Quantitative Imaging of Living Biological Samples Using PeakForce QNM

It is a well-known fact that determining the mechanical properties of living cells ex vivo can indicate the organism’s health from which they were extracted. Particularly in the force mode, AFM is a powerful diagnostic and investigational tool. Force spectroscopy has a lot of disadvantages such as less resolution, speed of acquisition, and it does not provide the needed quantitative information. PeakForce QNM has been developed by Bruker to offer informative data at high resolution with remarkable ease of use.

AFM and Cell Mechanics

Since its development, AFM is a tool of choice to image super soft biological samples, especially with the emergence of TappingMode™ and force spectroscopy and the fact that it is one of the few microscopy techniques that allows observation of cells under near-physiological conditions. AFM is often used to correlate elastic behavior and cell migration or division. The vast majority of these studies are based on TappingMode, single-force curves, or force-volume measurements.

TappingMode offers the advantage of applying negligible nominal, friction and shear forces, and phase imaging reflects the energy dissipated between the tip and the sample during each tap on the surface. Force volume is another powerful technique based on force measurements achieved on a matrix of points defined by the user. Stiffness and the adhesion between the tip and the sample can be extracted from each force curve. In case the tip is functionalized with a molecule of interest, specific unbinding events can also be identified on the retraction curve. To overcome these bottlenecks, Bruker has developed PeakForce QNM.

Easy, High-Resolution Quantification of Sample Mechanical Properties

PeakForce QNM enables direct extraction of quantitative nanomechanical information from biological samples without damaging the sample. It’s based on Peak Force Tapping technology, during which the probe is oscillated in a similar fashion as it is in TappingMode, but at far below the resonance frequency (1 or 2 kHz depending on the tool). Each time the tip and the sample are brought together, a force curve is captured. However, where the feedback loop maintains the tapping amplitude constant in TappingMode, Peak Force Tapping controls the maximum peak force on the probe. These forces can be controlled at levels much lower than contact mode and even lower than TappingMode allowing operation on even the most delicate biological samples.

Figure 1 shows the different force fields experienced by the probe during an approach-retract cycle, as well as all the information that can be extracted from the generated force curves. When the probe approaches the sample (figure 1a), it’s pulled down toward the surface by attractive forces, which are mainly capillary, Van der Waals and electrostatic forces. At point B, those negative forces become higher than the cantilever’s stiffness, which causes the tip to pull to the surface and then start indenting into the sample until the Z-position of the modulation reaches its maximum (point C). This position represents the maximum peak force value, which is used for the feedback control. After this point, the probe starts withdrawing until it reaches the pull-off point (the maximum adhesion point, which also corresponds to the minimum force). Then the tip continues retracting and reaches back to its original position (E) where (as in A) no more force field affects its motion.

Figure 1. Working principle of PeakForce QNM. While the probe is oscillated, a force curve is recorded for each pixel of the image. To distinguish between the different portions of the tip trajectory, this example was recorded by using a TAP150A probe, which is typically used to image rather stiff and poorly compliant samples. On biological samples, the typical peak force can be up to a thousand times lower.

This mechanics model assumes that the contact principle remains the same as in the Hertzian model but considers additional attractive interactions focused inside an annulus located outside of the contact area (figure 2a). In that case, and considering the contact between a sphere and an elastic half-space, the force is related to the deformation by:

where E* represents the reduced Young’s modulus, R the tip radius and d the deformation depth.

Eventually, the energy dissipated by the tip and the sample during each tap on the surface is obtained by integrating the area between the approach and the retraction curves.

Direct Quantification of AFM Signals on Biological Samples

When the probe is calibrated prior to the experiment, all the signals mentioned above will be directly quantitative. This calibration can be done as follows:

1. Engage a stiff part of the sample (like glass) and record a force curve from which the deflection sensitivity can be calculated.
2. Withdraw and calculate the spring constant using “Thermal Tune”.
3. Record a topography image of the Tipcheck sample to obtain a value of the tip radius R.
4. After entering the estimated R value, the deformation is adjusted on a sample of choice. The sample to be scanned should have similar mechanical properties as the biological sample that will be investigated during the experiment.

