Physik Instrumente PI Ultra High Resolution Microscopy WP Pi1114
PI_Ultra-High_resolution_Microscopy_WP_pi1114 Whitepapers & Success Stories
User Manual: Physik Instrumente Whitepapers & Success Stories
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WHITEPAPER – Ultra-High-Resolution Microscopy in a Modular System Dipl.-Phys. Gernot Hamann Piezo-Based Scanning Stages for Precise Sample Positioning and Measurement Ultra-High-Resolution Microscopy in a Modular System Physik Instrumente (PI) GmbH & Co. KG_Auf der Roemerstrasse 1_76228 Karlsruhe, Germany Tel. +49 721 4846-0, Fax +49 721 4846-1019 Email info@pi.ws, www.pi.ws Page 1 of 5 WHITEPAPER – Ultra-High-Resolution Microscopy in a Modular System Dipl.-Phys. Gernot Hamann In life sciences, chemical-pharmaceutical analyses or The same device can then provide and link molecular modern material sciences, the optical resolution and Raman and structural AFM information from the same information content of classical microscopy methods are sample region. no longer sufficient. In order to obtain the most comprehensive information on a sample, modular, highresolution microscope systems open up interesting opportunities, since different microscopy methods can be used either individually or in combination. As highprecision and dynamic sample positioning is indispensable in most application areas, piezo-based scanning stages are a good solution. Thanks to their compact design, they can easily be integrated in microscopes. For high-resolution optical information, the microscope can also be equipped with SNOM (scanning near field optical microscopy). As a result, precise optical, topographical and molecular analyses are possible, just as required by the respective application. This opens up opportunities for diverse of application areas. The application spectrum of modular high-precision microscopes ranges from pharmaceutical research and live cell analyses, through nanophotonics, forensics up to analyses in photovoltaic or semiconductor technology Close-Ups Below the Diffraction Limit . With conventional microscopy technology, the position resolution is limited to half of its wavelength due to diffraction effects on the objective. In contrast, scanning near-field optical microscopy can show far smaller structures. Here, a glass fiber couples laser light into a hollow measuring tip. This light emerges through a tiny opening at the tip with a diameter of less than 100 nm. If the opening of the measuring tip is brought closely to the sample surface, a spot far below the diffraction limit of classical microscopy can be illuminated. Depending on the geometry of the measuring tip and its opening, a lateral position resolution of up to around 60 nm can be attained, while with confocal (light) microscopy the value would be between 200 to 300 nm. For scanning the sample point by point, it is moved under the measuring tip by a piezo-driven, highresolution scanning stage. At each position, the camera Fig. 1 The modular microscopy systems from WITec make it possible to combine a confocal Raman microscope with atomic force microscopy (AFM) (Image: WITec GmbH) The ultra-high-resolution microscopy systems integrated in the microscope records the incoming light intensity and saves this value together with the position information, which is used to create the image. The from position resolution and accuracy of the image depend on WITec have a modular structure (fig. 1). This makes it the positioning accuracy and stability of the scanning possible to combine a confocal Raman microscope with stage as well as the optical and mechanical components atomic force microscopy (ATM), if required. of the microscope (fig. 2). Physik Instrumente (PI) GmbH & Co. KG_Auf der Roemerstrasse 1_76228 Karlsruhe, Germany Tel. +49 721 4846-0, Fax +49 721 4846-1019 Email info@pi.ws, www.pi.ws Page 2 of 5 WHITEPAPER – Ultra-High-Resolution Microscopy in a Modular System Dipl.-Phys. Gernot Hamann In the case of the AFM method, the measuring tip is also moved over the sample surface line by line in a defined grid. Forces are measured between a very thin measuring tip and the surface of the object, which then provides information on the topology of the surface. In addition, sample characteristics such as adhesion, stiffness or viscosity can be determined. The lateral resolution is 10 nm and below. The position of the sample is readjusted in the direction of the Z axis here as well. The variation of the Z position together with the relevant X and Y coordinates for the spatial resolution Fig. 2 For high-resolution optical information, the microscope can also be equipped with SNOM (scanning near field optical microscopy). With SNOM, far smaller structures can be shown than with conventional microscope technology (Image: WITec GmbH) Information on the Surface Topology SNOM simultaneously provides information on the surface topology: Since the distance between the measuring tip and the surface has to be kept constant and practically every surface has a certain roughness, the position of the sample must be readjusted in the Z direction. This readjustment is carried out by the scanning stage and provides topological information additionally to the optical SNOM image. then provide high-precision topology information on the samples (fig. 3). The Chemical Fingerprint Raman microscopy is based on a confocal, optical microscope combined with a Raman spectrometer. In the case of a confocal system, apertures are used to suppress light outside of the focal plane of the microscope. In this way, only light information from the focal plane is transferred to the spectrometer. In the spectrometer, this light is spectrally separated and detected. The sample is scanned point by point and line by line. The lateral resolution is approximately 200 nm with green excitation light. During the measurement, a complete Raman spectrum is recorded for each pixel. These Raman spectra act like a specific fingerprint for each type of molecule, so that the chemical components of a sample can be identified for each pixel and their distribution in the sample can be shown. Combining Raman imaging with AFM yields topographical information with high spatial resolution as well as molecular information on the sample surface. Since the corresponding images are recorded in succession (fig. 4), the requirements for the scanning stage are very high. Any drift would distort the correlation Fig. 3 Atomic force microscopy (AFM) provides precise information on the surface topology (Image: WITec GmbH) between the two images. Precise positioning in all three axes is indispensable for the accuracy of the image. Physik Instrumente (PI) GmbH & Co. KG_Auf der Roemerstrasse 1_76228 Karlsruhe, Germany Tel. +49 721 4846-0, Fax +49 721 4846-1019 Email info@pi.ws, www.pi.ws Page 3 of 5 WHITEPAPER – Ultra-High-Resolution Microscopy in a Modular System Dipl.