Autofluorescence Imaging of FFPE Tissue

The autofluorescence of protein-bound NADH and oxidized flavoproteins containing flavin adenine dinucleotide (Fp) of formalin-fixed-paraffin-embedded (FFPE) tissue slides (8 µm) was imaged and quantified, as previously described [47 (link)]. NADH signals were collected using bandpass filters with excitation (Ex) at 370–400 nm, and emission (Em) at 414–450 nm. The Fp signals were acquired with Ex: 450–488 nm and Em: 500–530 nm bandpass filters using a Zeiss wide-field microscope (Axio Observer.Z1/7, One North Broadway, White Plains, NY, 10601, USA). Tile images (pixel size 1.172 × 1.172 µm²) were taken using a 5×/0.16 NA objective with shading correction on the fly, followed by photostitching. The acquired images were processed with a customized Matlab^® (The MathWorks, Inc., 1 Apple Hill Dr, Natick, MA 01760, USA) routine to generate the redox images where the redox ratio Fp/(NADH + Fp) image was generated pixel-by-pixel from the NADH and Fp images and to obtain the mean value of each of the redox indices (NADH, Fp, the redox ratio) using global averaging [47 (link),48 (link),49 (link)]. Obvious fluorescent artifacts (in either channel) were removed during the imaging analysis.