|Used in:||2D polarisation single-molecule spectroscopy|
O. Mirzov, R. Bloem, P. R. Hania, D. Thomsson, H. Lin, and I. G. Scheblykin, "2D polarisation single molecule imaging of multichromophoric systems with energy transfer", Small, 2009, 5, 1877
Measuring of intensity and polarization of a single molecule fluorescence excited by differently polarized excitation gives us insights to internal organization of the studied molecule and its orientation in space. Changing of the excitation light polarization state can be done using λ/2 and λ/4 waveplates, while the fluorescence polarization can be probed by a linear polarization filter (analyser) placed in front of the detector.
Let us consider a simple chromophore (e.g. an organic dye) in which the absorption and emission dipoles are oriented in the same direction. We can obtain information about orientation of the transition dipole by measuring the fluorescence polarization (fig. 3, scheme 2). For the same purpose scheme 1 can also be used where anisotropy of absorption is measured. In the last case the orientation of the chromophore dipole is given by the excitation polarisation orientation when the chromophore absorbs the most leading to maximal fluorescence intensity.
However, usually we are interested in more complex systems like polymers and multi-chromophore nanoparticles. A polymer chain fluorescence can be excited by light with different polarization state (linear, circular etc.). The fluorescence emitted by the chain can be polarized or depolarized or something in between. The degree of polarization (referred to as modulation) of the absorption will depend on the configuration of the light absorbing chromophores within the molecule i.e. its structure. The polarization degree of emission also reflects chromophores' organization, but only those, which emit fluorescence.
Even if the modulation in absorption is low the modulation in emission could be high (fluorescence is highly polarized). This is a direct evidence of energy transfer in the system. It's also possible that energy transfer results in a change not only in fluorescence polarization degree but also in the angle between maximum absorption and emission. To see such effects we use scheme 3 where both the λ/2 waveplate and the analyzer are rotated with different frequencies by step motors while the fluorescence time transient is detected.
This technique gives all information about polarization of the light absorbing and light emitting states, which can be possibly obtained in experiments with linearly polarized light. It allows finding the relative angle between those orientations that gives the highest absorption and emission. If we know the orientations of the excitation polarization and emission polarization filters in the laboratory frame we can find absolute angles. The technique also allows to probe if energy transfer takes place or not within the system.
Note, that dichroic mirrors of the microscope may distort the chosen type of excitation light polarization depending on the polarization orientation. To make corrections for these distortions we use a Berek compensator.
We use two types of polarization setups, which we call one channel (scheme 3 in figure 3 and figure 4) and two channels configuration, figure 5.
To perform polarization-sensitive experiments according to scheme 3 a λ/2 waveplate is placed into linearly polarized excitation beam and an analyzer is set in front of the CCD camera. Berek polarization compensator was used to maintain the linear polarization of the excitation light at the sample plane after it passed the dichroic mirror and filters. By rotating the λ/2 plate by a step motor the polarization angle of the excitation light (φex) was rotated in the sample plane. The analyzer angle (φem) was also rotated by another motor with a different frequency. As a result, fluorescence intensity as a function of angles φex and φem was measured.
The setup described above we call "one-channel configuration". In this configuration blinking events can't be distinguished from fast shifts in polarization orientation. To be able to distinguish these two different processes we use a two channels configuration.
Here the emission polarization is split by a rotated analyser (wire-grid polarizer) to two components. One of them is transmitted (Itrans) and the other(Iref) is reflected by the analyser. Transmitted and reflected components have polarizations parallel and perpendicular to the analyser transmission axis respectively. The transmitted light gets reflected back by a mirror and it is passing the analyser again. The surface of the mirror is placed with a small angle relative the surface of the analyzer. Thus, we get two separated images on the CCD camera containing complementary polarization information. The benefit of this configuration is that the total intensity is recorded in that sense a shift in polarization orientation makes light go from one channel to the other. Thus recorded data can be normalized to total fluorescence intensity (Itrans + Iref) and blinking events be accounted for.