-Phys. Gernot Hamann Fig. 4 Topology of a PEET-PET polymer film on a glass substrate recorded with AFM (left), the Raman spectrum (right) and the false color representation of the Raman image (center). Raman spectra act like a specific fingerprint for each type of molecule, so that chemical components present in a sample can be identified for each pixel and their distribution in the sample can be shown (Image: WITec GmbH) Positioning with a Very High Position Resolution and Dynamics Since the positioning system used for scanning provides the spatial resolution, its resolution must be in the sub- For these reasons, WITec decided on a piezo-based scanning stage (fig. 5) from PI (Physik Instrumente) for positioning. It is designed for working distances of 100 or 200 µm in the axes of the scanning plane and 30 µm in the Z axis. It allows a position resolution of better than 2 nm and provides the best conditions for use in modular microscopes for all three methods. As there are no classical mechanical components that could cause friction or mechanical backlash in the drive, very high motion resolution is possible with the piezo drives. the dynamics are high: The faster the topography Capacitive Sensors and Digital Electronics Ensure Stability tracking in the Z direction, the faster the positioning in Stability and path accuracy during scanning are crucial the X and Y axis can be. Consequently measurement when combining Raman imaging with AFM, since the times are shorter and temperature drift, which would measurements can take a few minutes and any drift increase with time, is reduced. So the high dynamics would distort the recordings. In addition, the active also benefit the accuracy. guiding nanometer range. At the same time, the requirements for using capacitive sensors increases path accuracy: The sensors measure any deviation in the axis lateral to the direction of motion. Undesired crosstalk of the motion (for example from external forces or mechanical crosstalk) into another axis can be detected in this way and actively compensated in real time. A digital controller provides the necessary control. It is specially adapted to the piezo-based scanning stage and guarantees a good linearity, also for dynamic operation. Fig. 5 The piezo-based scanning stage is essential for sample positioning. It is designed for working distances of 100 or 200 µm in the axes of the scanning plane and 30 µm in the Z axis. It allows a position resolution of better than 2 nm. Capacitive sensors and digital electronics provide maximum stability The digital electronics work with a high clock rate. This is decisive for an accurate assignment of the position values of the piezo scanner and the recording camera. If it were too slow or inaccurate, there would be a loss of resolution and distortions (jitter) during the assignment. Physik Instrumente (PI) GmbH & Co. KG_Auf der Roemerstrasse 1_76228 Karlsruhe, Germany Tel. +49 721 4846-0, Fax +49 721 4846-1019 Email info@pi.ws, www.pi.ws Page 4 of 5 WHITEPAPER – Ultra-High-Resolution Microscopy in a Modular System Dipl.-Phys. Gernot Hamann The piezo-based scanning system is an essential part in methods, high-precision microscopes. As a result of its compact sciences and life sciences. Europe, the USA and the dimensions, the scanning stage can be easily integrated Asia/Pacific region are the most important sales markets, in microscopes where installation space is usually with users coming from research and industry. limited. Author are found in nanotechnology, materials PI (Physik Instrumente) in Brief In the past four decades, PI (Physik Instrumente) with headquarters in Karlsruhe, Germany has become the leading manufacturer of nanopositioning systems with accuracies in the nanometer range. With four company sites in Germany and ten sales and service offices abroad, the privately managed company operates globally. Over 700 highly qualified employees around the world enable the PI Group to meet almost any Dipl.-Physicist Gernot Hamann, Business Development requirement Manager for Microscopy at PI (Physik Instrumente) positioning WITec in Brief in the field technology. of All innovative key precision technologies are developed in-house. This allows the company to control every step of the process, from design right down to Since its foundation in 1997, WITec GmbH (Ulm, shipment: precision mechanics and electronics as well Germany) has developed into a market leader in high- as resolution microscopy systems (Raman, AFM and elements are manufactured by our subsidiary PI Ceramic SNOM) and generated average growth rates of annually in Lederhose, Germany, one of the global leaders for 10%. With 52 employees worldwide, the company is piezo actuator and sensor products. PI miCos GmbH in represented by subsidiaries in the USA and Asia, and Eschbach near Freiburg, Germany, is a specialist for has regional offices in Spain and Japan. The main positioning systems for ultrahigh vacuum applications as application areas for the modular high-performance well as parallel-kinematic positioning systems with six microscopes, allowing the combination of different degrees of freedom and custom-made designs. position sensors. The PI headquarters in Karlsruhe, Germany: More than 350 employees work on high-resolution drive systems and positioning systems Physik Instrumente (PI) GmbH & Co. KG_Auf der Roemerstrasse 1_76228 Karlsruhe, Germany Tel. +49 721 4846-0, Fax +49 721 4846-1019 Email info@pi.ws, www.pi.ws Page 5 of 5 required piezoceramic
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