Scanning Microwave Microscopy for Quantitative Imaging of ... · PDF file 04 | Keysight |...
Embed Size (px)
Transcript of Scanning Microwave Microscopy for Quantitative Imaging of ... · PDF file 04 | Keysight |...
Keysight Technologies Scanning Microwave Microscopy for Quantitative Imaging of Biological Samples Including Live Cells
Introduction The Scanning Microwave Microscope (SMM) merges the nanoscale imaging capabilities of an atomic force microscope (AFM) with the high-frequency broadband (from MHz to GHz) impedance measurement accuracy of a vector network analyzer (VNA) (Figure 1). The typical frequency range of the combined SMM is between 1-20 GHz (Huber et al. 2010). It allows characterizing electric, dielectric, and magnetic properties of materials at microwave frequencies with nanometer lateral resolution. Using the microwave signal, impedance nanoscale imaging and doping profiling can be performed. Typically the SMM is operated in reflection mode, whereby the ratio of the reflected and incident electromagnetic waves, the so called S11 scattering parameter, is measured by the VNA at each pixel of the AFM tip-sample contact point. As such a microwave image is generated pixel by pixel, simultaneously to the topographical image. Imaging speeds are fast resulting in a typical acquisition time of one minute per image. There are mainly two different imaging modes in SMM. The first is quantitative dopant profiling by means of differential capacitance (also called dC/dV), which is a widely used technique for semiconductor failure analysis and detecting leakages in solid state devices with nanometer resolution (Huber et al 2012). The dC/dV mode relies on a low frequency (kHz) modulation of the GHz S11 signal using the Dopant Profile Measurement Module (DPMM). It allows tuning the semiconductor depletion zone with low frequency and probing the doping concentration through the native oxide interface at GHz frequency. The second SMM mode which is mainly used in life science is complex impedance imaging and it is based directly on the scattering S11 signal at GHz frequency (Gramse et al 2014).
Figure 1. SMM sketch (upper panel) and equivalent electric circuit at the tip/sample interaction (lower panel). SMM impedance matching network consists of half-wavelength resonator, 50 Ω shunt resistor, and coaxial cable. The lower panel presents a 3D geometrical model of a cell in contact with the fully metallic AFM probe. Equivalent electric circuit is composed of cantilever stray capacitance (Cstray), tip/cone capacitance (Ccone), and tip/sample capacitance (Cpar) in parallel with cell conductance (loss, 1/Gpar). A thin layer of native SiO2 on top of doped silicon (Si
++) forms the substrate.
03 | Keysight | Scanning Microwave Microscopy for Quantitative Imaging of Biological Samples Including Live Cells - Application Note
Complex impedance imaging with nanometer precision and low impedance values has proved to be a powerful tool for materials science applications (Hoffmann et al 2014; Kasper et al 2014a). In particularly, the capability of sub-surface imaging of buried nanoscale structures has been demonstrated, as well as conductivity measurements of 2D layers (Gramse et al 2015). Recently, the high frequency characterization of biological materials has attracted considerable interests, as SMM represents a convenient non-invasive evanescent imaging technique that complements AFM (Tuca et al 2016). While AFM measures the surface topography, the SMM can see inside materials (eg cell nucleus and organelles) using the electromagnetic wave penetration capabilities at GHz frequency. In the following we present calibrated complex impedance measurements of bacteria and cells, complex permittivity measurements of single bacteria at 20 GHz, and live cell imaging in liquid media. Furthermore we present augmented SMM solutions where the standard SMM mode is extended with other electronic measurement devices from Keysight (eg the dielectric probe kit, or the electronic calibration unit ECal) that opens new and advanced SMM measurement capabilities. Finally, we combine SMM with the Keysight EMPro 3D modeling capabilities at GHz frequencies that can be used to calculate E-fields and complex impedance values inside cells in order to assist quantitative SMM data interpretation.
