Combining Optical Upright Microscopy & AFM: Biomaterial Workstation BioMAT. The atomic force microscope (AFM) is a flexible instrument that can be used for imaging, measuring forces and elastic properties and manipulating a variety of samples, at high resolution. The applicability of AFM is further extended in combination with light microscopy as optics deliver more bulk details and by fluorescence, compositional contrast. To date, the combination of AFM and light microscopy has been limited to samples on transparent substrates, where AFM has top-down access to the sample and an inverted light microscope has bottom-up access to the same area [1, 2].
A Combination of Techniques
However, there are many areas of research in which the combination of AFM with optical microscopy on opaque samples would be a powerful tool. Topics such as bacterial growth on metallic surfaces, bionics and surface chemistry, fluorescent polymers and coatings, i.e. areas from both life and material science could be addressed with such a combination of techniques. The main problem limiting the effective combination of these techniques on non-transparent surfaces has been providing access for both techniques at the same spot on the sample surface. To reach the full capabilities of optical microscopy, objective lenses with an extremely short working distance are required, leaving no space for AFM access to the same position. JPK Instruments has developed a solution to allow integration of upright optical microscopy and AFM, named the BioMAT Workstation (fig. 1).
Portable Shuttle Stage
The workstation spatially separates the upright optical microscope from the AFM to assure that neither of the two techniques is compromised. The key element of the BioMAT Workstation design is the portable shuttle stage on which the sample is loaded. The transfer of this shuttle stage from the upright optical microscope to AFM and vice versa allows precise positioning of the sample on both microscopes such that the same area is imaged by both systems. This transfer can be repeated as often as necessary, allowing the sequential measurement of optics and AFM.
For combining upright light microscopy with AFM first the BioMAT Workstation has to be aligned, matching the AFM scan rage to the field of view of the optical microscope.
To do this, a transparent reference sample displaying a cross-hair structure, which can be imaged with both systems is used (fig. 2). With the workstation's integrated inverted optics, the AFM tip can be coarsely aligned with respect to the cross hair on the reference sample (fig. 3). This coarse alignment involves adjusting the position of the AFM head on top of the BioMAT Workstation so that the AFM tip position matches the center of the reference cross. By exchanging the reference sample with the sample of interest, the same area of the sample can be imaged sequentially with the two separate microscopes.
One area of research where combining light microscopy and AFM on opaque substrates would be useful is the investigation of bacteria with metal surfaces. Thiobacteria can leach mineral sulfides from various metals. During this process of bioleaching the bacteria, which mostly belong to species of thiobacillus ferrooxidans are in close contact to the mineral sulfides, forming a monolayered biofilm. AFM is particularly suitable for investigation of such biofilms as it allows the visualization and characterization of biological samples under physiological conditions with high spatial resolution. The option to combine AFM with fluorescence microscopy is a powerful means to correctly interpret and validate the topographic images obtained by AFM with the help of corresponding images of fluorescently labelled structures. Since almost all sulphur containing minerals are opaque such samples are the perfect application for the BioMAT Workstation. Here, thiobacillus ferrooxidans was grown on a piece of compressed sulphur (sample courtesy Prof. Sand, University Duisburg-Essen). For fluorescence microscopy the bacterial DNA was stained using DAPI. Fluorescence images were acquired with a Zeiss AxioImager A1m equipped with a 100 x Acroplan water immersion objective. After imaging on the AxioImager, the specialized sample holder was transferred to the BioMAT Workstation and AFM imaging was conducted in intermittent contact mode in fluid using the NanoWizard II.
As contrast in AFM is based on structure, the images of bacteria on the surface of the elementary sulphur are complex. One can see steps and structures in the sulphur substrate, as well as groups of bacteria on the surface. By comparing the AFM image with the fluorescence image of the same area it is clear which surface features correspond to bacteria (fig 4). When it is clear exactly which region of the surface contains bacteria the surface properties of the metal and the biofilm can be characterised in high resolution with the AFM.
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