Polarization as a new dimension in super-resolution microscopy

Abstract

The physical phenomenon of fluorescence has a number of fundamental dimensions, e.g., intensity, wavelength, time, and polarization. In particular, the fluorescence polarization effect— first discovered in 19261—arises from the transverse nature of light waves (i.e., from dipole orientations). Various fluorescence anistropy techniques have previously been developed to study the dipole orientation of fluorophores. For example, fluorescence polarization microscopy (FPM) is used extensively for biological imaging applications. In this technique, the angle of a fluorophore is measured so that the orientation and structural details of a targeted protein can be resolved. Conventional FPM methods, however, are limited because of the presence of many molecules within the diffraction-limited volume. This means that the fluorescence polarization information is collected from dipoles with many different orientations. The idea of using super-resolution to improve imaging resolving power was first proposed in 1995.2 This idea has since been realized, i.e., with a photobleaching-photoactivation process used to separate molecules (with a resolution of about 20nm) in the time domain.3 Previously developed super-resolution microscopy approaches, which extend vision beyond the diffraction limit, are mostly based on the intensity, wavelength, and temporal dimensions of fluorescence. Although the fourth dimension of fluorescence, i.e., polarization, can also be used to modulate fluorescence (without restriction to specific fluorophores), this mode of super-resolution microscopy has only recently been investigated. Indeed, a new technique—sparse deconvolution of polarization-modulated fluorescent images (SPoD)—was first developed in 2014 (with which a resolution of 5nm was demonstrated at 1 frame/second).4 Although super-resolution can be achieved with this technique, the dipole orientation information is lost during the SPoD reconstruction and an interesting debate—whether or not fluorescent Figure 1. Illustration of the super-resolution dipole orientation mapping (SDOM) technique. (a) Two fluorophores, 100nm apart, with different dipole orientations are shown in red and green. When excited by rotating polarized light they emit periodic signals. The emission ratio between the two molecules can be modulated accordingly and used to separate them in the polarization domain. The SDOM procedure provides a super-resolution image of the effective dipole intensities (compared with an unresolved wide-field image). Arrows indicate the different dipole orientations. Scale bar denotes 200nm. (b) SDOM result for two fluorophores, superimposed on top of a super-resolution image, where the two molecules cannot be separated. (c) The same data shown in a 3D coordinate system (XY is the plane of the super-resolved intensity image and is the dipole orientation). In this perspective, the two molecules can be completely resolved.7