Combining 3D single molecule localization strategies for reproducible bioimaging

Abstract

We developed a 3D localization-based super-resolution technique providing an almost isotropic 3D resolution over a 1 µm range with precisions down to 15 nm. The axial localization is performed through a combination of point spread function (PSF) shaping and supercritical angle fluorescence (SAF), which yields absolute axial information. Using a dual-view scheme, the axial detection is decoupled from the lateral detection and optimized independently. This method can be readily implemented on most homemade PSF shaping setups and provides drift-free, tiltinsensitive and achromatic results. Its insensitivity to these unavoidable experimental biases is especially adapted for multicolor 3D super-resolution microscopy, as we demonstrate by imaging cell cytoskeleton, living bacteria membranes and axon periodic submembrane scaffolds. We further illustrate the interest of the technique for biological multicolor imaging over a several µm range by direct merging of multiple acquisitions at different depths.Despite recent advances in localization-based super-resolution techniques, 3D fluorescence imaging of biological samples remains a major challenge, mostly because of its lack of versatility. While photoactivated localization microscopy (PALM) and (direct) stochastic optical reconstruction microscopy ((d)STORM) can easily provide lateral a localization precision (i.e. the standard deviation of the position) down to 5-10 nm [1,2,3,4], a great deal of effort is being made to develop quantitative and reproducible 3D super-localization methods. The most widely used 3D SMLM technique is the astigmatic imaging, which relies on the use of a cylindrical lens to apply an astigmatic aberration in the detection path to encode the axial information in the shape of the spots, achieving an axial localization precision down to 20-25 nm [5]-though the precision varies with the axial position. Other Point Spread Function (PSF) shaping methods are also available [6,7,8], but their implementations are not as inexpensive and straightforward. Still, all PSF shaping methods including astigmatic imaging suffer from several drawbacks such as axial drifts, chromatic aberration, field-varying geometrical aberrations and sample tilts. These sources of biases often degrade the resolution or hinder 1 All rights reserved. No reuse allowed without permission.(which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.The copyright holder for this preprint . http://dx.doi.org/10.1101/385799 doi: bioRxiv preprint first posted online Aug. 6, 2018; colocalization and experiment reproducibility. Axial measurements can also be performed thanks to intensity-based techniques like Supercritical Angle Fluorescence (SAF) [9,10,11,12,13,14], which relies on the detection of the near-field emission of fluorophores coupled into propagative waves at the sample/glass coverslip interface due to the index mismatch. Combined with SMLM, this technique, called Direct Optical Nanoscopy with Axially Localized ...

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“…This quad-view system (dual-cam sCMOS cameras, Orcaflash v4, Hamamatsu) provided 3D nanoscopy information with high isotropic localization precision (15×15×15 nm, over an axial range of ∼1 mm). Axial information was obtained by super-critical angle fluorescence (SAF) and the point spread function (PSF) deformation with a strong astigmatism (DAISY) ( Cabriel et al, 2019 ). A total of 20,000 frames at 50 ms exposure time were acquired and used for single-molecule detections and to reconstruct a nanoscopy image.…”

Multicolor localization microscopy and point-spread-function engineering by deep learning

“…The likely source of the size-similarity is the imperfect chromatic-correction present in Apo objectives that causes a focal shift between different colors (Fig. 21), as has been observed by others as well [28].…”

“…In an experimental sample, the problem of defocus is further complicated by the wavelength-dependent focal length of even chromatically corrected objectives which can make the PSFs appear more similar in size (Appendix 5.8. ), also observed by Cabriel et al [28]. Unlike a comparison of the Gaussian-fit size, the net utilizes any differences in the PSFs, and therefore any chromatic aberrations in the optical system can help break the ambiguity between the two shapes.…”

“…The PSF of any imaging system depends on the emitter's wavelength, due to diffraction and chromatic aberrations [28]. To test first whether we could discriminate between two types of emitters imaged on a grayscale camera, we prepared a thin sample containing green and red quantum dots (Qdots) with emission peaks at 565 and 705 nm, and imaged it using an epifluorescence microscope ( Fig.…”

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“…To perform fluorescence nanoscopy experiments, a SAFe360 module (Abbelight, France) was added to the camera port of the microscope. This detection module couples single-molecule localization microscopy (SMLM), supercritical angle fluorescence (SAF), and astigmatism ( 60 , 61 ) in a dual-view setup coupled with scientific complementary metal oxide semiconductor (sCMOS) cameras (Orcaflash v4; Hamamatsu). Prior to each acquisition, bright-light and diffraction-limited images were acquired.…”