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.