Single Molecule Blinking Traces

At the end of my last post, I mentioned that I had taken a break from trying to measure the fluorescence lifetime of individual molecules of the organic dye Rhodamine 560, since I was unable to get a lifetime curve with a high enough signal to noise ratio at single molecule concentrations. Instead, I returned to a technique called single molecule blinking, which allows us to observe changes in emission intensity while an individual molecule is under continuous excitation.

Establishing the appropriate settings for this phase of experimentation was not as difficult as expected, although I had to switch from using the laser in pulsed mode to using in continuous wavelength (CW) mode. This was because another project in the lab had used the laser in CW mode when studying another organic dye. Although the settings were not identical, they gave me a starting point, which sped up the control tests significantly. Once the laser power settings were established, a clear “image” of the dye molecules on a glass slide could be generated by the detection of photon counts at each point. From these images, I could set up the laser to focus on each potential molecule to observe emission patterns.

Over the course of about a week and a half, I gathered around 80 blinking traces. Using a program in Matlab called change point detection (CPD), I could input a threshold to determine whether each blinking trace showed appropriate intensity changes to be considered a “good” molecule. Finally, I used another Matlab code to try and fit the molecules’ behavior to various mathematical functions.

Other members of my group have gone through this process with other dyes, from establishing experimental settings to statistically analyzing the data. In the past, I have contributed to small parts of the process, but getting to go through the steps more independently was a very helpful experience.