Measuring Fixed Cells with AFM and Scanning Ion Conductance Microscopy

The preservation of cell or cellular components in a life-like state by preserving the vital physical and chemical properties of the cells is one of the main aims of cell fixation for in-vitro studies. Cell fixation is also helpful in immunostaining by enabling antibodies to access intracellular structures [1].

When compared to the live cell, the fixed cell preserves a more consistent architecture over the entire cell surface [2]. Yet, no systematic assessment or correlation exists between variations in the mechanical characteristics of fixed and live cells. Cell fixation protocols can possibly be optimized, and valuable insights into assisting the cell fixation process can be gained by observing different states of cell fixation and the living cells. There are a number of fixation agents for cross-linking cytoplasmic proteins and cell membranes, where paraformaldehyde (PFA) is most extensively used for tissue and cell samples [1–3].

The function of PFA is to cross-link molecules covalently, bonding them together and forming an insoluble meshwork that modifies the mechanical characteristics of the cell surface. The mechanical characteristics of live and fixed cell structures cross-linked with PFA were analyzed by measuring the surface fluctuation and elastic modulus by using Scanning Ion Conductance Microscopy (SICM) and Atomic Force Microscopy (AFM), respectively [3–6].

Experimental Setup

Cell Sample

The cell sample that was used contained mouse fibroblast L929 cells (from ATCC, USA) cultured in Dulbecco’s modified Eagle medium (DMEM; from Invitrogen Life Technique, USA) supplemented with 10% fetal bovine serum (from Thermo Fisher Scientific, USA) along with 1% streptomycin/penicillin (from Invitrogen Life Technique, USA) in a humidified atmosphere at a temperature of 37 °C with a CO2 concentration of 5%. Cells with densities of 1 x 104/mL were put on a cell culture petri dish (from NUNC, Denmark) with a diameter of 35 mm, washed three times using phosphate buffered saline (PBS; from Sigma-Aldrich, USA), and treated using a 4% PFA solution for 5 minutes. PBS was used to rinse the fixed cell samples thrice before performing the SICM and AFM experiments.

Setup of AFM and SICM

The cell imaging setup included the Park NX-Bio scanning probe microscope from Park Systems, Korea. This microscope is fitted with an inverted optical microscope (from Nikon Corp., Japan) designed exclusively for biological applications. The AFM has the ability to collect information related to the mechanical characteristics of a sample, and SICM provides the cell surface information of soft material samples.

All experiments carried out on live cells were performed within a live cell chamber. The live cells’ chamber conditions were regulated to 95% humidity, 5% CO2, and 37 °C. The chamber had the environment required to support living cell cultures.

AFM Measurements for Young’s Modulus of Cells

Force curve measurements were obtained by using the AFM to estimate Young’s modulus of the cells. A commercial cantilever (BL-AC40TS; from Olympus, Japan) with a nominal spring constant of <0.09 N/m was employed. The use of a cantilever with a small spring constant facilitates a comparatively large deflection by applying lesser force and enables reliable collection of data related to the structure of the cell surface. The thermal vibration technique was used to carry out the spring constant calibration of the AFM cantilever. Measurements were carried out using 50 force curves, each of which contained 512 data points (see Figure 1a).

Optical images of L929 cell for AFM (a) and SICM (b). Each probe was positioned at the apex of the single cell.

Figure 1. Optical images of L929 cell for AFM (a) and SICM (b). Each probe was positioned at the apex of the single cell.

The force curves were analyzed by implementing a Hertz model using the Park XEI imaging analysis program from Park Systems. The AFM tip’s shape was presumed to be a four-sided pyramid with half cone angle α. Consequently, the force (F) exerted on the cantilever is denoted as:

In the above equation, E is Young’s modulus, δ is the indentation (depth), and ν is the Poisson’s ratio, which was set as 0.5. The half cone angle α was set as 35°. The maximum loading force was 8 nN and the AFM scan rate was set at 1 μm/second.

SICM Measurement for Cell Imaging and Fluctuation Analysis

SICM involves using ion current that flows between an electrode positioned within a nano-pipette and an external electrode placed in a bath solution (see Figure 1b). A feedback signal provided by the ion current regulates a steady tip-to-sample distance and enables the nano-pipette to scan topographical information.

In spite of a low lateral resolution of ~30 nm [7], SICM can offer valuable topographical measurements without requiring mechanical forces to be applied onto the sample surface. The height images of the live and fixed L929 cell are illustrated in Figure 2. At first sight, the two cases look similar, but the fixed cell’s surface is somewhat rougher because of cross-linking of cell membrane proteins.

(a, c) Live and (b, d) 4% PFA treated cell surface imaging by SICM.

Figure 2. (a, c) Live and (b, d) 4% PFA treated cell surface imaging by SICM.

SICM imaging and ion current-distance (I-D) curve experiments were conducted by fabricating nano-pipettes with an inner diameter of 80 nm from borosilicate capillaries (with 0.6 mm inner diameter and 1.0 mm outer diameter; from World Precision Instruments, USA) with the help of a CO2-laser pipette puller (from Sutter Instruments, USA).

