Results and Discussion

The obtained raw data was processed as described above in the methods section. The step size for polar and azimuthal angles for calculating the model patterns was chosen as 5^◦ and then pattern matching was performed in order to obtain the orientations of each single molecule. Thereafter, MIET calibration curves were calculated for a dipole oriented at various polar angles in a thin polymer assuming a polymer thickness of 20 nm, and the height based of each single molecule was obtained. Figure 4.24 shows the calibration curves as well as the height of the molecules for the fitted polar angles (0^◦, 5^◦, ..., 90^◦). The density of the molecules, as can be seen from this figure, is not uniform over the entire thickness of the polymer film. Also, the number of molecules for each orientation is not constant, as can be seen from the density of points on each curve in the figure. Therefore, we plotted the average orientation as a function of the axial position, shown in figure 4.24. The plot shows that close to the interfaces the dipoles are orientated almost parallel, whereas in the middle, the dipoles assemble in all possible orientations. The plot also shows an inhomogeneous distribution of molecules across the polymer.

CHAPTER 4. SM ORIENTATION 4.2. SMMIET WITH RADIALLY POLARIZED EXCITATION

Figure 4.22: Left column shows the intensity image with all the photons recorded in the top and the patterns recognized in the bottom, whereas the right column shows the same for the case of time-gated photons. The reconstructed image with the patterns for the time-gated analysis has been enhanced by 1.5× to make the weak patterns more visible. Comparing the bottom figures, 4 more molecules were recognized and one artifact was removed in the right image after gating the photons.

The scale bar marks a length of 2µm. The plots in the right-top figure shows the intensities in the pixels corresponding to the same line in raw data with and without time-gating. The signal-to-noise enhancement was roughly 2 times after the gating.

There are several reasons for observing such a distribution of molecules. First, since the molecules were introduced following the casting of the polymer film by spin-coating, one would expect that the concentration of the molecules is low in the bottom layers of the polymer film. On the other hand, the top of a spin-coated polymer is not smooth and the height variations can be in the order of±2 nm. This might explain the presence of only a few molecules higher than 12 nm. Also, the molecules close to the gold surface (in the bottom of the polymer) are quenched more than the molecules at larger distances, making them dimmer. Therefore, one has poor signal-to-noise ratios for the molecules close to the bottom interface, which makes it difficult for the pattern matching algorithm to detect, contributing to the overall distribution that we observe here. Moreover, the relative intensity of a parallel dipole is higher as compared to a vertical dipole in the bottom. This can be seen from figure 4.25 which shows the collection efficiency of parallel and vertical dipoles as a function of height which was calculated by the

4.2. SMMIET WITH RADIALLY POLARIZED EXCITATION CHAPTER 4. SM ORIENTATION

Figure 4.23: MIET calibration curves of a Rhodamine 6G molecule’s lifetime at various heights in a 20 nm thick PVA polymer on top of a layered substrate for it’s various orientations. The details of the substrate are described in the methods section above. Also the distribution of the axial positions of the molecules together with their orientations is shown along these curves.

fraction of the energy propagating into the collection cone of a 1.49 N.A. objective, using equation (2.145). Based on the trend seen in the curves, the chance of detecting a photon from a vertical dipole is low at the bottom of the polymer film which can be a contributing reason for the observed average orientation distribution. Although these might be a few reasons to explain the distributions seen in the figure, a complete understanding of the distribution and the orientations of the molecules requires modeling the diffusion and transport of the dye molecules into the pores of the thin film in the presence of centrifugal forces which is beyond the scope of this thesis.

There are several limitations for performing smMIET experiments using a radially polarized laser scanning. Orientation estimation using pattern matching algorithms provides reasonable results only when there are no artifacts such as blinking/bleaching or any overlap of intensity patterns. This limits the selection of dyes and the conditions of experiments in order to ensure photostability. Since each single molecule pattern spreads over an area of ∼ 1µm×1µm, the concentration of fluorophores must be low enough in order to avoid any such overlap. Therefore, this technique is applicable only for a sparse distribution of labeled entities. The MIET calibration curves can be calculated, as shown above, for fixed dipole orientations or for the case where the dye has free rotational freedom and the rotational diffusion time is shorter than the average fluorescence lifetime so that it can be assumed to be isotropic emitter. Combining scanning with radially polarized excitation with superresolution techniques such as STORM or PALM means that one is limited to perform scans over a small area in order to achieve a fast frame

CHAPTER 4. SM ORIENTATION 4.2. SMMIET WITH RADIALLY POLARIZED EXCITATION

Figure 4.24: The distribution of Rhodamine 6G molecules and their average inclination as a function of height above the surface.

Figure 4.25: Total collection efficiency of a parallel and vertical dipole as a function of height above the SiO₂ spacer in a thin polymer film. The values represent the amount of power emitted into the collection cone of a 1.49 N.A. objective normalized to the total emission power of a dipole in free space.

rate and resolve multiple molecules fluorescing randomly. However, a major challenge for scanning an area repeatedly stems from the positioning inaccuracy and the drift one induces during scanning with a piezo stage. An probable solution to this problem is to use galvo scanning mirrors before the objective or two fast electro-optic deflectors before the linearly polarized laser is converted into a radially polarized beam. However, this