Video-rate remote refocusing through continuous oscillation of a membrane deformable mirror

AbstractThis paper presents the use of a deformable mirror (DM) configured to rapidly refocus a microscope employing a high numerical aperture objective lens. An Alpao DM97-15 membrane DM was used to refocus a 40×/0.80 NA water-immersion objective through a defocus range of −50 to 50 μm at 26.3 sweeps per second. We achieved imaging with a mean Strehl metric of > 0.6 over a field of view in the sample of 200×200 μm2 over a defocus range of 77 μm. We describe an optimisation procedure where the mirror is swept continuously in order to avoid known problems of hysteresis associated with the membrane DM employed. This work demonstrates that a DM-based refocusing system could in the future be used in light-sheet fluorescence microscopes to achieve video-rate volumetric imaging.

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High-efficiency, large-area lattice light-sheet generation by dielectric metasurfaces

Nanophotonics ◽

10.1515/nanoph-2020-0227 ◽

2020 ◽

Vol 9 (12) ◽

pp. 4043-4051

Author(s):

Fenghua Shi ◽

Jing Wen ◽

Dangyuan Lei

Keyword(s):

Spatial Resolution ◽

Temporal Resolution ◽

High Efficiency ◽

Numerical Aperture ◽

Light Sheet ◽

Field Of View ◽

Objective Lens ◽

High Temporal Resolution ◽

Light Sheet Microscopy ◽

Illumination Efficiency

AbstractLattice light-sheet microscopy (LLSM) was developed for long-term live-cell imaging with ultra-fine three-dimensional (3D) spatial resolution, high temporal resolution, and low photo-toxicity by illuminating the sample with a thin lattice-like light-sheet. Currently available schemes for generating thin lattice light-sheets often require complex optical designs. Meanwhile, limited by the bulky objective lens and optical components, the light throughput of existing LLSM systems is rather low. To circumvent the above problems, we utilize a dielectric metasurface of a single footprint to replace the conventional illumination modules used in the conventional LLSM and generate a lattice light-sheet with a ~3-fold broader illumination area and a significantly leveraged illumination efficiency, which consequently leads to a larger field of view with a higher temporal resolution at no extra cost of the spatial resolution. We demonstrate that the metasurface can manipulate spatial frequencies of an input laser beam in orthogonal directions independently to break the trade-off between the field of view and illumination efficiency of the lattice light-sheet. Compared to the conventional LLSM, our metasurface module serving as an ultra-compact illumination component for LLSM at an ease will potentially enable a finer spatial resolution with a larger numerical-aperture detection objective lens.

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

10.1101/2020.06.29.177782 ◽

2020 ◽

Author(s):

Wenjun Shao ◽

Kivilcim Kilic ◽

Wenqing Yin ◽

Gregory Wirak ◽

Xiaodan qin ◽

...

Keyword(s):

Numerical Aperture ◽

Field Of View ◽

Objective Lens ◽

Wide Field ◽

Volumetric Imaging ◽

Zebrafish Larvae ◽

Mouse Cortex ◽

New Variant ◽

Intermediate Image

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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