SMM complex impedance for biological applications
Complex impedance calibration: basic workflow An SMM S11 complex impedance calibration workflow has been recently developed where no calibration sample is required (Figure 2). From the measured S11, calibrated capacitance and resistance images of metallic, dielectric, biological or semiconducting materials are obtained. For bio-applications silicon substrates are used to adhere cells, bacteria, viruses or membranes. S11 approach curves in order to calculate the three error coefficients in a standard S11 black box calibration. A low frequency EFM approach curve is used to measure the capacitance change with the distance when the cantilever approaches the sample surface. In the case of bio-samples the approach curves are done on the silicon substrate. Using this black-box calibration procedure the complex S11 images are converted to impedance images (Figure 2 & 3).
Figure 2. SMM sketch (upper panel) and complex impedance calibration (lower panel). The main SMM compo- nents are the AFM cantilever with standard laser deflection readout, the impedance matching network as well as the VNA for GHz signal readout. The VNA measures the S11 amplitude and phase images. Based on a standard VNA three error parameter black-box calibration the conductance and capacitance images are derived.
Impedance matching network
Vector network analyzer
Complex S11 - parameter
04 | Keysight | Scanning Microwave Microscopy for Quantitative Imaging of Biological Samples Including Live Cells - Application Note
After calibration, the topography crosstalk between the topography image and the capacitance image is removed, allowing the subtraction of the cantilever based stray capacitance from the local AFM tip capacitance. The stray capacitance is thereby extracted from the linear part of the S11 approach curve, and the topography-influenced values are subtracted pixel-by-pixel from the raw capacitance values. The impedance calibration method is available as a script in Pico Café (Keysight PicoCafe). Figure 3 shows the calibration script graphical user interface, which allows selecting the most appropriate equivalent electrical network model.
Figure 3. Graphical User Interface (GUI) of the Picoscript for the automated complex impedance calibration with SMM. The upped panel shows the GUI window that allows importing the EFM/SMM approach curve to calibrate the image. The lower panel shows an example of calibrated Capacitance (fF) and Conductance (µS) images obtained using the script. The script allows the user to select the most appropriate equivalent electrical RC model (red circles on the right). The advantage of this calibration workflow is that no specific calibration sample is required and that the calibration can be performed on the bare conductive substrate (eg silicon) where the cells and bacteria are adhered.
SMM calibration script
Calibrated images shown in the script GUI
05 | Keysight | Scanning Microwave Microscopy for Quantitative Imaging of Biological Samples Including Live Cells - Application Note
Complex impedance of bacteria: capacitance and complex permittivity The SMM was used to measure Escherichia coli (E. coli) bacteria in air and in liquid using intermittent contact mode for imaging soft materials (Figure 4) (Tuca et al 2016). Quanti- tative SMM calibration is achieved resulting in complex impedance images of bacteria in air including capacitance (aF; attoFarad) and conductance (μS; microSiemens) images. E. coli bacteria were immobilized onto a silicon substrate and imaged with the SMM either in the dry state or in buffer solution. Figure 4, upper panel, shows an ensemble of bacteria spread over the surface of Si and SiO2 pillars while the lower panel shows two individual bacteria on Si. The topography images show E. coli bacteria with lengths of 2-3 μm and a height of 300-350 nm. The SMM raw data includes the VNA amplitude image showing a good contrast between the bacteria, SiO2 pillars and the Si substrate. The calibrated capacitance images show individual bacteria with a capacitance ranging from 20-100 aF depending on the tip diameter and the frequency. The capacitance of the bacteria as well as the SiO2 pads is lower than the capacitance of the substrate. This is consistent assuming a simple parallel plate capacitor model where the capacitance decreases with increasing distance of the tip and the substrate. The conductance channel shows no variation between bacteria and substrate (data not shown), which is expected from the non-conductive SiO2 pads and indicates that the bacteria have no electrical loss under the measurement conditions.
In a subsequent study we quantified the electric permittivity of single bacterial cells at microwave frequencies and nanoscale spatial resolution (Biagi et al 2016) (Figure 5). Calibrated complex impedance and admittance images have been obtained at 19 GHz and analyzed with a methodology that removes the non-local topographic cross-talk contributions and thus provides quantifiable intrinsic dielectric images of the bacterial cells. Results for single E-coli provide a relative electric permittivity of ε = 4 in dry conditions and ε = 20 in humid conditions, with no significant loss contributions. The ability of microwaves to penetrate the cell membrane opens an important avenue in the microwave label-free imaging of single ce