2.3.1. Confocal Scanning Laser Microscopy (CSLM)
Confocal microscopy was performed using Fluorescein isothiocyanate (FITC) and Nile Red to dye the gelatin HG and RBW-OG, respectively. FITC and Nile Red were added to each phase prior to gelation at 0.2 and 0.15 g/kg, respectively. Each bigel sample was cut into disks and kept cool before being placed on a glass bottom slide. Micrographs were acquired on an SP5 X MP Confocal Microscope (Leica Microsystems Inc.; Buffalo Grove, IL, USA) using a 40x objective magnification with no zoom. Excitation and emission wavelengths for FITC were 495 and 505-540 nm, respectively, and 552 and 565-700 nm for Nile Red, respectively. Images were 1024 x 1024 pixels and acquired at 200 Hz. Signals were overlaid in Leica LAS AF Lite software (Leica Microsystems Inc.; Buffalo Grove, IL, USA). A sample with no dye was viewed as well (results not shown) to confirm that no autofluorescence was occurring from the bigel’s natural constituents. A total of 5-7 images were obtained of each replicate.