C apochromat 40x 1.2w

As described previously, heat treatment of the gels was performed with the DSC instrument (cf. Section 2.3), the lids of the DSC pans were peeled and gels were carefully removed from the pans. A 10 ​μL drop of Rhodamine B (diluted in water at 0.001%) was placed in the well of a chambered coverglass and the gel sample was positioned on top of this staining drop. Gels were observed with a Zeiss LSM 880 inverted confocal laser scanning microscope (Carl Zeiss AG, Oberkochen, Germany) with an excitation wavelength of 543 ​nm. Fluorescence was measured at 565–600 ​nm and images were acquired with a water immersion objective C-Apochromat 40x/1.2 ​W (Carl Zeiss AG, Oberkochen, Germany). Three replicates were prepared and observed for each experimental condition. The open source ImageJ software and the image processing package Fiji were used to visualize and process the images (Schindelin et al., 2012 (link)). Image processing was the same for all samples: a “despeckle” filter was used first, then contrast was enhanced (saturated pixels 5%) and finally outliers were removed (radius 2 pixels, threshold 50, bright outliers). Processed images were binarized and areas where clear aggregates were visible were selected. D_f of each selected area was determined with the fractal box count tool of the software (box sizes 2,3,4,6,8,12,16,32,64 and black background).