On most of the tested samples, a Sneddon fit was used to extract the Young’s modulus by capturing a HSDC (High Speed Data Capture) file on a scan line at a very high resolution. When the force and height profiles are compared, the non-desired parts (force curves captured on a portion of the sample that is not of interest, such as glass) can be excluded manually. The remaining force curves can be exported as a single file, post-processed by an external program, and the average Young’s modulus can be calculated by considering different contact theories, such as the the Sneddon model.

This mechanical theory considers the contact between an elastic half-space deformed by a rigid conical indenter (figure 2b), determining that the load is proportional to the square of the penetration depth. The indentation depth and the tip radius are related by:

Figure 2. Contact mechanics in AFM. In a, the DMT fit is based on a Hertzian assumption but states that the adhesion forces are focused outside the contact area. This is well adapted to high-density polymers and poorly deformable samples. In b, the Sneddon fit considers the tip as an infinite conical indenter, which is well adapted to soft (biological) and deformable samples.

On such samples, a wide range of AFM probes have been tested and the recommendation is given in figure 3. The softest existing probes on the market are OBL-B, which have a nominal spring constant of 0.006 N/m and are thus appropriate to investigate super soft living cells, such as neurons.

Figure 3. Range of compliance of various biological samples and corresponding AFM probes recommended for Peak Force Tapping. Depending on their type, eukaryotic cells can exhibit very different mechanical properties. Neurons can be extremely soft (down to 1kPa) whereas bone cell can be as robust as bacteria. In order to properly probe the cell properties, selecting the right spring constant and thus sensitivity is mandatory.

Imaging Biological Samples with PeakForce QNM

Marine biological samples are often composed of a mixture of soft and rigid components. A sample of water taken from the Adriatic Sea was put on a glass slide and investigated by PeakForce QNM. Other than very relevant observations on living diatoms, some cell wall remnants were also found in the suspension. Figure 4 gives an example how those structures look. The 3D-topography profile reveals a characteristic waffle-like structure with pores 100 nm in size and an average height of 20 nm. The adhesion channel shows a marked contrast between the bottom of the pores (about 50 pN in average) and the rest of the cell wall (less than 20 pN). However, the most informative channels are the elasticity and the deformation data. On both channels the three portions of the frustule are distinguished, each exhibiting clearly different mechanical properties: the center of the pore (average Young’s modulus of ~300 kPa and average deformation of ~7 nm), the ring around the pore (~75 kPa and ~25 nm) and the core part of the cell wall, which seem to have intermediate mechanical properties (~200 kPa and ~10 nm).

Figure 4. Imaging of phytoplankton cell wall with a BioScope Catalyst AFM. Top left: electron microscopy image of a diatom, sample courtesy of Dennis Kunkel, Astrographics. Most of the PeakForce QNM channels provide a remarkable contrast and high-resolution features.

Additional experiments were carried out on Escherichia coli K12 bacteria. Unlike most of E. coli species, K12 strains are able to multiply in the intestine and are particularly resistant to antibodies. One of their other characteristics is that they possess pili (see figure 5a) that typically retract under depletion conditions or any stressing environment. Until now, imaging those bacteria alive with the AFM, in any mode, has been a considerable challenge and a historically elusive result.

Figure 5 shows high-resolution images of such living bacteria, easily obtained in less than one hour. As can be seen on the 3D-representation of the height channel (figure 5b), the pili are no longer visible, which can be explained by the fact that extracting from their suspension medium and spreading them on a dish induces a stress that causes those pili to retract. Figure 5c shows the DMT modulus channel. By using a Sneddon fit, the average Young’s modulus was determined to be 183 kPa, which perfectly matches previous observations.

Figure 5. E. Coli K12 bacteria imaged by PeakForce QNM on a BioScope Catalyst AFM. In a, the structure of the strain is drawn. In b, an AFM 10x10μm 3D-height representation of a cluster of bacteria is shown. In c, Young’s modulus channel (z-scale: 0-4GPa) is depicted. This is the first time that such bacteria has been imaged alive by AFM.

