Mapping large areas of life science samples

article
Josef A. Käs1, Thomas Fuhs1 & Torsten Mueller2 and André Koernig2

1. Faculty of Physics and Earth Science, Peter Debye Institute of Soft Matter Physics, Soft Matter Physics Division, University of Leipzig, Leipzig, Germany

2. JPK BioAFM Business, Bruker Nano GmbH, Berlin, Germany

Biography
Dr Thomas Fuhs, AFM Group Leader and his research focuses on the physics of tissues. Using AFM-based micro-rheology and high-resolution fluorescent imaging, he aims to achieve a deeper understanding of the mechanics of cells and how the mechanics of cells correlate with the aggressiveness of tumors and the outcome of neurodegenerative diseases.
 
Acknowledgements
The Vero cell samples were provided by Prof. A. Hermann and his group (Humboldt University, Berlin).
 
Corresponding author
Dr. Thomas Fuhs
AFM group leader
University of Leipzig, 
Faculty of Physics and Earth Science, Peter Debye Institute for Soft Matter Physics, Soft Matter Physics Division
 
Linnéstraße 5, 04103 Leipzig, Germany
Room 329
Telephone: (+49 341) 97 32583
Fax: (+49 341) 97 32479
 
INTRODUCTION
 
Crucial parameters that affect cell adhesion, morphogenesis, cell differentiation and cancer invasion include the molecular interactions between cells and their extracellular matrix environment, their 3D topography and the corresponding mechanical properties1-3. AFM (atomic force microscopy) is an advanced multi-parametric imaging technique which delivers 3D profiles of the surfaces of molecules and cells in the nm-range. It also enables the characterization of nanomechanical properties (adhesion, elasticity etc.) and the visualization of structural changes taking place at the molecular level. 
 
The mapping of tissue samples and thicker multi-cellular layers as well as the nanomechanical characterization of the extracellular matrix and its embedded cells requires a (semi)automated AFM approach for a millimeter range in x,y with nm–resolution. Large variations in the topography of tissue samples, combined with fluidity and stickiness of membrane and extracellular proteins, require a larger flexibility in the z-axis of the AFM3.
 
MATERIALS AND METHODS
 
Working closely with life science users in the ever-growing AFM community, the JPK BioAFM team has developed a highly versatile solution: HybridStage. It is equipped with three scanner units that can move either the tip or the sample. A standard NanoWizard AFM head has an xyz tip scanner with a scan range of 100 x 100 x 15 µm3. The HybridStage houses an xyz sample scanner with a number of optional scan ranges: 100 x 100 x 100 µm3, 200 x 200 x 200 µm3 or 300 x 300 x 300 µm3. These may be individually selected depending on the individual application. The system also features a motorized unit for large sample movements (20 x 20 mm2). This wide choice of scanners together with optical tiling and multi-region AFM probing enables multi-parameter characterization of soft samples over a large area. It also provides additional optical data sets on either inverted and upright microscopes respectively (figures 1, 2). 
 
Figure 1. Top, The HybridStage integrated into a JPK NanoWizard AFM system on an inverted Nikon microscope
 
Figure 2. Above, JPK HybridStage on a Zeiss upright AxioZoom V.16 fluorescence macroscope with an Andor EMCCD camera for force mapping experiments on non-transparent tissue samples
 
RESULTS
 
We will report a number of examples to illustrate the benefits of using a configuration like the HybridStage.
 

SINGLE CELL FORCE SPECTROSCOPY AND MEMBRANE TETHERS

The most frequently reported activities in the field of Single Cell Force Spectroscopy (SCFS) include the study of the adhesion of individual cells on (bio)materials, extracellular matrix proteins and the characterization of cell-cell interactions4
 
Due to the effect of membrane tethering, the pulling length required to separate cells from a substrate frequently exceeds several tens of microns5. However, the flexible, modular design of the HybridStage with its options of 3D sample scanners can meet this demand. 
 
Figure 3 illustrates a detachment curve of a fibronectin functionalized cantilever from a Vero cell. After reaching the cell surface at a piezo height of approx. 60 µm, separation is completed at a final piezo height of 105 µm. The membrane tube formations, with force plateaus of 1 – 15 µm in length, are the main reason for a total separation distance of about 45 µm in this example.
 
