Figure 5

iDISCO, CUBIC, SCALE, and PEGASOS clearing alter the fluorescent emission spectra of fluorophores and fluorescent dyes in cell-free solutions. Raw plate reader-based emission spectra visualize the differences in the amplitude between measurements in PBS and iDISCO, CUBIC, SCALE, and PEGASOS clearing (a–d). Emission data normalized to their peak intensity highlight the shift of the emission peaks to altered wavelengths (e–h). Bar graphs summarize the direction and magnitude of emission peak wavelength shifts per fluorophore and clearing agent in cell-free solutions (i) and in mouse brain tissue (j). Bar graphs visualize the shifts depicted in Figs. 4 and 5 and represent the shifts of average emission curves generated from at least n = 8 independent measurements and wells per datapoint. DAPI was excited at 405 nm (a,e), Alexa Fluor 488 at 488 nm (b,f), Alexa Fluor 568 at 555 nm (c,g), and Alexa Fluor 647 at 639 nm (d,h). The gaps in spectra surrounding the excitation wavelength are due to technical limitations in the plate reader (see “Methods” section). Error bars indicate standard deviation. n = 8 independent wells for each datapoint.