Oblique-plane single-molecule localization microscopy for tissues and small intact animals

Kim, Jeongmin; Wojcik, Michal; Wang, Yuan; Moon, Seonah; Zin, Emilia A.; Marnani, Nadia; Newman, Zachary L.; Flannery, John G.; Xu, Ke; Zhang, Xiang

doi:10.1038/s41592-019-0510-z

Cited by 89 publications

(68 citation statements)

References 36 publications

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“…As compared to the previously reported results (9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20)(21)(22), the achievable FOV (5.8 mm×4.9 mm×0.7 mm) is an order of magnitude higher, the largest FOV in OPM so far to the best of our knowledge.…”

Section: Discussionsupporting

confidence: 42%

“…To improve the resolution, increase volumetric imaging speed and enable flexible sample mounting/positioning protocols, different variants of LSFM /SPIM have emerged over the recent two decades (9)(10)(11)(12)(13)(14)(15). Among them, oblique plane microscopy (OPM) provides an attractive optical design and provides excellent balance among the above three aspects.…”

Section: Introductionmentioning

confidence: 99%

“…Among them, oblique plane microscopy (OPM) provides an attractive optical design and provides excellent balance among the above three aspects. In contrast to conventional LSFM that has two objective lens, OPM uses only single objective lens but applies an off-axis oblique light sheet excitation and a remote focusing system to capture the light sheet in a nearorthogonal angle (9)(10)(11)13,14). This "single objective lens layout" of an OPM liberates the sample placing space, and can be set up in the existing inverted, or upright microscopes.…”

Section: Introductionmentioning

confidence: 99%

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Wide field-of-view volumetric imaging by a mesoscopic scanning oblique plane microscopy with switchable objective lens

Shao

Kılıç

Yin

et al. 2020

Preprint

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AbstractConventional light sheet fluorescence microscopy (LSFM), or selective plane illumination microscopy (SPIM), enables high resolution 3D imaging over a large volume by using two orthogonally aligned objective lenses to decouple excitation and emission. The recent development of oblique plane microscopy (OPM) simplifies LSFM design with only one single objective lens, by using off-axis excitation and remote focusing. However, most reports on OPM has a limited microscopic field of view (FOV), typically within 1×1 mm2. Our goal is to overcome the limitation with a new variant of OPM to achieve mesoscopic FOV. We implemented an optical design of mesoscopic scanning OPM to allow using low numerical aperture (NA) objective lens. The angle of the intermediate image before the remote focusing system was increased by a demagnification under Scheimpflug condition such that the light collecting efficiency in the remote focusing system was significantly improved. We characterized the 3D resolutions and FOV by imaging fluorescence microspheres, and demonstrated the volumetric imaging on intact whole zebrafish larvae, mouse cortex, and multiple Caenorhabditis elegans (C. elegans). We demonstrate a mesoscopic FOV up to ~6× 5×0.6 mm3 volumetric imaging, the largest reported FOV by OPM so far. The angle of the intermediate image plane is independent of the magnification. As a result, the system is highly versatile, allowing simple switching between different objective lenses with low (10x, NA 0.3) and median NA (20x, NA 0.5). Detailed microvasculature in zebrafish larvae, mouse cortex, and neurons in C. elegans are clearly visualized in 3D. The proposed mesoscopic scanning OPM allows using low NA objective such that centimeter-level FOV volumetric imaging can be achieved. With the extended FOV, simple sample mounting protocol, and the versatility of changeable FOVs/resolutions, our system will be ready for the varieties of applications requiring in vivo volumetric imaging over large length scales.

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Section: Discussionsupporting

confidence: 42%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Wide field-of-view volumetric imaging by a mesoscopic scanning oblique plane microscopy with switchable objective lens

Shao

Kılıç

Yin

et al. 2020

Preprint

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“…There are several ways in which light-sheet microscopy schemes can yield super-resolution with reduced phototoxicity. Superresolution in live samples has been demonstrated using lightsheet microscopy by simply combining this illumination geometry with SRM techniques such as SMLM (142)(143)(144) and RESOLFT (145). However, the employed SRM methods still require high-intensity illumination, and thus such composite techniques do not exploit the inherent low phototoxicity of light-sheet imaging.…”

Section: Hardware Developments For Improved Livecell Imagingmentioning

confidence: 99%

Between Life and Death: Reducing Phototoxicity in Super-Resolution Microscopy

Tosheva¹,

Yuan²,

Pereira³

et al. 2019

Preprint

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Super-Resolution Microscopy enables non-invasive, moleculespecific imaging of the internal structure and dynamics of cells with sub-diffraction limit spatial resolution. One of its major limitations is the requirement for high-intensity illumination, generating considerable cellular phototoxicity. This factor considerably limits the capacity for live-cell observations, particularly for extended periods of time. Here, we overview new developments in hardware, software and probe chemistry aiming to reduce phototoxicity. Additionally, we discuss how the choice of biological model and sample environment impacts the capacity for live-cell observations. Phototoxicity | Photodamage | Super-Resolution Microscopy | Fluorescence Correspondence: s.culley@ucl.ac.uk, r.henriques@ucl.ac.uk Tosheva et al. | 1-12Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted:

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“…Remote lateral scanning of the light-sheet and detection planes was achieved using a rotating polygon mirror [11] and galvo mirrors [12,13]. Folding the remote-refocussing system about a small tilted mirror placed in the focal plane of the second microscope objective [14,15] can be used to increase the numerical aperture of the third microscope objective in the remoterefocussing system [16]. The NA of the third microscope objective can also be increased by using a microscope objective with an NA that approaches unity but with a very small working distance and a front element shaped to allow its close approach to the second microscope objective [17].…”

Section: Introductionmentioning

confidence: 99%

Dual-view oblique plane microscopy (dOPM)

Sparks

Bakal

Behrens

et al. 2020

Preprint

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We present a new folded dual-view oblique plane microscopy (OPM) technique termed dOPM that enables two orthogonal views of the sample to be obtained by translating a pair of tilted mirrors in refocussing space. Using a water immersion 40× 1.15 NA primary objective, deconvolved image volumes of 200 nm beads were measured to have full width at half maxima (FWHM) of 0.35±0.04 μm and 0.39±0.02 μm laterally and 0.81±0.07 μm axially. The laterally integrated z-sectioning value was 1.33±0.45 μm using light-sheet FWHM in the frames of the two views of 4.99±0.58 μm and 4.89±0.63 μm. To qualitatively demonstrate that the system can reduce shadow artefacts while providing a more isotropic resolution, a multi-cellular spheroid approximately 100 μm in diameter was imaged.

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Oblique-plane single-molecule localization microscopy for tissues and small intact animals

Cited by 89 publications

References 36 publications

Wide field-of-view volumetric imaging by a mesoscopic scanning oblique plane microscopy with switchable objective lens

Wide field-of-view volumetric imaging by a mesoscopic scanning oblique plane microscopy with switchable objective lens

Between Life and Death: Reducing Phototoxicity in Super-Resolution Microscopy

Dual-view oblique plane microscopy (dOPM)

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