Figure 3. Force distance curve of a Vero cell in contact with a fibronectin coated tipless cantilever (50 μg/ml for 30 minutes; TL-2 – NanoWorld) Force settings: 3D piezo scanner, 100 x 100 x 100 μm3, 1nN, z-speed 10 μm/s, contact 10 seconds. The graph of the force curve (lower right) shows the zoomed-in data highlighted in the black box. (The inset image shows an optical microscope image of a layer of Vero cells).
 
One of the challenges of a long-ranged z-movement for the tip and sample scanner is that either the cantilever or the sample may move out of the optical focus. To overcome this, a new piece of software has been developed. Known as JPK SlimFocus (figure 4), it enables the synchronization of the objective lens motion with the current z-scanner (figure 5).  The control software allows manual adjustment of the z-position using a slider and the setting of two marker positions, e. g. for the tip/sample. During AFM experiments, the optical focus motion can be synchronized with the z-scanner currently being used up to 150 µm.
 
Figure 4. Left,  JPK SlimFocus unit equipped with an objective lens mounted on a microscope turret. The sample stage is above the lens.
 
Figure 5. Right, The SlimFocus software takes into account the height corrections needed if the objective lens and sample are surrounded by different media (liquid/air)6
 
OPTICAL TILING AND MULTI-REGION IMAGING 
 
Sometimes, if the x,y piezo scan range and the optical observation window (typically 100 µm) are much smaller than the sample size (in the range of mm) to be investigated with the AFM, a conflict may arise.  This is compensated by a clever design feature: In order to enlarge the microscopic optical overview, the motorized sample scanner executes a repetitive pattern, acquiring the corresponding optical images of the sample which are then tiled and displayed in the SPM software (figure 6). When tiling starts, the file chooser opens an automatically named sub-folder. The calibration file and the image file are saved in the sub-folder and are easily accessible for data processing. 
 
Figure 6. Screenshot of the NanoWizard control software. Right: Live camera image with Vero cells in liquid on an inverted microscope with optical phase contrast. The shadow of the retracted cantilever is visible. Left: The first optical image is calibrated in x,y with piezo accuracy using the AFM scanner (scan range marked in green, here 100 x 100 μm2). A “Tiling Tool” button allows the user to draw a tiling rectangle on the surface. The software automatically considers the number of optical images required after selecting the tiling area (here 4 x4 images for 500 x 500 μm2).
 
For multi-region AFM imaging, the software interface asks the user how many measurement positions they wish to examine. They may then select points of interest before a measurement starts. This is displayed in the data viewer with tiled optical images. When the measurement begins, the positions are processed sequentially. Several different types of measurement definitions are available: single points, lines and rectangular regions. This multi-scan option is also available for most measurement modes, e.g. for force mapping, spectroscopy, AFM imaging, Quantitative Imaging (QI™) and multiple DirectOverlay™ snapshots. An example of multi-scan QI imaging of mammalian Vero cells on an inverted optical microscope is shown in figure 7. The experimental steps were as follows: first, DirectOverlay was performed to correlate the cantilever tip position with the corresponding optical image; second, optical tiling with 4 x 4 images on 500 x 500 µm2 was carried out (see figure 6) to increase the region of interest; then third, a multi-scan range was selected. For this specific example, 4 x 4 QI scans, with an individual map size of 50 µm, were performed (frames are labeled in magenta). In figure 7, 13 of the 16 maps have already been executed. The green frame on position 14 indicates the current map position. Using the “advanced force oscilloscope”, multiple data viewers can be activated, e. g. height (of the sample) at force setpoint, adhesion (between cantilever and sample), slope (as an indication of sample stiffness) and reference force height(s) at a defined value of the force setpoint. In the specific example shown in figure 7, two different data viewer channels are displayed (height at 100% of the setpoint and reference height at 80% of the setpoint). To obtain a better contrast of cellular structures, the reference height is indicated as a pixel difference image. This versatility has been of particular benefit when collecting large quantities of data.
 
Figure 7. Screenshot of the NanoWizard HybridStage control software. Adherent Vero cells in phase contrast optical microscope, imaged in PBS buffer, under 37°C temperature control in a JPK PetriDishHeater™. Top: Optical tiling over 500 x 500 µm2 consists of 4 x 4 optical images. The selected multi-scan region is indicated by the magenta colored frame: 4 x 4 maps with 50 x 50 µm2 size. Top left: Height channel maps. Top right: Pixel difference map of the same region. Bottom: Advanced force oscilloscope and a force curve for a specific index position. QI mode settings: Force 0.23 nN, z- length 300 nm, 256 x 256 pixels. A PFQNM-LC-A cantilever with a tip height of approx. 19 µm and blunt tip (radius 70 nm) was used.
 
