Combining fluorescence and atomic force microscopy
Integration of optical and atomic force microscopy (AFM) is a powerful tool to obtain comprehensive information on a variety of samples. Especially combining fluorescence microscopy and the AFM technique provides complementary information: the fluorescence about the location of labelled molecules not detectable by transmission light microscopy, the AFM finally on the topology of the sample.
The design of the NanoWizard®II AFM (JPK Instruments, Berlin) allows its integration into inverted optical devices providing different optical techniques like epifluorescence. The use of the DirectOverlay feature available for the JPK SPM software enables real optical integration, not only by detecting the position of the cantilever within the otpical image but also by getting rid of optical distortions caused by the use of lenses. DirectOverlayTM can be performed using a wide range of cameras or even with confocal techniques, but the most convenient way is to use cameras that can be controlled by the SPM software. For basic applications like phase contrast or DIC the DFK 31AF03 camera from Imaging Source that is usually provided by JPK is a reasonable solution. For advanced applications as sensitive fluorescence techniques JPK offers software integration of Jenoptik cameras as the
ProgRes® MFcool and
ProgRes® CFcool.
In this report the combining of sensitive fluorescence detection and AFM, and the way how these techniques can complement each other are described by different applications.
Fluorescence detection and high resolution AFM imaging of nano structures
Imaging structures in the nanometer range as beads or quantum dots is an exemplar for the use of AFM technique. To get a first impression of the composition of the sample and to localize the particles or rather to find the optimal sample position, sensitive fluorescence microscopy emerges as a useful tool.
In the first example 40 nm red fluorescent spheres were deposited on a mica substrate. The spheres were focused and an optical image taken using the
ProgRes® MFcool (exposure time 1 sec). After the optical system was calibrated to the SPM software using the DirectOverlayTM feature an interesting scan region was specified. AFM scanning of this region revealed clusters of beads, appearing as high fluorescent spots within the optical image, but also single beads showing only weak fluorescence.
In the next example sensitive fluorescence detection was used to localise quantum dots deposited on mica in a low concentration to finally resolve their topology and composition by AFM imaging. In low concentrations quantum dots are almost impossible to be visualised by eye. Then it helps to use sensitive fluorescence detection as shown here using the monochrome camera
ProgRes® MFcool.
Since relatively long exposure times (around 7 sec) are necessary to take high quality images the use of the binning option is very helpful. The light information of several pixels is summarised and the exposure time automatically decreased to keep the chosen brightness of the raw image constant. If for instance 3fold binning is chosen the resolution decreases by summarising squares of 3x3 pixels. But the exposure time also decreases to the same extent, allowing to focus the right plane. When the optimal focus and sample region is found the maximum resolution (no binning, low gain, high exposure time) can be used to get high quality images. To take high resolution and quality images it is recommended not to use binning for such small structures like quantum dots, where the size of the structure is similar to the pixel size. When using the binning option anyway, it is essential to use the same binning for the calibration images, since the overlay feature is based on the correlation of the pixel position of the cantilever tip to the pixels within the snapshot of the sample.
The blinking nature of quantum dots makes it difficult to localize all dots in one region at the same time. However, using optics it is possible to distinguish between areas of high and low quantum dot concentration and the optical image helps to orientate and choose an adequate scan region.
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Imaging cellular structures
To localize cellular structures down to the molecular level fluorescence labeling is an indispensable technique. Topographic information of the corresponding structures or the cellular compartments hosting the structures is another refinement that can be provided by AFM imaging.
Here L929 fibroblasts and MC3T3 osteoblasts were stained for f-actin using AlexaFluor546-and FITC-phalloidin respectively. L929 cells were grown and fixed on glass slides, the MC3T3 cells on coverslips for the use with the CoverslipHolder. To prevent photo bleaching, focus and adequate sample position were adjusted using fluorescence detection in binning mode. High quality images for the overlay were taken before calibration images. Applying phalloidin staining the actin stress fibers could be visualized using fluorescence microscopy. Additional topographical information could be derived by AFM imaging, also revealing the fine structure of the filaments and thus complementing the optical image.
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Conclusion
As shown in this report, combining sensitive fluorescence and atomic force microscopy can be provided by integrating the JPK NanoWizard®II AFM and the
ProgRes® MFcool into the fluorescence setup. The DirectOverlay feature allows for real optical integration and provides optical and topographic information within one region. Fluorescence labeled cellular components like the cytosceleton or trafficking molecules can be optically tagged and the morphology of the corresponding region characterised by AFM imaging. Investigating structures in the nanometer range that cannot be optically resolved, fluorescence can help to roughly detect the structures and thus serve as an orientation tool for the search for interesting regions to be finally resolved by AFM imaging.