Fig 1.
Schematics of coverslip method and nanosheet wrapping mount.
(A) In a conventional coverslip method, an objective lens can only be moved until it is in contact with the coverslip. WD, working distance of an objective lens; NFP, nominal focal plane. (B) If the coverslip is replaced by a polymer nanosheet with a thickness of around 100 nm, an extra distance of 170 μm for the movement of an objective lens is obtained, namely a deeper maximum NFP can be expected.
Fig 2.
(A) Schematic of nanosheet wrapping process. A tissue specimen is placed on a coverslip in advance. The nanosheet is transferred from the surface of water to wrap the coverslip with the help of a wire loop, which is then reversed and bonded to a perforated bottom dish for observation. (B) Photos of a typical wrapping process. From (i–iii): a CYTOP nanosheet floated on the surface of water (arrows indicate the corner of nanosheet); the same nanosheet supported by a wire loop in the air; and an agarose gel (~2 mm thickness) wrapped with a nanosheet. Scale bar, 1 cm.
Fig 3.
Mechanical robustness and chemical resistance of CYTOP nanosheet.
(A) The recorded force during the process of a magnet approaching, where the applied force is constantly increased until the nanosheet breaks (purple line, and smoothing is shown in red). Compared to the control group without wrapping (gray line), the stretchable distance of a nanosheet, ΔD, is about 0.6 mm. (B) Snapshots taken in sequence (i–iii) during the test process, which are 40 mN is applied under the elastic region of nanosheet, 116 mN is applied to achieve a maximum bearable force of nanosheet, and the moment that nanosheet is broken with a further magnet approaching. (C) Contact angles with a variety of liquids on a silicon substrate supported CYTOP nanosheet (thickness of 130 nm), including water, mineral oil, immersion oil Type NF, and the contact angle measured after in contact with NF immersion oil for 1 h. (D) The same contact angle measurement is conducted on a silicon substrate supported PDMS nanosheet (thickness of 130 nm) as a comparison.
Fig 4.
Coverslip-free imaging on a model tissue.
(A) With a conventional setup, a coverslip supported agarose gel embedded with 2 μm green fluorescent beads is observed from the upper surface of coverslip to a depth of 400 μm, and a 1 μm thick polystyrene film loaded with Nile red is applied as a height reference at z = 0. (B) Imaging on a pristine agarose gel with nanosheet wrapping mount. The corresponding schematics of optics conditions are shown above, and yellow dashed lines indicate the maximum depth of focal plane in each condition. Imaging area in xy plane is 212×212 μm2, and depth is labelled at the side.
Fig 5.
Coverslip-free imaging on a model tissue with adjusted optics conditions.
(A, C) With nanosheet wrapping mount, imaging tests with an agarose gel treated with LUCID, and further modified with RapiClear are conducted. The corresponding schematics of optics conditions are shown above, and yellow dashed lines indicate the maximum depth of focal plane in each condition. Imaging area in xy plane is 212×212 μm2, and depth is labelled at the side. (B, D) Spatial resolutions at different depths of nanosheet wrapping mount. 200 nm NPs are embedded in agarose gel and images are obtained at every 50 μm. Scale bar, 500 nm; and depth is labelled below. Representative NPs at each depth are arbitrarily chosen (n = 3 independent tests; and 10 NPs are analyzed for each test), and FWHMs along the intensity profile in x-axis and z-axis are measured as shown in histograms (mean ± SEM).
Fig 6.
Nanosheet wrapping imaging on a brain slice.
(A, B) With nanosheet wrapping mount, a 1 mm thick thy1-EYFP-H mouse brain slice is observed from the surface of nanosheet to a depth of 400 μm, and a 1 μm thick polystyrene film loaded with Nile red is applied as a height reference at z = 0. Imaging tests with brain treated with LUCID, and further modified with RapiClear are conducted (n = 5 independent tests; and representative images are shown). The corresponding schematics of optics conditions are shown above. Imaging area in xy plane is 212×212 μm2, and depth is labelled at the side. Images of individual axon fibers at representative depths are extracted and shown at right. Scale bar, 15 μm; and depth is labelled at upper right. (C, D) Magnified views of dendrites are trimmed from projection images (maximum intensity projection) of Fig 6A, Fig 6B at both superficial (depth: 20±10 μm) and deep (depth: 220±10 μm) regions within volume of a 30×10×3 μm3 cube. The normalized fluorescence intensity along the yellow lines with a distance of 5 μm is measured to demonstrate the edge of dendrites, and yellow arrows indicate some representative dendritic spines with fine structures.