Imaging Cells with a Combination of Fluorescence Microscopy and AFM

Imaging cells using a combination of AFM and optical techniques provides a better understanding of the structural constitution of their surface and helps with the functional labeling of specific components.

This article demonstrates a few examples of combining AFM and optical microscopy techniques for epi-fluorescence, DIC and phase contrasts with a JPK NanoWizard^® AFM integrated on a Zeiss Axiovert 200 inverted optical microscope.

In this analysis, hydrogen peroxide (H₂O₂) was used to promote apoptotic blebbing in the cells. Integrating AFM with optical microscopy not only helps in understanding the changes in the surface of the membrane during vesicle formation, but also helps in investigate the underlying structure of the actin cytoskeleton.

Mouse Melanoma Cells labelled with YFP

A mouse melanoma secondary cell line (B16) was used to better illustrate the concept. A yellow-fluorescent protein (YFP) was used to label the actin in the B16 cell line. The cells exiting in the cell line are not stationary, and hence, do not stick properly as they grow. As a result, the cells were fixed prior to imaging and then treated with 2% glutaraldehyde for a period of 45 seconds to ensure that the glutaraldehyde does not contribute fluorescence. Next, the cells were fixed for a period of 20 minutes in 4% paraformaldehyde.

Figure 1 shows the optical images of a B16 cell. Here, the top picture is a phase contrast image, which was acquired by means of a 20x water immersion objective, while the YFP fluorescence of an area close to the cell edge can be observed in the bottom panel. A 63x oil immersion lens (NA 1.2) was used to acquire the latter image

Figure 1. Optical images of a mouse melanoma cell. Upper image –phase contrast, 20x water immersion lens. Lower image –fluorescence from YFP-labelled actin, 63x oil immersion lens. The region covered by the fluorescence image is marked with a box in the upper image.

The actin monomers are labeled by YFP, which means both the actin filaments and cytoplasm make a contribution to the fluorescent signal. Dulbecco’s PBS solution was used for AFM imaging of the cells. Irregular contact mode was employed in this imaging technique with triangular silicon nitride cantilevers that had a spring constant of 0.3 N/m. The AFM sensor moves back and forth across the sample surface when in intermittent contact mode, and the scanner is controlled using the oscillation amplitude.

With this mode, topography images of the surface of the cell as well as images of the amplitude signal are obtained. The latter highlights the intricate details of the surface of the cell.

Figure 2. B16 mouse melanoma cell imaged using JPKNanoWizard^® mounted on Zeis Axiovert 200 inverted opticalmicroscope. Upper panel – YFP fluorescence. Lower panel –montage of two AFM amplitude signal images of the same region. Arrowheads – examples of actin filaments seen in bothpanels. Ring – example of rounded features on the cell surface, seen only in the AFM images. Scale bar 2µm in both panels.

A comparison of AFM and optical images of the B16 mouse melanoma cell edge is shown in Figure 2. A YFP fluorescence image is seen in the top image, and a montage of a couple of AFM amplitude signal images of the same area is seen in the bottom image. As the tip scans across the cell surface, the AFM amplitude signal corresponds with the topographic gradient.

This means the images seem to be shadowed, revealing finer details of the surface. The sample area is marked similarly as in Figure 1. To facilitate better comparison with the AFM images the fluorescence image was rotated. In both set of panels, the scale bar measured 2 µm.

In the fluorescence image, both the actin filaments and the cytoplasm are weakly labeled since all actin was labeled by the YFP labels. Both optical fluorescence and AFM images revealed the larger actin filaments. Whilst other details of the surface of the cell can be seen in the AFM images, they are not seen in the fluorescence image. This was the case for the rounded features highlighted in a white ring (Figure 2). On the left side of the AFM image, surface structure appears differently from the layout of the actin cytoskeleton beneath them.

Fibroblasts

For further analysis, glass coverslips were used for growing mouse dermal fibroblasts. To fix these cells, 2% of glutaraldehyde was used for a period of 45 seconds, followed by 4% of paraformaldehyde for about 20 minutes, similar to the mouse melanoma cells. Fluorescent labeling was applied to the actin filaments and this was achieved using phalloidin-FITby to incubate the cells overnight at 4 °C temperature.

Once the cells have been imaged in Dulbecco’s PBS solution, a FITC filter set and a 63x oil immersion lens were used to collect fluorescence images.

Figure 3. Optical image of a mouse dermal fibroblast cell. Fluorescence from FITC-phalloidin labelled actin. The area marked with the white box is shown in Figure 4

An optical image of a separated fibroblast is shown in Figure 3, displaying the fluorescence from the YFP-labeled actin filaments. When compared to the YFP in Figure 1, the actin is more intensely labeled, and the fibroblast’s cytoskeleton is also more developed in comparison to the melanoma cell. As a result, the actin cables are clearly observed in the fluorescence image.

