantilever. The deflection is measured by a reflecting laser spot from the cantilever surface into a four quadrant diode. Tender, fragile, particulate or adhesive surfaces are scanned in phase imaging or tapping mode with an oscillating probe tip. In this way, neither the tip nor the sample can be contaminated during scanning, and no force is exerted on to the surface. Thus, material contrasts are visible. In addition, an electrochemical measuring mode can provide information of the electrochemical properties combined with topographic information. Magnetic Force Microscopy (MFM) The silicon tip is replaced by a permanent magnet, so a change in the magnetic interaction can be imaged. Chromosome

4 detection aperture detectors monochromator Nanoscopic Analysis VIS-camera SPM-Unit Zeiss Universal- Microscope-Spectral- Photometer UMSP 80 with near field unit SIL-SNOM schematic setup light sources Scanning Near Field Optical Microscopy (SNOM) and Near Field Optical Spectroscopy (SNOS) The diffraction limit of wide-field microscopy prevents resolving features smaller than half of a wavelength. Near field optical microscopy is able to extend the range of optical measurements beyond the diffraction limit. During the last years, Scanning Probe Microscopy has developed as a valuable tool to image different surface interactions even in the nanoscale. The combination of this high resolution technique and the chemical information of spectroscopy makes scanning near-field optical microscopy and -spectroscopy attractive for the characterization of the morphology and the chemistry of surfaces and cell structures. a b Apertureless SNOM: Solid Immersion Lens (SIL) Aperture-based SNOM: Pyramidal silicon tip with pinhole ( nm wide) SIL-SNOM Example Images. a: monocyte cell structures, b: test pattern for lateral resolution evaluation Topography and optical information can be acquired simultaneously by scanning near field optical microscopy (SNOM). For this purpose, either an apertureless solid immersion lens (SIL) or an aperture-based probe (pinhole pyramid) is positioned in the light path. Using a SIL enables different contrast modi and even the possibility of near field spectroscopy (SNOS): - Reflection-, Photon tunnelling-, Fluorescence contrast modi - Fluorescence Lifetime Measurements - ~30 nm x ~30 nm x ~0.2 nm resolution limit - VIS-, NIR- and Raman Spectroscopy

5 Nanoscopic Analysis Witec alpha300 system with near field unit and VIS-, snirand Raman spectrometer SIL-SNOM Contrast Modi SNOM and SNOS with Aperturebased and Aperture-less Probes Our unique, multimodal microscopy system allows SNOM and SNOS in various modi and combinations: - Use of aperture-less probes (SIL) or aperturebased probes (pinhole pyramids) - Measurement in reflexion or in transmission - SNOS: VIS-, NIR-, Raman-, Back Scattering- and Fluorescence Spectroscopy Reflectance-SNOM (R-SNOM) SNOM setup with SIL in the Witec alpha300 microscope. Photon-tunnelling SNOM (P-SNOM) The combination of AFM, near field (SNOM, SNOS) and far field techniques (e.g. Raman Imaging, confocal Microscopy) on the same instrument allows us to analyze the same sample position with various measuring setups. a b c Fluorescence SNOM (F-SNOM) SIL SNOM images in different contrast modi. a: R-SNOM, b: P-SNOM of a RAM chip (40 x 40 µm). c: F-SNOM - Intrinsic fluorescence of a flax cross section (100 x 100 µm), excitation: 395 nm 440 nm

6 Nanoscopic Analysis Scanning Near Field Optical Spectroscopy (SNOS) Using a Solid Immersion Lens (SIL) for SNOS measurements has plenty of advantages: Surface topography of human metaphase chromosomes High transmission efficiency Illumination and collection of light with a single probe Good signal/noise ratio in spectroscopy and fluorescence measurements. Optical and topographical information at the same time Suitable as near field solution for materials science as well as for life sciences. a b Optical near-field image of human metaphase chromosomes a: SIL-SNOM image of a human chromosome. Near field spectra were recorded at the points marked as p, q and c. b: Near field spectra of a human chromosome, using a Solid Immersion Lens and VIS-Spectrometer. Optical near-field image of an Al 2 O 3 -membrane-surface Near field VIS spectrum of an Al 2 O 3 -membrane-surface