Abstract
Super-resolution (SR) fluorescence microscopy that breaks through the diffraction barrier has drawn great interest in biomedical research. However, obtaining a high precision three-dimensional distribution of the specimen in a short time still remains a challenging task for existing techniques. In this paper, we propose a super-resolution fluorescence microscopy with axial localization capability by combining multifocal structured illumination microscopy with a hybrid detection PSF composed of a Gaussian PSF and a double-helix PSF. A modified reconstruction scheme is presented to accommodate the new hybrid PSF. This method can not only recover the lateral super-resolution image of the specimen but also retain the specimen's depth map within a range of 600 nm with an axial localization precision of 20.8 nm. The performance of this approach is verified by testing fluorescent beads and tubulin in 293-cells. The developed microscope is well suited for observing the precise 3D distribution of thin specimens.
Highlights
Super-resolution (SR) techniques which overcome the diffraction limit in optical microscopy have met increasing interests in biomedical imaging [1,2,3]
The SR-SIM has been implemented with diffractionlimited excitation focus, including image scanning microscopy (ISM) [11,12], multifocal structured illumination microscopy (MSIM) [13], re-scan confocal microscopy (RCM) [14], and Received 18 Feb 2020; revised 2 May 2020; accepted 4 May 2020; published 14 May 2020
The emitting fluorescence from the specimen is collected by the same objective and relayed onto the panel of liquid crystal spatial light modulator (LC-SLM) (1920×1080 pixels, Pluto II, HoloEye Photonics AG, Germany) through a 1:1 4f system
Summary
Super-resolution (SR) techniques which overcome the diffraction limit in optical microscopy have met increasing interests in biomedical imaging [1,2,3]. Even though the axial resolution can be enhanced (∼300 nm) by generating three-dimensional structured illumination and filling in the “missing cone” of spatial frequencies [9], it is still inferior to the lateral resolution (∼100 nm) Another approach to improve the axial resolution was achieved by combining SR-SIM with I5M [10], with which near-isotropic resolution (∼100 nm) in three-dimension can be produced. It requires two opposing objective lenses, which greatly increases the difficulty of system calibration and sample-holding, limiting its widespread application in biomedical imaging. ISM and MSIM based on PSF engineering (termed refocusing after scanning using helical phase engineering, RESCH) [17,18,19,20] were developed by employing post-acquisition refocusing algorithms [17,21], enabling faster acquisition of the super-resolution images in 3D with a more modest spatial resolution
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