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Simultaneous atomic force and fluorescence measurements on the nanoscale

Combining time-resolved photon counting, scanning optical techniques, and atomic force microscopy provides new tools for single-molecule investigations.
30 January 2007, SPIE Newsroom. DOI: 10.1117/2.1200701.0584

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).

Figure 1. Combined setup for simultaneous detection of fluorescence and force. An atomic force microscope (AFM) and inverted optical microscope are placed on a vibration isolation platform. The AFM can be positioned using a microscope stage and piezo translators. The sample plate can also be moved independently. A pulsed laser provides optical excitation. Either four-channel single-photon counting detectors or a spectrograph equipped with a CCD camera detects the fluorescence. L1, L2: Lenses. DB: Dichroic beam splitter. PB: Polarizing beam splitter. IF: Interference filter. APD: Avalanche photodiode. MFD: Multiparameter fluorescence detection.

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.

Figure 2. Combined force and fluorescence data obtained in a simultaneous experiment. The force curve (blue line) shows a single DNA molecule being stretched and ruptured. The fluorescence intensity curve exhibits an abrupt signal increase upon DNA rupture (red curve). The concentration of SYBR Green I fluorescent dye in the buffer solution is 0.6–1 µM. The excitation power is ~0.5kW/cm2.

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.

Dr. Suren Felekyan and Carl Sandhagen are acknowledged for their help with the realization of this project. This work is supported by the BMBF (Bio-Future Award, Grant No. 0311865) and Volkswagen-Stiftung.

Alexander Gaiduk, Ralf Kühnemuth, Matthew Antonik and Claus A.M. Seidel
Heinrich-Heine-University Duesseldorf
Institute of Physical Chemistry II, Germany