Significance Fluorescence microscopy stands as one of the most potent tools for probing cellular structures and dynamic processes. Recent advances in super-resolution imaging techniques have surpassed traditional diffraction limits, achieving nanometer-scale resolution in live biological samples. However, the complex variation in refractive indices within biological specimens often reduces imaging performance due to aberrations induced by the sample itself. AO, a technology designed to correct wavefront errors, when integrated into super-resolution microscopy, can significantly enhance the imaging resolution, depth, and speed of super-resolution systems. In pursuit of further augmented imaging performance in super-resolution microscopy, researchers continually innovate applications of AO within the realm of super-resolution techniques. It is foreseeable that AO will experience rapid development in the field of super-resolution microscopy, substantially enhancing the imaging capabilities of super-resolution systems. This underscores the significant importance of research into AO within the context of super-resolution microscopy.
Progress Initially, the application of AO in fluorescence microscopy and its classical methodologies are presented; This is followed by a categorization and summation of AO's applications in super-resolution microscopic imaging in recent years. Aberration measurement is broadly categorized into two approaches of direct wavefront sensing and indirect wavefront sensing. In direct wavefront sensing, Wavefront Sensors (WFS) are commonly employed to directly measure phase aberrations in the received wavefront. Notably, the Shack-Hartmann Wavefront Sensor (SH-WFS), due to its compact size, low cost, simplicity, and ease of operation, is frequently utilized. Indirect wavefront sensing calculates aberrations indirectly via the intensity distribution of images produced by the microscope. One method of this indirect measurement utilizes phase retrieval, reconstructing the pupil function by imaging fluorescent beads and extracting aberrations from it; Another relies on image quality metrics, imposing aberration biases on each mode and determining correction parameters by maximizing image quality measures such as brightness, contrast, sharpness, and resolution. The widespread application of AO in microscopy has significantly improved the imaging performance of microscopic systems, greatly aiding in the detection and correction of aberrations within fluorescence microscopy. Subsequently, a brief introduction is provided on the types and principles of super-resolution microscopy. The spatial resolution of traditional optical microscopes is limited by the diffraction limit, whereas super-resolution microscopy elevates the resolution by an order of magnitude. The three principal types of super-resolution microscopy are Single-Molecule Localization Microscopy (SMLM), Structured Illumination Microscopy (SIM), and Stimulated Emission Depletion Microscopy (STED). SMLM achieves nanometric precision in positioning by utilizing the signal sparsity of activated fluorescent markers, temporally separating microscopic structures that are spatially challenging to divide. SIM, a wide-field technique, uses high-frequency structured illumination to transpose the spatial frequency of the sample into the passband of the microscope's diffraction limit. STED, a point-scanning method, employs point spread function engineering to generate a “doughnut-shaped” hollow focus spot to deplete the fluorescence groups, causing stimulated emission depletion and producing an effective sub-diffraction volume of fluorescent excitation. Finally, the application of AO across various super-resolution microscopic imaging techniques is discussed, addressing the imaging characteristics and underlying optical principles of each method. The conclusion synthesizes the trajectory of super-resolution microscopy's development with the latest advancements in AO technological innovation, providing a prospective summary.
Conclusions and Prospects Originating in astronomy and later flourishing in the realm of microscopic imaging, AO technology has become an indispensable component of super-resolution microscopy due to its formidable capacity to correct wavefront aberrations. Higher spatial resolution, greater imaging depth, additional imaging dimensions, and faster imaging speeds represent the perpetual goals of scholars in the super-resolution field. The author aims to provide a reference for the integration of AO technology with the super-resolution microscopy domain by detailing the application of AO in super-resolution microscopic imaging, with the expectation that the use of AO technology will become even more widespread and profound in the field.
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