Results and Discussion

Young’s Modulus of Live and Fixed Cells

The stiffness of live and fixed cell surfaces was determined by obtaining force spectroscopy measurements from live cells, fixed cells, and also a solid substrate. Figure 3a illustrates the resultant force-distance curves. Fixed cells exhibit a considerably steeper force curve slope than live cells.

Average force-distance curves (a) and ion-current-distance curves (b) of the solid substrate (black), 4% PFA treated (fixed) cell (red), and live cell (blue).

Figure 3. Average force-distance curves (a) and ion-current-distance curves (b) of the solid substrate (black), 4% PFA treated (fixed) cell (red), and live cell (blue).

Moreover, when compared to live cells, the force needed for surface indentation is greater for fixed cells. The average Young’s modulus values presented in Table 1 indicate that the stiffness of fixed cells (77.95 kPa) was higher compared to that of the live cells (8.11 kPa). These AFM cell stiffness measurements indicate that the acting filamentous structures had a strong influence on the stiffness.

Table 1. Young’s modulus value

Paraformaldehyde Concentration (%) Young’s Modulus
Mean SD
Live cell (0) 8.11 kPa 2.93
Fixed (4) 77.95 kPa 8.16
Solid Substrate 3.94 MPa 0.11

Another prominent fact is that PFA treatment has an impact on the cross-linking of cell surface proteins, such as the F-actin filaments. Moreover, cell stiffening was observed to correspond to protein cross-linking [8]. In particular, it can be assumed that the PFA fixation process has a direct link to an increase in cell stiffness based on the number of randomly distributed cross-linking sites available on the cell surface [9].

Surface Fluctuations of Live and Fixed Cells

Cell surface fluctuations were detected by obtaining the I-D curves carrying out SICM measurements on fixed cells, live cells, as well as a solid substrate. In the case of solid substrates (petri dish), I-D curves demonstrate the steepest slope; however, they possess a broader slope while untreated. In the case of PFA-treated cells, I-D curves fall in between the two values.

Then, it can be said that the live cells exhibited more activity than the fixed cells. The process of PFA treatment leads to smaller surface fluctuations for fixed cells, which induces cross-linking of proteins between the cytoplasmic proteins and the membrane.

Conclusion

In this article, live cells and cells fixed with PFA have been compared to show a basic mechanical difference. Scanning ion conductance microscopy and atomic force microscopy measurements demonstrated an evident transition in elastic modulus and surface fluctuation of cells upon being exposed to PFA. Upon performing complete fixation with PFA, there was a decrease in cell surface fluctuation compared to a live cell, while there was a five-fold increase in Young’s modulus.

These results offer an important understanding of the reaction of cells to chemical treatment with PFA. Apart from the conventional understanding of the chemical impact of PFA on cells, this article has unraveled the influence of PFA on the mechanical characteristics of the surface of cells. Although it is presumed that cell membranes are flexible and variable, in certain conditions (specifically chemical treatment), changes take place on the morphological and biological levels.

Observations such as these offer a strong motivation for performing further analyses of cell surface fluctuations as a vital prerequisite for gaining insights into the cell functions in relation to cell dynamics. Scanning probe microscopy methods, particularly SICM and AFM, evidently serve as critical tools to carry out quantitative studies of both live and fixed cells.

References

  1. Lanier, L. and N. Warner, Paraformaldehyde fixation of hematopoietic cells for quantitative flow cytometry (FACS) analysis. Journal of immunological methods, 1981. 47(1): p. 25–30.
  2. Yamane, Y., et al., Quantitative analyses of topography and elasticity of living and fixed astrocytes. Journal of electron microscopy, 2000. 49(3): p. 463-471.
  3. Binnig, G., C.F. Quate, and C. Gerber, Atomic force microscope. Physical review letters, 1986. 56(9): p. 930.
  4. Korchev, Y.E., et al., Scanning ion conductance microscopy of living cells. Biophysical Journal, 1997. 73(2): p. 653.
  5. Cappella, B. and G. Dietler, Force-distance curves by atomic force microscopy. Surface science reports, 1999. 34(1): p. 1–104.
  6. Mizutani, Y., et al., Nanoscale fluctuations on epithelial cell surfaces investigated by scanning ion conductance microscopy. Applied Physics Letters, 2013. 102(17): p. 173703.
  7. Rheinlaender, J., et al., Comparison of scanning ion conductance microscopy with atomic force microscopy for cell imaging. Langmuir, 2010. 27(2): p. 697–704.
  8. Hopwood D. Theoretical and practical aspects of glutaraldehyde fixation. InFixation in histochemistry, Springer, Boston, MA. 1973: p. 47–83.
  9. Tanaka KA, et al., Membrane molecules mobile even after chemical fixation. Nature Methods. 2010 Nov; 7(11):865.

This information has been sourced, reviewed and adapted from materials provided by Park Systems.

For more information on this source, please visit Park Systems.

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