Quanta 600f

Polymer thin films for spectroscopic study were prepared on glass substrates using spin-coating method at 4000 rpm for 45 seconds. The thicknesses of the PMMA and PS films are 200 nm and the thickness of the SU-8 films is 800 nm. We further baked the film samples on a hot plate at 180 °C for 1 minute to evaporate extra solvent.
PMMA nanostructure samples were created on glass substrate using E-Beam lithography nanopatterning. A 200 nm-thick polymethyl methacrylate (950PMMA, MicroChem) layer was spin-coated (Laurell WS-650-23) on an indium-tin-oxide coated glass cover slip at 4000 rpm for 45 seconds. It was further baked on a hot plate at 180 °C for 1 minute to evaporate extra solvent. After patterning with an E-beam lithography system (FEI Quanta 600F) as a high-resolution negative resist, the unexposed PMMA was dissolved in acetone for 1 minute and then cleaned with distilled water and air-dried. We patterned several structures, including “NU” logo with an approximate 100-nm gap between letters and PMMA grid patterns with periodicity of 200, 250, 300 and 400 nm.

The initial SEM sample preparation steps followed the protocol for TEM sample preparation as described above. Following the graded dehydration series, samples were dried using the Tousimis Autosamdri 815 critical point dryer (Tousimis, Rockville, MD, USA), and then sputter-coated with 10 nm of platinum using the EMS 150T-ES Sputter Coater. Images were acquired with a FEI Quanta 600F environmental scanning electron microscope (FEI, Hillsboro, OR, USA). The STEM sample preparation procedure was similar to the TEM protocol as described above. Following the soaking of the midguts in the gold-nanoparticle suspension, samples were collected and processed for TEM as described. Scanning transmission electron microscopy of the TEM preparations (STEM) was performed in high angle annular dark field (HAADF) image mode on a ThermoFisher Tecnai F30 Twin 300 kV TEM operated at 200 kV. The intensity of STEM-HAADF imaging was proportional to the atomic number Z1.7, enabling gold-nanoparticles to be identified as bright particles in the sample.

An in situ deformation-cathodoluminescence (CL) measurement system is built in a field-emission environmental scanning electron microscope (ESEM), combined a home-made tensile-bending setup and a spatially-resolved CL spectroscope with a liquid nitrogen cooling stage (Figure S2). The ESEM (FEI Quanta 600 F) has a resolution of 1.2 nm, and the CL spectroscope (GATANMONO3PLUS) has a precision of ~0.66 nm. The measurement conditions include: an accelerating voltage of 5~30 kV, a beam current of 10⁻⁸~10⁻¹⁰ A, a working distance of ~12.6 mm, and a PMT detector with a grating of 1200 l/mm for collecting CL spectra with a high precision. For collecting the whole cross-sectionally spectra, the beam energy of 15∼30 keV was used to obtain a strongly spectral intensity. For collecting the cross-section resolved spectra across the radial direction from the outer tensile edge to the inner bending edge of the fiber, the beam energy of 5∼10 keV was used to obtain a highly spectral resolution via a point-by-point linescan irradiation of the e-beam. The scan step was less than ∼50 nm for the ZnO.

Cell, tissue, and scaffold fiber morphology were assessed from SEM images (three predefined locations/sample). The formalin-fixed samples were placed in 0.25% glutaraldehyde (1 h), dehydrated in an ordered series of ethanol dilutions, and dried in vacuo overnight. After visualization of the cell and tissue morphology in low vacuum using a 10 kV electron beam (Quanta 600F, FEI, Hillsboro, OR, USA), half of the samples were decellularized in 4.6% sodium hypochlorite (15 min), washed in H₂O (2 × 5 min), and dried in vacuo overnight to visualize the scaffold fiber morphology. Together with the non-decellularized samples, samples were gold-sputtered and visualized in high vacuum using a 10 kV electron beam.

Microstructure of cross-sections and surface area of CNCs nanocomposite film was investigated by FE-SEM (FEI Quanta 600F, Corvallis, OR, USA). The fractured sample obtained from the mechanical measurements was used for imaging the cross-section morphology. Prepared sample was mounted on aluminum stub with the cross-section oriented up and coated by gold palladium alloy sputter coater (Cressington Scientific Instruments Ltd., Watford, Hertfordshires, UK) to improve the interface conductivity. Digital images were collected at an accelerating voltage of 4.0 kV.