Spotlight: How to... get more out of scanning probe microscopy data
Isabelle Cauwet, Applications Engineer at Digital Surf, looks at the benefits of using software to enhance microscopy data.
Most recent scanning probe microscopes (SPM), including atomic force microscopes (AFM) and scanning near-field optical microscopes (SNOM), come equipped with software for acquiring data. However, more often than not, when it comes to post-processing and further investigating data, the tools available are fairly limited.
Specialised software tools for SPM data analysis bring solutions to this problem, allowing users to prepare and correct data as well as perform many different, and sometimes complex, analysis sequences.
Surface data measured with an SPM instrument often contains artefacts that must be removed before analysis can be carried out. In particular, anomalous scan lines caused by a shift during measurement – dust on the tip or a loss of signal – are a fairly frequent occurrence. SPM instruments also frequently have a non-linear coupling between the lateral plane and the Z-axis causing unwanted bowing in the image.
One way of overcoming this problem is to process data using specially designed tools, for instance line-by-line levelling tools available in software such as MountainsSPIP. Users can automatically realign shifted horizontal lines on the sample surface. This tool can also be set up to ignore structures above the surface (bumps) and below the surface (holes).
Furthermore, a correct lines tool can be used, particularly in the case of the presence of dust on the sample. Deleted lines are replaced with lines calculated by interpolation from neighbouring zones.
Other options for data correction include tip deconvolution, removing isolated artefacts and normalisation and de-noising.
Particle analysis – characterising micro-structure
Particle analysis is used in research and industry across many fields ranging from quality control of structured materials such as metal alloys, to characterisation of micro and nano-structure assemblies. Being able to quickly identify and quantify features in an image or on a surface brings better understanding of surface properties.
Feature detection can be performed using the appropriate detection method – threshold, watershed, edge or circle detection. In the case of multi-signal files, any layer of data can be used.
When it comes to quantification, over 70 different parameters – including area, perimeter and diameter – can be calculated for the sample as a whole or for any individual particle. The tool is also interactive – clicking on a particle in the sample results brings up its parameters.
Achieving correlative analysis through co-localisation
In many studies, exploring a sample beyond the limitations of one single instrument technology can be useful or even fundamentally necessary.
Data from 3D optical profilers, atomic force microscopy, scanning electron microscopy, fluorescence, Raman, infrared and other microscopes, can be combined to gain better insight into the sample studied. In this case, it is important to have a tool that effectively manages data at different scales and aligns and overlays image and surface data automatically.
This is the case with the MountainsSPIP software, as shown in a study led by the Université Paris-Sud, France, on cell morphology of streptomyces, filamentous soil bacteria capable of storing excess carbon as triacylglycerol (TAG), a potential source of biodiesel.
By co-localising a nano-infrared absorption image and atomic force microscopy 3D topography, scientists were able to reveal size and location of small TAG vesicles (pockets of fat). Furthermore, particle analysis was carried out in order to calculate area, diameter and aspect ratio parameters for all vesicles.
Handling force spectroscopy data
Characterising molecular interactions at the nanoscale using atomic force microscopy (AFM) is a key application in the fields of materials and life sciences. Analysis software contains features for viewing, processing and analysing force curves and force volume images.
In ongoing research at the University of Limoges, France, the software is being used to probe innovative kinds of ceramic material, known as bioceramics, particularly useful in the repair and reconstruction of bone. One current focus is the impact of bioceramic surface topography and composition on protein adhesion forces.
To measure this, a fibronectin protein was grafted onto the cantilever tip of an AFM. This tip was put into contact with a calcium phosphate bioceramic substrate made from silicated hydroxyapatite powder synthesised by an aqueous precipitation method. Once contact between the functionalised tip and the substrate was obtained, the cantilever was lifted and the adhesive force of fibronectin measured. Force mapping was performed with spectroscopy mode in air.
The force value was then calculated for each point of the surface at the adhesion event cursor and displayed on a parameter map. Cursors were created on the parameter map to display and compare individual force curves in neighbouring zones.