2D polarisation single-molecule spectroscopy

This technique has the following projects (and possibly other techniques) related to it:

• Polarisation single molecule spectroscopy of conjugated polymers
• Single molecule spectroscopy of bioobjects

The idea of polarisation single-molecule spectroscopy consists in modification of the basic version of Single-molecule spectroscopy setup with two rotating polarisers (more precisely, a λ/2 plate in the excitation channel and a transmission/reflection polarisation analyser in the fluorescence channel). This allows to record the fluorescence intensity of a single molecule as a function of two arguments: excitation polarisation plane orientation angle φ_ex and allowed emission polarisation plane orientation angle φ_em. More thoroughly the experimental setup and measurement technique is described on the Polarisation single-molecule spectroscopy setup page. A summarizing scheme of the setup is given below in Fig.1:

Fig.1. Experimental setup. Back to top.

The raw experimental results of this technique are "movies" of the samples with fluorescing molecules. The sample image is split into two, corresponding to the parallel (I_||) and perpendicular (I_⊥) polarisation components. These movies are recorded while rotating the polarisers. To trace the polarisation angles for every frame of the movies, synchronized flashes of light were projected on the CCD detector and recorded into the movies, too. Thus, the next step after the experiment was to extract these flashes and the fluorescence intensity traces of the molecules. This was achieved using home-made software BrightStat (written by Oleg Mirzov).

Fig.2. Software for single-molecule fluorescence signal extraction. Back to top.

The fluorescence intensity (I_||(n) and I_⊥(n), where n is the frame number) and polarisation signal traces obtained in the previous step are still not easy to interpret. So, the next step is to convert them to something more readable.

Fig.3. Experimental single-molecule fluorescence signal traces. Back to top.

The traces from the previous step were chopped into pieces that cover the "phase space" for φ_ex and φ_em from 0 to π. These pieces were replotted as 2D color plots I_||(φ_ex,φ_em), I_||/I and I(φ_ex,φ_em), where I=I_||+I_⊥. After that the series of plots were summed up to produce averaged 2D plots with higher signal to noise ratio.

Fig.4. Experimental 2D plots. Back to top.

These polarisation 2D plots are much more informative and easy to look at than the raw intensity traces. To see the meaning of different shapes on the plots it is helpful to study the following simple examples (Fig.6). The examples concern simple model systems of a few linear dipole absorbers/emitters with or without energy transfer between them.

Fig.6. Model results for some special cases. Back to top.

It was discovered that any experimental 2D plots obtained with this technique can be successfully simulated as 2D plots of a simple model system shown in Fig.5.

Fig.5. Formal model for describing polarisation properties of the molecule and simulating 2D plots. Back to top.