Figure 4. Comparison of optical and AFM images of afibroblast using JPK NanoWizard^®. A: fluorescently labelled actin (phalloidin-FITC). B: montage of two AFM amplitudesignal images. C: montage of two AFM topography images.Scale bar 2µm in all, height range of AFM image is 1.8µm.

In Figure 4, the images reveal the area of the cell edge highlighted with a box (Figure 3). In order to enable an easier comparison with the AFM images of the same region, the optical fluorescence image (A) was turned around. The delicate protrusions at the cell edge highlighted with white arrowheads are not clear in the fluorescence image (A), but can be resolved in the AFM images (B, C).

Other features such as the large feature labeled with a black arrowhead at the surface of the cell can be seen only in the AFM images. Here, both optical and AFM images provide a host of complementary data regarding the submembranous structures and cell surface.

The AFM images not only provide 3D data regarding the cell shape, but also reveal fine details at the surface of the cell. Both structural and optical data helps in detecting cellular components underlying specific structures of the cell membranes.

Treated Fibroblasts

The apoptotic effects of H₂O₂ on fibroblasts has been used to show how different data from combined microscopy methods can be effectively applied. H₂O₂ of low concentration was used for treating the mouse dermal fibroblast cells to trigger a cell death cascade, and 100 µM H₂O₂ was used to incubate the treated cells for a period of 15 minutes, 30 minutes and an hour.

The cells were then fixed followed by fluorescent labeling of the filamentous actin, as illustrated.

Figure 5. Combination of optical and AFM images of fibroblasts treated with hydrogen peroxide. Image series A-C shows control cells (no H₂O₂ treatment), D-F shows cells that have been treated with 100µM H₂O₂ for 30 minutes before fixation, and G-I shows cells that have been treated for one hour before fixation.

In Figure 5, epi-fluorescence from FITC-phalloidin labeled actin filaments (B,E,H) and phase contrast images (A,D,G) reveal the same scan area for each case. Also, for a each case, AFM topography images (C,F,I) are 20 µm x 20 µm scans for the area labeled with a box in previous optical images.

In both cases, the overall height scale for C and F was found to be 2.3 µm and the overall height scale in I was noted to be 9 µm. In contact mode, AFM imaging was performed with unsharpened DNP cantilevers with a spring constant of  0.06 N/m.

The results summary is shown in Figure 5 for control cells without H₂O₂ treatment (image series A-C) and for cells treated with H₂O₂ for a period of 15 minutes (image series D-F) or 30 minutes (G-I). In images along individual row, different imaging methods are observed for the same cell sample. The same sample area is seen in the epi-flurorescence (B, E, H) and phase contrast (A, D, G) images.

The scan region for the AFM images was observed to be 20 x 20µm in individual case. In all three images (A-C), distinct actin cables can be observed for the control cells that were not treated with H₂O₂. Vesicles can also be viewed in the phase contrast image, and these structures correspond with the protrusions on the surface of the cell in the AFM topography images, for instance the one circled in C.

The actin filaments begin to retract from the cell boundaries following treatment with the 100 µM H₂O₂ for a period of 30 minutes. Through the fluorescence image (E), a slight staining was seen at the edge of the cell; however, the AFM image (F) clearly reveals the finer details.

At the edge of the cell (F), a relatively flat membrane region is present that features a fine mesh structure supporting it. However, the distinct actin fibers do not extend to the cell boundary. In the case of cells treated with H₂O₂ for 60 minutes, rearrangement of the actin cytoskeleton as well as a significant change is seen in cell morphology. The cells become rounder, blebs begin to form on the surface, and the actin cables are minimized drastically.

The blebs form bulk 3D structures: the overall height scale for the AFM images in C and F is measured to be 2. 3µm, and the height range for the image (I) is measured to be 9µm. The fluorescence image (H) reveals the actin cytoskeleton beneath the bleb structures.

Conclusion

Combining AFM with different optical imaging methods opens up many possibilities for analyzing cell morphology and responses. These combinations of methods extend the scope of data that can be acquired regarding function and structure of cells. With these unique approaches, certain molecules on the surface of the cell or the underlying cell components can be imaged to study their association with the morphology of specific structures, including the cell surface.

Such experiments can be performed either on living cells or under different physiological conditions, and provide a range of possibilities for studying the link between cellular function and physical structures.

This information has been sourced, reviewed and adapted from materials provided by JPK Instruments AG - Scanning Probe Technologies.

For more information on this source, please visit JPK Instruments AG - Scanning Probe Technologies.