Imaging beyond the diffraction limit barrier has attracted wide attention due to the ability to resolve previously hidden image features. Of the various super-resolution microscopy techniques available, a particularly simple method called saturated excitation microscopy (SAX) requires only simple modification of a laser scanning microscope: The illumination beam power is sinusoidally modulated and driven into saturation. SAX images are extracted from the harmonics of the modulation frequency and exhibit improved spatial resolution. Unfortunately, this elegant strategy is hindered by the incursion of shot noise that prevents high-resolution imaging in many realistic scenarios. Here, we demonstrate a technique for super-resolution imaging that we call computational saturated absorption (CSA) in which a joint deconvolution is applied to a set of images with diversity in spatial frequency support among the point spread functions (PSFs) used in the image formation with saturated laser scanning fluorescence microscopy. CSA microscopy allows access to the high spatial frequency diversity in a set of saturated effective PSFs, while avoiding image degradation from shot noise.
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Stockton, Patrick_A ; Field, Jeffrey_J ; Squier, Jeff ; Pezeshki, Ali ; Bartels, Randy_A ( , Optica)
Optical diffraction tomography (ODT) is an indispensable tool for studying objects in three dimensions. Until now, ODT has been limited to coherent light because spatial phase information is required to solve the inverse scattering problem. We introduce a method that enables ODT to be applied to imaging incoherent contrast mechanisms such as fluorescent emission. Our strategy mimics the coherent scattering process with two spatially coherent illumination beams. The interferometric illumination pattern encodes spatial phase in temporal variations of the fluorescent emission, thereby allowing incoherent fluorescent emission to mimic the behavior of coherent illumination. The temporal variations permit recovery of the spatial distribution of fluorescent emission with an inverse scattering model. Simulations and experiments demonstrate isotropic resolution in the 3D reconstruction of a fluorescent object.