4.6.3. Light and ESEM Microscopy

For image acquisition in light microscopy cells were seeded at a density of 50,000 cells/well on 24-well plates pre-coated or not with F1, F2, F3, and F4 *C. reniformis* FSs, prepared as described in paragraph 4.4, and allowed to adhere for 16 h in complete medium. At the end of the experiment, the cells were washed with PBS to remove floating-unattached cells and stained with crystal violet by standard procedures (0.1% crystal violet in methanol for 30 min, followed by extensive washing with water). For image acquisition, an inverted optical microscope (IX53 Olympus, Tokyo, Japan) was used equipped with a CCD camera (U-LH100HG Olympus, Tokyo, Japan) and the relative software.

For ESEM observation of mammalian cells adhering to F1 and F3-derirved SCMs, 50,000 L929 fibroblasts or NCTC keratinocytes were seeded or not onto 0.25 cm<sup>2</sup> ethanol-sterilized membranes and incubated for 3 day at 37 ◦C in complete medium. At the end of the experiment SCMs were washed with PBS and fixed with with a mixture of 2% paraformaldehyde and 2.5 % glutaraldehyde 7.4 pH for 30 min, washed with PBS, extracted from the well plates, and mounted on a plastic support.

Specimens of F1, F2, F3, and F4 SCMs alone and of cells adhering to SCM-F1 and F3 were dehydrated by passing through a series of ethanol alcoholic solutions with an increasing concentration of up to 100%. The dehydrated membranes were further dehydrated at critical point, avoiding the use of acetone due to the presence of the plastic support, and then graphite was covered and observed. Observation and acquisition of the images of cells adhering to SCM-F1 and F3 were performed with an ESEM Vega3–Tescan, type LMU (Tescan Brno s.r.o., Brno, Czech Republic) equipped with a microanalyzer system EDS-Apollo\_x and EDS Texture And Elemental Analytical Microscopy software (TEAM). Observation and acquisition of the four SCMs per se were performed with a FESEM Zeiss SUPRA 40 VP (Carl Zeiss AG, Oberkochen, Germany) and its associated software. The showed results are representative of three independent experiments.

Physical measurements of the fibrillar diameter and of the pore areas observed in the collagen membranes was performed on the images that were obtained by the FESEM analysis of the various membranes, using the ImageJ free software (Rasband, W.S., ImageJ, U. S. National Institutes of Health, Bethesda, MD, USA, https://imagej.nih.gov/ij/, 1997–2016). Means ± S.D. were calculated on at least 10 measurements of fibril diameter or pore areas performed on each membrane.
