Discussion and Outlook

The error of the observed single-molecule lifetime values is less than 0.1 ns, which corre-sponds to an axial localization accuracy of less than 2 nm for horizontal dipoles. Since the error of lifetime variation follows Poisson statistics, a reduction in the spread of the lifetime distribution can be achieved by increasing the number of collected photons per molecule, for example, by using suitable oxygen-scavenging protocols to reduce the rate

CHAPTER 3. SMMIET 3.2. PROOF OF PRINCIPLE EXPERIMENTS

Figure 3.10: An illustration showing the application of smMIET for structural biology. The pro-tein/macromolecule is labelled site-specifically with two labels much similar to FRET. Independent lifetime and orientation measurements are perfomed to obtain the heights of the two labels on each immobilized complex. Carrying out simple statistics, one can derive the distance between the two sites.

of photobleaching [125]. In our current experiments, we detected on average 369, 767, 1002, and 1031 photons per individual molecule for spacer thickness values of 20 nm, 30 nm, 40 nm, and 50 nm, respectively.

The physical basis behind smMIET is the energy transfer from the excited molecule to surface plasmons in the metal and it is thus quite similar to FRET. Unlike FRET where three relative orientation angles between donor emission and acceptor absorption dipoles are needed, which are inaccessible by using any independent measurement, here we need the out-of-plane orientation α of the emitting molecule with respect to the metal surface which can be obtained using the methods mentioned above. Moreover, the distance range over which smMIET works is much larger than FRET and as can be seen from figure 3.6, goes upto 100 nm. Therefore, in order to find its application in structural biology, one would need both the lifetime and orientation for nanometer-precise distance measurements. In our current measurement scheme, the fundamental limitation is that we have no means of measuring the orientation (polar angle) of the molecule simultaneously with the intensity and lifetime. As can be seen from figure 3.6, the relation between distance and lifetime is strongly orientation dependent. There are several options to achieve this, including defocused imaging [120, 126] scanning with radially polarized light [76], or detecting separately sub- and supercritical fluorescence emission [127]. However, all these methods require significant extensions and/or mod-ifications of a conventional confocal laser scanning microscope, some of which we will investigate in the forthcoming chapter.

In combination with such orientation measurements, smMIET can determine dis-tance values of single molecules from a surface with nanometer resolution. Already with our nonoptimized (in terms of photobleaching) measurements we could estimate the distance with accuracy higher than 2.5 nm. Although smMIET achieves this reso-lution only along one single axis, this method will open new fascinating possibilities for structural biology. For example, for determining the intramolecular distance between two fluorescent labels in a macromolecule, as shown in figure 3.10, one can envision

us-3.2. PROOF OF PRINCIPLE EXPERIMENTS CHAPTER 3. SMMIET

ing smMIET to measure the absolute height differences between both labels for a large number of macromolecules immobilized on a surface. Next, one could apply simple statistics to obtain the absolute distance between the labels.

In the forthcoming chapters we will enlist a few existing techniques for measuring the orientations of single molecules. The main focus will be to point out the most feasible method for obtaining the orientations together with the lifetime information in order to extend smMIET as a versatile tool for structural biology.

4 Single-Molecule Transition Dipole Imaging

Abstract

An electronic transition between two molecular energy levels is a redistribution of tron density over the molecule’s structure following the interaction with the local elec-tromagnetic field. Molecules that have a preferred direction for such a redistribution show a classical dipole behavior and this direction defines the excitation or emission transition dipole moment. Almost all organic dye molecules behave as electric dipole oscillators. In this chapter, we introduce two well-known methods, one for imaging the excitation transition probability, and the other for the emission transition probability of single emitters. Both of these methods are used for determining the complete three di-mensional orientations of these two vectors in space. We apply them for the study of the excitation and emission properties of Carbon Nanodots (CNDs) that are novel fluores-cent probes gaining popularity in bioimaging. We show that the CNDs are single dipole emitters similar to organic dyes. Thereafter, we present the first experimental method for determining the geometry of the two transition dipoles and their three-dimensional orientationssimultaneously for each individual emitter. This directly gives us the angle γ in between both the vectors. We perform experiments on two dye molecule species, and the results show a non-negligible γ. We speculate that this arises due to a signifi-cant rearrangement in the backbone structure of the molecule following the excitation as a result of vibrational relaxations before the emission occurs. The feasibility of these two methods for smMIET experiments is also discussed.

CHAPTER 4. SM ORIENTATION

Parts of this chapter and some figures have been published in the following journal articles:

1. Ghosh,S.; Chizhik, A.M.;Karedla, N.; Debaliuk, M.O.; Gregor,I.; Schuhmann,H.;

Seibt,M.; Bodensiek,K.; Schaap, I.A.T.; Schulz,O.; Demchenko,A.P.; Enderlein,J.;

Chizhik,A.I. “Photoluminescence of Carbon Nanodots: Dipole Emission Centers and Electron-Photon Coupling” Nano Letters 14 5656-5661 (2014)

2. Karedla, N.; Stein, S.; H¨ahnel, D.; Gregor, I.; Chizhik,A.I.; Enderlein, J. “Si-multaneous Measurement of the Three-Dimensional Orientation of Excitation and Emission Dipoles” Physical Review Letters115 173002 (2015)