Measuring and mapping mechanical properties of live cells is crucial in today's biological research. Although atomic force microscopy is mainly used to produce a 3D profile of the scanned surface, it can provide much more information.
Tapping mode avoids tip and sample damage caused by friction and shear forces, thus enabling qualitative mechanical property mapping through phase imaging. Force spectroscopy and force volume are the conventional techniques used for making quantitative measurement of mechanical forces at the nanometer scale. However, slow acquisition speed and a lack of automated tools have limited the popularity of these techniques.
PeakForce QNM from Bruker
The aforementioned limitations are addressed by Bruker with the release of its PeakForce QNM (Quantitative Nanomechanical Property Mapping), which provides better results with respect to speed, resolution, user-friendliness, and quality of delivered information. PeakForce QNM is built upon Bruker's PeakForce Tapping mode, which employs peak force for feedback control and oscillates the probe at about 1kHz.
This article discusses the recent developments in mapping the properties of soft samples like cells and gels with PeakForce QNM and force volume, and the application of the latest NanoScope and NanoScope Analysis features to gather and study the information obtained from these techniques.
Comparison of PeakForce QNM and Force Volume
Force spectroscopy, force volume and PeakForce QNM are all valuable techniques for exploring cell mechanics. However, PeakForce QNM is ideal for high-resolution imaging or relatively high-speed imaging to study material properties. The key differences between force volume (or force spectroscopy) and PeakForce QNM are as follows:
-
Force volume employs linear ramping, while PeakForce QNM employs a sinusoidal modulation of the base of the cantilever corresponding to the sample surface, thus enabling acquisition of thousands of ramps per second.
-
The higher ramp rate of PeakForce QNM facilitates the acquisition of in-depth material property maps in much less time when compared to force volume
-
The normal force of the tip-sample interaction is controlled by PeakForce QNM through the detection of the peak force of each tap and feeding of the data into a continuously running feedback loop. The force control benefits obtained from the earlier taps and from the fact that the sinusoidal waveform makes the tip velocity to reach zero when the tip reaches the peak enables ultra-low interaction force down to 10pN.
-
PeakForce QNM typically shows more stability than force volume.
These qualities make PeakForce QNM ideal for material property mapping in many cases. Moreover, it is possible to separate the force spectroscopy and force volume taps by some distance because the triggering is treated independently. However, it is necessary to keep the PeakForce QNM taps as close as possible to obtain the optimum feedback performance.
Novel Features of Bruker’s Newly Introduced Nanomechanics Package
The new features of Bruker’s recently introduced Nanomechanics package are listed in Figure 1. This package adds key functionality, which enables users to easily connect between methods of single force spectra, force volume, PeakForce QNM, while including some key capabilities for exploring soft materials like cells.
For PeakForce QNM, the nanomechanics package includes the capability to operate at a broad range of frequencies and amplitudes. The addition of Sneddon cone model of elastic deformation enables calculation of modulus based on a conical or pyramidal tip shape rather than the parabolic (spherical) tips of the DMT model.
The PeakForce Capture enables simultaneous saving of a force curve at every pixel in the image in addition to gathering PeakForce QNM material property maps.
| |
PeakForce QNM |
Quantitative Force-Volume Mapping |
Single Force Curves |
| Measurement |
Continuous high-speed, sinusoidal force distance curves are measured while raster scanning. Tip-sample force is directly controlled using a continuous feedback loop. Curves are analyzed in real-time to generate modulus and adhesion maps. |
Single force curves are measured at points on a 2D grid. Tip-sample force is controlled by discrete force triggering at each point. Curves are analyzed in real-time to generate modulus and adhesion maps. |
Single force curves are measured at discrete points, targeted either manually or using “Point and Shoot” on optical or AFM images. |
| Offline Analysis |
New PeakForce Capture function captures a force curve at each pixel. The entire image of curves can be reprocessed offline, e.g., with different indentation models, to obtain updated property maps. |
Because every force curve is captured, the entire image can be reprocessed offline, e.g., with different indentation models, to obtain updated property maps. |
A full suite of force curve analysis tools is available, including baseline correction, filtering, indentation analysis, and adhesion peak finding. All functions may be automated for batch analysis of multiple curves. |
| Benefits |
Feedback-controlled sinusoidal drive uniquely enables the highest speed and highest lateral resolution property mapping with excellent precision force control. PeakForce Capture provides full access to force curves for additional offline analysis. |
Technique performs highly accurate force measurements, and is widely used and cited for property mapping. |
Highly accurate discrete force measurements can be precisely targeted using “Point and Shoot.” |
| Disadvantages |
None |
The lateral resolution is typically lower and image aquisition slower. Increasing the ramp rate results in overshoot of the force trigger, an unavoidable issue with mapping modes that use triggered, linear ramps. |
None |
| Ideal Use Case |
Technique is best for high-speed, high-resolution property mapping on biological samples with corresponding high-resolution topography. |
Technique serves as a comparison to modern PeakForce QNM technique. It is also ideal for special cases where loading rate dependence is critical (e.g., extracting kinetic parameters of binding/unfolding). |
Technique is best for cases where it is more valuable to have a few measurements on many cells instead of many measurements on a few cells. |
(A)
.jpg)
(B)
.jpg)
(C)
Figure 1. (A) Comparison of force volume, PeakForce QNM and single-force measurements. (B) Typical AFM tip- often neither a sphere nor a cone perfectly describe its shape. Comparison between Hertzian (DMT) sphere and Sneddon cone models of elastic deformation. (C) Nanomechanics features in NanoScope Analysis enable the user to modify the force parameters, flatten the baseline when necessary, and choose between various models depending on the nature of the tip and sample.
