Ultra 55

The morphology
of the hydrogels
was analyzed by field emission scanning electron microscope (FESEM)
(Zeiss ULTRA 55, Carl Zeiss Microscopy) with an accelerating voltage
of 1.5 kV. The samples were lyophilized after swelling in liquid water
at 37 °C for 24 h to constant weight and freezing at −80
°C overnight. The cross-section was observed in the lyophilized
samples, which were previously immersed in liquid nitrogen and cryofractured
for FESEM observation. Finally, the samples were coated with a carbon
layer using a sputter coating (EM MED020, Leica). The percentages
of Ca and Zn ions were obtained with an Energy Dispersive X-ray Spectrometry
(EDX, X-Max N, Oxford Instruments) mounted on the Zeiss ULTRA 55 FESEM
(accelerating voltage 15 kV).

The scattering and luminescence spectra contain three different stages (stage 1: Si NS on glass/Si substrates; stage 2: system-1/-2; stage 3: system-1/-2 (annealed)), which were measured by using a dark-filed optical microscope (BX53, Olympus, Tokyo, Japan) equipped with a spectrometer (SR-500i-B1-R, Andor) and a color charge coupled device (CCD) (01-QIClick-R-F-CLR-12, QIMAGING, Surrey, BC, Canada). The 100× objective lens with a numerical aperture of 0.8 was utilized to collect the scattering light and the luminescence signal excited by an fs oscillator (Mira-HP, Coherent, San Francisco, CA, USA). The integration time for the scattering spectra measurement was 0.6 s. The integration time for the luminescence spectra measurement was 2 s and the gain coefficient was set to 20 times. The surface morphology and the element mapping distribution images of the samples were analyzed using the scanning electron microscope (SEM, ZEISS Ultra 55, Carl Zeiss, Oberkochen, Germany) and energy-dispersive spectroscopy (EDS, ZEISS Ultra 55, Carl Zeiss, Oberkochen, Germany). The crystallinity of the sample was analyzed using the transmission electron microscope (TEM, JEM-2100F, JEOL, Tokyo, Japan).

Physically processed and enzymatically digested corneal dECM microparticle (EdECM) size and morphology were recorded using a field-emission scanning electron microscopy (Zeiss Ultra-55, Oberkochen, Germany) operating at a voltage of 5 kV (Figure 2). Briefly, lyophilized physically processed or EdECM microparticles were sputter-coated with gold/palladium to achieve a 10-nm coating and visualized under low vacuum conditions. Elemental analysis by energy dispersive X-ray spectroscopy was also performed to determine molecular constituents of EdECM samples (Zeiss Ultra-55, Oberkochen, Germany).