The overall goal of this analysis of Blinking Surface-enhanced Raman Scattering is to investigate the behavior of single molecules on metal nano-particle surfaces. This method can help answer key questions in interface and coral science about working behavior of solid liquid interfaces The main advantage of this technique is that many data can be gathered because approximately a dozen blinking spots can be simultaneously observed in one video. To begin this procedure, wash two glass microscope slides with liquid detergent and tap water.
Rinse the slides with distilled water and allow them to air dry. Next, apply an aqueous solution of 0.1 percent poly-l lysine to one dry slide. Allow the solution to sit on the slide for two to three seconds.
Then, use a compressed air blower to remove the excess solution and dry the slide. Next, apply a suspension of colloidal silver nanoparticles to the slide. Allow the suspension to sit for two to three seconds.
Dry the slide with compressed air. Then, use a hydrophobic barrier pen to completely enclose a 26 millimeter by 26 millimeter area on the slide. Allow the barrier to dry.
Drop distilled water onto the slide to fill the enclosed area. Cover the slide with the second clean dry glass slide to prevent water evaporation. Place the slide assembly on the sample stage of the inverted microscope.
Illuminate the slide with white light through a dark field condenser. Focus on spots of various colors by changing from a 2x objective lens to a 60x objective lens. Next, use a diode pumped solid state continuous wave 532 nanometer laser, a band pass interference filter, and a lens to illuminate the sample with an attenuated beam at a 30 degree angle relative to the sample surface.
Move the laser illumination to the center of the view, and slightly adjust the z direction of the sample stage, to refine the focus on the spots. Observe the aggregates as monotone spots against a uniform background. Next, turn off the laser, and insert a long pass-edge filter after the objective lens.
Illuminate the sample with the laser at a 30 degree angle relative to the sample surface, through the interference filter and the external lens. Move the stage in the x and y directions to find the blinking spots. Then, use a deep cool digital CCD camera with a frame time of 61 to 120 milliseconds to acquire a 20 minute long video of the spots.
To begin the analysis, open the acquired video in the CCD camera software. Click and drag to select areas with and without blinking spots. Perform a temporal analysis to derive a signal intensity time profile for the spots and dark areas.
Acquire profiles for all blinking spots and dark areas of interest in this way. Then, generate probability distributions for bright and dark events relative to their durations. Multicolored blinking spots were observed from SERS of silver nano aggregates, prepared with poly-l lysine coated slides.
The blinking signal is attributed to the random walk single molecules at silver nano aggregate junctions. The signals from the single nano aggregate showed varying intensities over time, unlike the blinking fluorescence of a quantum dot. The threshold for bright events was set to three standard deviations above the baseline intensity, and the bright and dark event durations were determined for each spot.
The probability distribution of bright events was plotted against event duration as a line and a log log graph, whereas the probability distribution of dark events was plotted against duration as a curve. As the dark event durations were generally longer than the bright even durations, the power law exponents for bright events tended to be smaller than those for dark events. Shorter truncations times for dark events indicated a quicker molecular random walk, a higher energy barrier from a non-emissive state to an emissive state, or a combination of both.
After watching this video, you should have a good understanding of how to observe and analyze blinking SERS phenomena.
