Confined diffusion and dynamics of single molecules
Abstract
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J. Hohlbein and M. Steinhart
Several approaches to reduce the detection volume below the confocal volume have been reported in literature. We present a conceptually different method, which reduces the effective detection volume by an order of magnitude in a conventional confocal detection scheme by infiltrating solutions containing probe molecules into highly ordered porous materials. We use ordered porous alumina with a pore diameter of 25 nm and a pore depth of 30 µm, which we mount on the microscope in such a way that the pores are parallel to the long axis of the focal volume (Fig. 2). Within the geometric confinement of the pores, quasi one-dimensional diffusion of the fluorescent molecules through the focus of the confocal microscope occurs. The probe molecules within the pores stay up to 20 times longer within the detection volume than freely diffusing molecules.
Figure 1. Schematic diagram of a standard Scanning Confocal Optical Microscope. Additionally, pulsed diode lasers can be used in order to probe the fluorescence lifetime of the molecules.
Figure 2. Schematic diagram of a porous alumina membrane mounted on a Scanning Confocal Optical Microscope. The microscope focus can be adjusted to an area within the membrane. The membrane can be moved along the z-direction (optical axis and long axis of the membrane pores) using a linear actuator.
Figure 3 shows transient intensity traces of the dye Alexa Fluor 488 confined to self-ordered porous alumina (red line) and in free solution (blue line). The mean intensity is lowered within the membranes, indicating that the average number of dye molecules in the focal volume is reduced compared to the free solution. The intensity traces of 10nM Alexa Fluor 488 within the pores show a pronounced burst-like behaviour that can be attributed to single dye molecules passing the focal volume. The curves in Figure 4 are autocorrelation functions obtained from Fluorescence Correlation Spectroscopy (FCS) measurements, representing the diffusion times of single fluorescent molecules and the mean particle number within the focal volume. The amplitudes of the curves are inversely proportional to the mean particle number (the larger amplitude the lower the mean particle number). The upper four curves show measurements for different concentrations of Alexa Fluor 488 inside self-ordered porous alumina, the lower four curves measurements in free solution, respectively. The black lines represent the corresponding fitting functions, assuming a one-dimensional diffusion within the membrane and a three-dimensional diffusion within free solution, respectively. The mean diffusion time is up to 20 times longer within the membranes.
Figure 3. Intensity traces of the dye Alexa Fluor 488 within the membrane (red line) and in free solution (blue line). The mean intensity is lowered within the membranes, indicating that the average number of dye molecules in the focal volume is reduced. The intensity traces of 10nM Alexa Fluor 488 within the pores show a pronounced burst-like behaviour that can be attributed to single dye molecules passing the focal volume.
Figure 4. Autocorrelation functions obtained from Fluorescence Correlation Spectroscopy (FCS) measurements of Alexa Fluor 488 for different concentrations. Upper four curves: measurements inside nanoporous alumina; lower four curves: measurements in a free solution. The black lines represent the corresponding fitting functions, assuming a one-dimensional diffusion within the membrane and a three-dimensional diffusion within free solution, respectively. The mean diffusion time is up to 20 times longer within the membranes.