Fluorescence nanoscopy by polarization modulation and polarization angle narrowing

Excitation using rotating polarized light and detection of periodic signals from rectangular nanoareas allows widef-ield super-resolution imaging of biological structures in cells and in tissue with reduced background. When excited with rotating linear polarized light, differently oriented fluorescent dyes emit periodic signals peaking at different times. We show that measurement of the average orientation of fluorescent dyes attached to rigid sample structures mapped to regularly defined (50 nm)2 image nanoareas can, in combination with application of the SPEED (sparsity penalty-enhanced estimation by demodulation) deconvolution algorithm, provide subdiffraction resolution (super resolution by polarization demodulation, SPoD). Because the polarization angle range for effective excitation of an oriented molecule is rather broad and unspecific, we narrowed this range by simultaneous irradiation with a second, de-excitation, beam possessing a polarization perpendicular to the excitation beam (excitation polarization angle narrowing, ExPAN). This shortened the periodic emission flashes, allowing better discrimination between molecules or nanoareas. Our method requires neither the generation of nanometric interference structures nor the use of switchable or blinking fluorescent probes. We applied the method to standard wide-field microscopy with camera detection and to two-photon scanning microscopy, imaging the fine structural details of neuronal spines.