MULTI-SCAN TISSUE MAPPING 
 
In order to obtain a better understanding of the development and progression of cancer, analyzing changes in the mechanical properties of cells and their surrounding extracellular matrix material appears to be crucial3. Using the example of human cervix tissue, the benefits of the HybridStage and the mode of operation, in combination with an upright macroscope, are presented here. 
 
Because of its long working distance, the upright macroscope allows the collection of optical images with or without the AFM head. Furthermore, replacement of the AFM head does not require a readjustment of the sample. The excellent replacement accuracy of the head with a mounted cantilever is in the range of < 2µm in x,y,z. Once the region of interest is found in the optical image, it can be used to position the AFM probe with respect to the area of interest (figure 8). For these studies, a HybridStage with a 3D piezo sample scanner of 300 x 300 x 300 µm3 was used in conjunction with the motorized stage to perform a multi-scan map of 1000 x 1000 µm2 with 5 x 5 single maps of 200 x 200 µm2 and a pixel distance of 10 µm each (figure 9). All individual maps were analyzed using the batch processing option for indentation experiments in the data processing software. Conveniently, the data may also be exported, analyzed and composed using a third-party software e. g. Matlab. For example, figure 10 summarizes the frequency distribution of the apparent Young’s modulus of the multi-scan shown in figure 9.    
When performing a multi-scan force map over a larger area on a very rough sample with varying heights, there may be damage to the cantilever or sometimes the AFM measurements may not yield usable data. Also, when looking at very soft samples with sticky surfaces, the cantilever only separates from the sample surface after several tens of micrometers. This may result in a noticeable deformation of the sample or removal of the membrane tube.
 
Figure 8. Zeiss upright AxioZoom V.16 (lens 0.5x) fluorescence macroscope was used for the overview transmission light image (left) and fluorescence image (right) of human cervix tumor tissue. Fluorescence staining was performed with Hoechst 33258 for the nucleus labelling. The white frames illustrate the 1000 x 1000 µm2 AFM mapping area. The optical images were done without an AFM head to obtain better optical quality for the fluorescence image.
 


Figure 9. Overview of apparent Young’s modulus multi-scan map for 1000 x 1000 µm2 (compare figure 8) with 5 x5 individual force maps of 200 x 200 µm2. The single force maps were analyzed using the NanoWizard DP software. The indentation experiments were performed at a 3 nN setpoint force with a Nanoworld-Cont cantilever with a 6 µm bead which was separately glued at the tip apex. 
 
Figure 10. Histogram of the apparent Young’s modulus of the data displayed in Fig. 9 with a unimodal frequency distribution. A typical indentation depth at 3 nN setpoint force was around 1µm – 3 µm.
 
Here too, the z-range must be increased in order to successfully complete the measurement. To solve this technical difficulty, the software can set the height of both the piezo-retract and the motor-retract. In this way, an individual map and entire scan can be optimally performed. Figure 11 shows that the tissue sample examined is very rough and has a very varied height.  Even in the individual map of 200 x 200 µm2, the height difference is approx. 80 µm. Despite this, the multi-scan could still stably perform the measurement without having to stop. 
 
Figure 11. Height profile of a single map of 200 x 200 µm2 of the cervix tissue (map from a top right corner of the multi-scan area).
 
SUMMARY AND CONCLUSIONS
 
We have illustrated that the HybridStage is a versatile tool for advanced, multi-parameter AFM characterization of samples in the range from mm to nm and with pN resolution. It is particularly versatile in that it has scanner units that can move the tip/sample. It is possible to study a wide range of samples including biomaterials, cells and microaggregates, embryos and tissues, model organisms in developmental biology (zebra-fish, C. elegans, etc.) and implants. The increased z-piezo range, equipped with wide-ranged motorized sample motion makes this ideal for the nanomechanical characterization of tissues required in cancer, nanomedicine and developmental biology. 
 
We believe the HybridStage has freed experiments from the constraints of the traditional AFM piezo range. Large-range tiling of optical images has provided a clear visual overview allowing a fast setup of optically guided experiments and the direct selection of the optical features for investigation. Navigatation around the sample helps us to easily set up and collect a list of interesting features for multi-scan, or even map force responses over greatly extended scan ranges. This, a modular, piezo-based sample scanner stage combined with motorized XY sample movement is an excellent system which gives direct access to anything in the field of view of the microscope.
 
REFERENCES
 
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