Monitoring Cell Dynamics in Real Time

All living cells are dynamic, changing shape due to rearrangement of their cytoskeleton scaffold and spreading and migrating on the cell culture substrate. These processes and the mechanical changes that accompany them can be monitored using the PeakForce QNM. In another set of experiments, PeakForce QNM was used to investigate glioblastoma cells. Glioblastoma are by far the most common and malignant form of brain cancer. Living glioblastoma cells have been imaged by PeakForce QNM on the BioScope Catalyst and maintained alive for the time of the experiment by the use of the PSI. This technology allows the user to apply a very gentle to moderate force on the sample, depending on the information needed. When applying a very light force on the sample, the topmost features of the cell (glycocalyx, protrusions) can be probed. On the other hand, a slightly higher force is required to sense the organelles and the cytoskeleton located underneath the plasma membrane. Probing the real mechanical properties of the sample also requires indentation of the sample (and thus flex in the cantilever) by at least a hundred nm. Figure 6a shows a typical high-resolution image obtained on living glioblastoma while applying a moderate force (~300 pN).

Figure 6. Images of living glioblastoma cells by PeakForce QNM and the BioScope Catalyst AFM. In a, 40x40μm height image recorded at a moderate force shows both topmost and internal structures. In b, 15x15μm 3D overlay of topography and deformation channels is shown. Bruker’s BioScope Catalyst with Perfusing Stage Incubator offers the best balance of living cell imaging for long-term experiments.

Keratinocytes are the major components of the outermost layer of the human skin. Studying such cells by AFM helps researchers understand the process of skin cancer or other impairments.

HaCat is an immortal cell line of human keratinocytes that is widely investigated in cytology and also represents a good candidate to explore the potential of PeakForce QNM. The cells were exposed to an oxidative agent capable of inducing a stress. In response to this chemical aggression, the cells tend to transform and synthesize so-called actin stress fibers. A typical medium- resolution image is shown in figure 7.

Figure 7. 75x75μm BioScope Catalyst and PeakForce QNM image of living HaCat cells under oxidative stress. The cells react by rapidly synthesizing stress fibrils to establish contacts with adjacent cells. Such dynamic processes can also be tracked by using this technique.

In PeakForce QNM, a force curve is made for each pixel of the image, thus the resolution is the same on all the channels. This example illustrates how easy and fast (384x384 pixel resolution images can be captured in 6 to 9 minutes) it is to directly and in a quantitative manner probe changes in topography and mechanical properties of living cells in response to drug treatments.

Overlaying AFM and Optical Channels

Another of the current key challenges for biological applications is to be able to get optical and AFM information simultaneously. Bruker’s exclusive Microscope Image Registration and Overlay (MIRO™) feature can be used to easily import optical/fluorescence images into NanoScope® software and overlay them with AFM images. After a short calibration, the user can select the location to make the AFM scan. Thus the sample automatically can be moved to the desired position and the AFM image can be captured pixel by pixel, and fully integrated into the optical image.

Figure 8 shows an overlay achieved on living endothelial cells. The fluorescence image (double-staining DAPI for nucleus and á-phalloidin for actin filaments) is set as the background and overlapped with an AFM image made of a mix of two channels: peak force error and Young’s modulus. The transparency was set at 50% so that a direct correlation can be made between the different parts of the cells (visible by AFM topography and fluorescence) and their corresponding mechanical properties (Young’s modulus AFM channel). In b, c and d the individual peak force error, Young’s modulus and deformation AFM images are represented. It can clearly be seen in the elasticity and deformation channels that on the edges of cells where the thickness is too low, the influence of the scaffold (glass) on the mechanical properties of the sample is non negligible, whereas on the core part of the cells, the average Young’s modulus is much more reliable (45.3 kPa). For a matter of clarity, only three AFM channels are shown here, but eight different signals can be displayed simultaneously.

Figure 8. Overlay of fluorescence and AFM images of living HUVEC cells created with MIRO on a BioScope Catalyst. The main benefit of MIRO is to enable the display of optical and AFM information simultaneously. When operating with functionalized probes, a “Point & Shoot” option can also be used to accurately trigger force measurement at desired locations without losing the ligand.

Conclusion

The applications shown above demonstrate that Peak Force Tapping is by far the most powerful and quantitative high-resolution AFM technique available today to probe quantitative chemical and mechanical properties of living biological samples with an acquisition speed comparable to TappingMode. The number of different mechanical properties that can be characterized exceeds that of other commonly used AFM modes. Its potential paves the way for many exciting new applications in the field of biology, especially in cancer research and cardiovascular diseases.

This information has been sourced, reviewed and adapted from materials provided by Bruker Nano Surfaces.

For more information on this source, please visit Bruker Nano Surfaces.