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.

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.