Modify force parameters enables the calibration of individual curves to be adjusted. Boxcar filter and Baseline correction facilitate the curves to be filtered to correct artifacts, while Indentation analysis enabling fitting of the curves with either Sneddon or DMT model with choices to incorporate adhesion, use approach or retract.
It is possible to automate all of these features to facilitate analysis of hundreds or thousands of curves and generate reports with statistics or histograms of data. The new MATLAB toolbox enables MATLAB to get direct access to NanoScope data files for highly complex analysis. This function makes researchers free from file parsing or ASCII exports so that they can remain focus on modeling and results.
Testing of PeakForce QNM on Bacteria
.jpg)
Figure 2. (A) PeakForce QNM (250Hz) Sneddon modulus data painted on 3D topography of E. coli bacteria. Brighter areas are stiffer with the brightest areas (substrate) ~50MPa. (B) Two force curves from the PeakForce Capture file showing the difference between substrate and cell. (C) Force volume Sneddon modulus image of the same bacteria collected at a ramp rate of 2Hz. (Standard DNP-A probe in water with 300nm modulation amplitude, Scan size 5µm.)
Figure 2A demonstrates a 3D topography of a pair of E. coli bacteria with the image brightness based on the Young’s modulus. This image was acquired using PeakForce QNM within in 10 minutes when compared to the force volume image shown in Figure 2C, which took nearly 35 minutes.
A pair of force curves obtained from PeakForce Capture is illustrated in Figure 2B. One was acquired on the top of the cell, while another one was acquired on the sample substrate as represented in Figure 2A. The accessibility for individual curves provides key opportunities for additional exploration and understanding.
Agarose Gel Analysis at Different Ramp Rates
In this example, agarose gels from 1%-5% wt. were prepared and imaged in PBS buffer to show the ability of PeakForce QNM and force volume to measure these soft samples. Figure 3 shows the analysis of agarose gels at various ramp rates.
Figure 3A shows the overlapping of typical approach curves acquired on the 3% agarose sample with ramp rates between 1Hz (force volume) and 250Hz (PeakForce QNM) on the same plot, illustrating the good agreement between the results obtained from the two methods, even over several orders of magnitude of ramp rate.
The same thing is depicted in a more statistically relevant manner in Figure 3C. The PeakForce QNM histogram yields much better statistics at minimal cost due to its 16,384 (128x128) measurements when compared to 256 (16x16) measurements for force volume.
.jpg)
Figure 3. Agarose gels at different ramp rates. (A) Individual curves collected on 3% agarose gel at different ramp rates between 1Hz and 250Hz. (B-D) Histograms of Sneddon modulus results on 1%-5% agarose gel respectively, comparing ramp rates of 1Hz with force volume (green) and 250Hz with PeakForce QNM (red). MLCT-D probe, k=0.047N/m, 1nN trigger, ramp size 300nm.
However, if there is a substantial time-dependent deformation mechanism in the sample to be analyzed, then the results of PeakForce QNM and force volume may differ. New analysis methods are required to quantify this time-dependent behavior. The new MATLAB toolbox in NanoScope Analysis is useful for scientists interested in developing these methods.
Plant Morphogenesis Analysis Using PeakForce QNM
Morphogenesis in plants mostly depends on meristems, a pool of stem cells localized in specialized plant tissues. In this example, confocal optical microscopy equipped with the BioScope Catalyst was used to image meristem cells, and the AFM measurements (rectangular area in Figure 4A) were carried out by selecting an area of interest by means of the MIRO software interface.
Fluorescence labeling of the cell walls using a lipophilic dye enabled easy detection of their location in the meristem correspondence to AFM modulus measurements, as shown in Figure 4A.
.jpg)
Figure 4. Typical application of PeakForce QNM imaging on living plant cells. (A) Projection from a confocal stack of an Arabidopsis Thaliana shoot apical meristem. Membranes were labeled with FM4-64. (B and C) PeakForce QNM images (top: 3D-height, topography only; bottom 3D- height with DMT modulus skin). The DMT modulus channel clearly indicated that the cell edges (anticlinal cell walls) were significantly stiffer than the rest of the cell. Circled areas show regions where the modulus and optical maps reveal the presence of anticlinal cell walls that are not detected when using topography alone.
The PeakForce QNM mode was used to image the same meristem cells, which are quite bumby with many relatively tall features. However, the ScanAsyst mode from Bruker can easily image this type of tissue with full automatic optimization of imaging parameters.
