Simultaneous atomic force and fluorescence measurements on the nanoscale
Single-molecule studies in chemistry, biology, and medicine frequently start with confocal optical microscopy in solution but then proceed to the imaging of fluorescence, or the visible light emitted, in complex biological samples. It is also common to combine investigations of fluorescence and atomic force microscopy (AFM), an imaging technique that uses a cantilever with a sharp tip to probe a surface.
The advantages of these combinations are straightforward. Confocal optical microscopy provides high optical sectioning within a sample. Time-correlated single-photon counting (TCSPC), a method of measuring fluorescence combined with optical imaging, delivers information about several fluorescence parameters (e.g., fluorescence decay and anisotropy). AFM combined with fluorescence detection and optical imaging allows changes in fluorescence and mechanical properties to be correlated with one another. The result is unique information about the structure and behavior of complex biological objects.1,2 For example, a molecule under controlled tension can be probed with fluorescence techniques to determine molecular orientation or photophysical activity. At the same time, proper labeling of stretched molecules makes it possible to study subnanometer intramolecular displacements using fluorescence resonance energy transfer between two fluorophores.3
Other researchers have detected changes in single-molecule fluorescence while nanomanipulating DNA-dye constructs with optical tweezers.4 In contrast to optical or magnetic tweezer experiments, the optics/AFM combination provides access to a larger force range and enables sample manipulation on a much larger length scale. Recently, Hards et al. achieved simultaneous lateral manipulation and wide-field fluorescence imaging of immobilized DNA molecules.5
In our lab we created a unique experimental setup with two microscopes placed together6 (see Figure 1). Advanced TCSPC allows us to detect the fluorescence signal with picosecond time resolution, and AFM provides subnanometer positioning control. We aim to localize an object of interest with wide-field optical imaging and then perform simultaneous force and multiparameter fluorescence detection with high time resolution (simultaneous force and fluorescence spectroscopy, or SFFS).
We characterize the influence of the AFM cantilever optical signal using multiparameter fluorescence information that is recorded at each point of the cantilever raster scan image. With this method, not only can we compare the intensity of the optical signal from the cantilever with the signal from a single dye molecule under the same experimental conditions, but we can also reconstruct fluorescence lifetime and anisotropy ‘maps’.6 Cantilevers made of Si3N4 are more fluorescent than those composed of Si, but they also possess more suitable mechanical properties for sensitive force measurements. Fluorescence and force spectroscopy measurements are synchronized, thus simultaneously monitoring fluorescence and force.
We applied the SFFS method to single DNA molecules attached between the surface and the AFM cantilever.7 Experiments were performed in buffer solution containing SYBR Green I fluorescent dye, which binds to DNA molecules. Figure 2 illustrates the utility of SFFS measurements: the fluorescence signal changes dramatically when a single DNA molecule mechanically ruptures. We can thus correlate the fluorescence signal with changes in the mechanical properties of a single DNA molecule.
In conclusion, we have performed the first simultaneous atomic force spectroscopy measurement of a single macromolecule with confocal fluorescence detection. Further work on these techniques would allow us to localize objects of interest for SFFS experiments by means of simultaneous AFM and confocal fluorescence imaging. That would enable us to address structural features precisely.
