Fourier domain heterochromatic fusion for single beam scanning super-resolution microscopy

Point spread function (PSF) engineering by emitter's response can code higher spatial frequency information of an image for microscopy to achieve super-resolution. However, complexed excitation optics or repetitive scans are needed, which explains the issues of low speed, poor stability and operational complexity associated with the current laser scanning microscopy approaches. Here we use the diverse emission responses of a fluorophore to improve the imaging quality and speed for super-resolution nanoscopy. By saturating the four-photon emission state of single upconversion nanoparticle using a doughnut-shape scanning excitation beam, the high-frequency information can be resolved through the doughnut emission PSF, while the complementary lower frequency information of an image can be simultaneously obtained from the Gaussian-like emission PSF, as a result of being over-saturated at the two-photon state of the same nanoparticle. With the Fourier domain heterochromatic fusion method, we show the fused optical transfer function (OTF) can cover both low and high-frequency information to yield the overall enhanced image quality. We show single upconversion nanoparticles can be super-resolved with a spatial resolution of 50 nm, 1/20th of the excitation wavelength. This work further suggests a new scope for developing nonlinear multi-colour emitting probes to improve the image quality and noise control in super-resolution nanoscopy.