Imaging three-dimensional, subcellular structures with high axial resolution has always been the core purpose of fluorescence microscopy. However, trade-offs exist between axial resolution and other important technical indicators, such as temporal resolution, optical power density, and imaging process complexity. We report a new imaging modality, fluorescence interference structured illumination microscopy (FI-SIM), which is based on three-dimensional structured illumination microscopy for wide-field lateral imaging and fluorescence interference for axial reconstruction. FI-SIM can acquire images quickly within the order of hundreds of milliseconds and exhibit even 30 nm axial resolution in half the wavelength depth range without z-axis scanning. Moreover, the relatively low laser power density relaxes the requirements for dyes and enables a wide range of applications for observing fixed and live subcellular structures.
KEYWORDS: Modulation, Simulations, Point spread functions, Light sources and illumination, Molecules, Microscopy, 3D image processing, Voxels, 3D modeling, Phase shifts
In recent years, modulated illumination localization microscopy (MILM) methods have been proposed to provide around two-fold improvement in lateral localization precision over conventional single molecule localization microscopy methods with the same photon budget. However, MILM with laterally modulated illumination was so far reported in two-dimensional imaging modalities. To fully exploit its three-dimensional (3D) imaging potential, we propose a 3D Single-Molecule Modulated Illumination Localization Estimator (3D-SMILE) that uses the raw data measured from MILM, which has enabled a high localization precision that reaches the theoretical Cramér-Rao lower bound (CRLB) in all three dimensions. 3D-SMILE is based an optimal joint fitting algorithm implemented on a graphics processing unit (GPU) for acceleration. We have shown in simulations that the average lateral localization precision of 3D-SMILE has been improved by more than 3.5 folds over 3D-SMLM over an imaging depth range of around 1.2 μm.
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