Biophysics and bioengineering
Piezo-based scanning stages for precise sample positioning and measurement
September 6, 2018
Dipl.-Phys. Gernot Hamann
In life sciences, chemical-pharmaceutical analyses or modern material sciences, the optical resolution and information content of classical microscopy methods are no longer sufficient. In order to obtain the most comprehensive information on a sample, modular, high-resolution microscope systems open up interesting opportunities, since different microscopy methods can be used either individually or in combination. As high-precision 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.
The ultra-high-resolution microscopy systems from WITec have a modular structure (fig. 1). This makes it possible to combine a confocal Raman microscope with atomic force microscopy (ATM), if required.
The same device can then provide and link molecular Raman and structural AFM information from the same sample region.
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, high-resolution scanning stage. At each position, the camera 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 position resolution and accuracy of the image depend on the positioning accuracy and stability of the scanning stage as well as the optical and mechanical components of the microscope (fig. 2).
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.
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 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 between the two images. Precise positioning in all three axes is indispensable for the accuracy of the image.
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-nanometer range. At the same time, the requirements for the dynamics are high: The faster the topography tracking in the Z direction, the faster the positioning in the X and Y axis can be. Consequently measurement times are shorter and temperature drift, which would increase with time, is reduced. So the high dynamics also benefit the accuracy.
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.
Capacitive Sensors and Digital Electronics Ensure Stability
Stability and path accuracy during scanning are crucial when combining Raman imaging with AFM, since the measurements can take a few minutes and any drift would distort the recordings. In addition, the active guiding 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. 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.
The piezo-based scanning system is an essential part in high-precision microscopes. As a result of its compact dimensions, the scanning stage can be easily integrated in microscopes where installation space is usually limited.
Dipl.-Physicist Gernot Hamann, Business Development Manager for Microscopy at PI (Physik Instrumente)
WITec in Brief
Since its foundation in 1997, WITec GmbH (Ulm, Germany) has developed into a market leader in high-resolution microscopy systems (Raman, AFM and SNOM) and generated average growth rates of annually 10%. With 52 employees worldwide, the company is represented by subsidiaries in the USA and Asia, and has regional offices in Spain and Japan. The main application areas for the modular high-performance microscopes, allowing the combination of different methods, are found in nanotechnology, materials sciences and life sciences. Europe, the USA and the Asia/Pacific region are the most important sales markets, with users coming from research and industry.
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 requirement in the field of innovative precision positioning technology. All key technologies are developed in-house. This allows the company to control every step of the process, from design right down to shipment: precision mechanics and electronics as well as position sensors. The required piezoceramic elements are manufactured by our subsidiary PI Ceramic in Lederhose, Germany, one of the global leaders for piezo actuator and sensor products. PI miCos GmbH in Eschbach near Freiburg, Germany, is a specialist for positioning systems for ultrahigh vacuum applications as well as parallel-kinematic positioning systems.