Abstract
The orientation of fluorophores can reveal crucial information about the structure and dynamics of their associated subcellular organelles. Despite significant progress in super-resolution, fluorescence polarization microscopy remains limited to unique samples with relatively strong polarization modulation and not applicable to the weak polarization signals in samples due to the excessive background noise. Here we apply optical lock-in detection to amplify the weak polarization modulation with super-resolution. This novel technique, termed optical lock-in detection super-resolution dipole orientation mapping (OLID-SDOM), could achieve a maximum of 100 frames per second and rapid extraction of 2D orientation, and distinguish distance up to 50 nm, making it suitable for monitoring structural dynamics concerning orientation changes in vivo. OLID-SDOM was employed to explore the universal anisotropy of a large variety of GFP-tagged subcellular organelles, including mitochondria, lysosome, Golgi, endosome, etc. We found that OUF (Orientation Uniformity Factor) of OLID-SDOM can be specific for different subcellular organelles, indicating that the anisotropy was related to the function of the organelles, and OUF can potentially be an indicator to distinguish normal and abnormal cells (even cancer cells). Furthermore, dual-color super-resolution OLID-SDOM imaging of lysosomes and actins demonstrates its potential in studying dynamic molecular interactions. The subtle anisotropy changes of expanding and shrinking dendritic spines in live neurons were observed with real-time OLID-SDOM. Revealing previously unobservable fluorescence anisotropy in various samples and indicating their underlying dynamic molecular structural changes, OLID-SDOM expands the toolkit for live cell research.
Highlights
Due to the diffraction limit, the spatial resolution of conventional wide-field fluorescence microscopy is about 200 nm, limiting its application in studying the fine structural changes, interactions, and function of subcellular
Similar to lock-in amplification widely applied in electronics, optical lock-in detection (OLID) has been demonstrated to be a powerful tool for contrast-enhanced imaging[37,38]
STED super-resolution microscopy has been employed to study the formation of dendritic spine[53]. To observe both the morphological plasticity and polarity of dendritic spines, here we reported the dynamic imaging of dendritic spines with a sub-diffraction resolution of 140 nm at 100 f/s frame rate using the frame-rolling method (Supplementary Notes 2, 5) in living neurons, using OLID-super-resolution dipole orientation mapping (SDOM) techniques (Fig. 6a)
Summary
Due to the diffraction limit, the spatial resolution of conventional wide-field fluorescence microscopy is about 200 nm, limiting its application in studying the fine structural changes, interactions, and function of subcellular. STED5,6 shrinks the point spread function (PSF) by filtering out the stimulated emission within the donut area with spectral-domain modulation. SIM expands the high-frequency space with structured illumination, resulting in a twofold increase in resolution relative to that of the wide field. For the sake of further improving the imaging performance and speed, Guan et al Light: Science & Applications (2022)11:4 deconvolution has been fully applied to a variety of computational imaging and the post-processing of superresolution imaging, such as 3D fluorescence imaging[7], confocal8, 3D SIM9, SOFI10, SMLM11, and STED12, providing a more powerful tool for dense samples imaging in the condition of weak fluorescence intensity.
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