Figure 4

iDISCO, CUBIC, and PEGASOS clearing alter the fluorescent emission spectra of fluorophores and fluorescent dyes in mouse brain tissues. Confocal microscopy-based emission spectra of Alexa Fluorophores 488, 568, 647, eGFP, and DAPI in PBS (a) vary from those in iDISCO (b), PEGASOS (c), and CUBIC (d). Colored outlines in b-d represent emission profiles of PBS shown in a) to facilitate visual comparison. The line graphs (e–h) represent the individual emission signals of cleared tissues shown in (a) through (d) with Alexa Fluorophore 488 in addition: Raw emission data visualize the differences in the amplitude between measurements in PBS and cleared tissues (e–h). Emission data normalized to their peak intensity highlight the shift of the emission peaks to altered wavelengths (i–l). DAPI was excited at 405 nm, eGFP and Alexa Fluor 488 at 488 nm, and Alexa Fluor 647 at 633 nm. Error bars indicate standard error of the mean and are at times too small to be visible. The reduction of fluorescence emission around 560 nm stems from the beam splitter in the light path (f–h and j–l; e,i used a different beam splitter without this gap). Spectra are shown as means measured in n = 25 regions of interest per fluorophore across 5 separate biological samples.