The topography in the area of interest is represented in 3D in Figure 4B, showing ~50 cells. The re-colored 3D topographic surface based on the elastic modulus map of the area is shown in Figure 4C, showing that the stiffness of the cell walls are much higher than the cell core.
This type of analysis demonstrates the capability of PeakForce QNM to explore the mechanical changes of plant cell walls during development, paving the way to relate local biophysical parameters to the global shape of the tissue in the existence of drugs, hormones or in a modified genetic background.
Advantages of PeakForce QNM in Live Cell Analysis
The faster, higher resolution mapping achievable with PeakForce QNM provides in-depth mapping of cell migration and cell division as they take place. Figure 5 illustrates the resolution possible on the lamellipodium of a mouse B16 cell imaged in HEPES buffer, which took 8.5 minutes. It would take nearly 9 hours to collect a similar force volume map at 2Hz ramp rate with the resolution.
The comparison of modulus maps computed from the Sneddon (cone) model utilizing the approach curves and the DMT (sphere) model utilizing the retract curves is shown in Figures 5B and 5C.
The results clearly show that the Sneddon model fits the data very well when compared to the DMT model.
.jpg)
Figure 5. Lamellipodium of a mouse B16 cell imaged in HEPES buffer. (A) 3D rendering of the lamellipodium topography showing actin fibrils. (B,C) Comparison between maps of DMT modulus and Sneddon modulus, along with individual curves and fits from the same point in the image. (D) Six additional data channels collected simultaneously, mapping the properties of the cell, such as deformation, dissipation, and adhesion. (Classic MLCT-D K=0.048N/m, tip with 35-degree half angle, R~30nm end radius, modulation amplitude of 200nm at 250KHz to minimize viscous background, setpoint 1nN.)
The comparison of the Sneddon modulus maps from Figure 5 with force volume results at 1Hz and 5Hz, and with PeakForce Capture results at 250Hz is illustrated in Figure 6. The 256x256 pixel map of Sneddon modulus measured concurrently with topographic imaging in Figure 6A and histogram of modulus on the lamellipodium at various ramp rates is depicted in Figure 6B.
Force volume images at 1Hz and 5Hz and PeakForce Capture image at 250Hz are illustrated in Figure 6C. The force volume images were captured at low resolution (16x16 pixels) for saving time; however, the resolution of the resulting modulus maps is not good enough to clearly show the actin fibrils in the cytoskeleton, which are observed in the PeakForce Capture and PeakForce QNM images.
.jpg)
Figure 6. Comparison of Sneddon modulus values obtained with PeakForce QNM and force volume at two different ramp rates. (A) 256x256 pixel map of Sneddon modulus calculated simultaneously with topographic imaging. Note actin fibrils clearly visible in cytoskeleton. (B) Histogram of modulus on the lamellipodium at different ramp rates. Green line is from 250Hz PeakForce QNM image, blue diamonds are force volume at 5Hz, red squares are force volume at 1Hz, Xs are from 250Hz PeakForce Capture data calculated offline. (C) Force volume images at 1Hz (top) and 5Hz (middle) and PeakForce Capture (PFC) image at 250Hz (bottom). All images are plotted with same data scale and color bar with range -50 to +300KPa for all.
Cancer Cell Analysis Using PeakForce QNM
Overexpressing some tumor suppressive factors can suppress the tendency of cancer cells to invade. However, this modification is expected to cause changes in mechanical properties. In this example, PeakForce QNM mode was used to analyze these changes and the results were reported in Figure 7.
It is possible to use PeakForce QNM either to image challenging cells at a high resolution, as depicted in Figures 7A and 7B, or to identify the mechanical properties, as shown in Figures 7C, 7D, and 7E. When compared to force volume, PeakForce QNM is more relevant for this type of measurement in terms of quality, resolution, and amount of delivered data, thereby providing great perspectives in cell mechanics analysis.
.jpg)
Figure 7. PeakForce QNM investigation of live U-251 Glioblastoma cells. (A and B) 20x20pm 512x512 3D-rendered height and peak force error images respectively of control cells. The loading force has been increased on purpose to reveal high-resolution details of the cell cytoskeleton (typical capture time = 35 minutes). (C, D and E) Peak force error, Young's modulus, and deformation maps (80x80µm 128x128 pixels) of cells transfected to overexpress TSF (typical capture time = 5-6 minutes). The right graphics summarize the measurements performed on control and transfected cells using traditional force volume and PeakForce QNM, showing good agreement between the techniques, but highlighting the significantly better statistics obtained with PeakForce QNM.
Conclusion
The structure and functional activity of biological samples are often affected by their mechanical properties. Hence, it becomes significant for biologists to measure and map the mechanical properties of biological samples. Force volume is designed for mapping with low ramp rates and with comparatively low resolution.
The high resolution and ramp rate of PeakForce QNM make it ideal for collecting and analyzing more data for better details and statistics. Furthermore, the new features of NanoScope and NanoScope Analysis enable users to easily collect, process and study the thousands of force curves in a typical force volume or PeakForce QNM map.
.jpg)
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.