Enabling Multiphoton and Second Harmonic Generation Imaging in Paraffin-Embedded and Histologically Stained Sections

Abstract

Nonlinear microscopy, namely multiphoton imaging and second harmonic generation (SHG), is an established noninvasive technique useful for the imaging of extracellular matrix (ECM). Typically, measurements are performed in vivo on freshly excised tissues or biopsies. In this article, we describe the effect of rehydrating paraffin-embedded sections on multiphoton and SHG emission signals and the acquisition of nonlinear images from hematoxylin and eosin (H&E)-stained sections before and after a destaining protocol. Our results reveal that bringing tissue sections to a physiological state yields a significant improvement in nonlinear signals, particularly in SHG. Additionally, the destaining of sections previously processed with H&E staining significantly improves their SHG emission signals during imaging, thereby allowing sufficient analysis of collagen in these sections. These results are important for researchers and pathologists to obtain additional information from paraffin-embedded tissues and archived samples to perform retrospective analysis of the ECM or gain additional information from rare samples.

FIG. 1.

Overview of the nonlinear femtosecond laser system used in this study. (A) Custom-built MP laser scanning MP/SHG imaging station. (B) Laser pathway setup with beam splitter to allow simultaneous acquisition of MP and SHG signals using PMTs with the detection of 425–509 and ≤425 nm, respectively. MP, multiphoton; PMT, photomultiplier tube; SHG, second harmonic generation. Color images available online at www.liebertpub.com/tec

FIG. 2.

(A) MP and (B) SHG signal contrast of porcine heart valves after excitation with 720–920 nm (GV SD = gray value standard deviation). Data are presented as mean ± standard deviation, n = 4. *Statistically significant increase in the emission signal contrast of rehydrated sections compared with paraffin sections at the excitation wavelengths indicated, p < 0.05. Merged MP and SHG images (C) before and (D) after rehydration. Scale bar equals 60 μm. Color images available online at www.liebertpub.com/tec

FIG. 3.

(A–C) Bright-field images of H&E-stained porcine leaflet sections, followed by (D–F) subsequent MP and SHG imaging of the same regions. Green represents collagen fibers, whereas red indicates elastic fibers. The same sections were destained and again subjected to (G–I) bright-field microscopy as well as (J–L) MP and SHG imaging. Scale bar equals 60 μm. H&E, hematoxylin and eosin. Color images available online at www.liebertpub.com/tec

FIG. 4.

(A) MP and SHG imaging of porcine leaflets while present in paraffin, deparaffinized rehydrated states, and destained H&E sections. Merged images represent the MP (red) and SHG (green) channels. Scale bar equals 60 μm. Bar graphs represent (B, C) MP and (D, E) SHG intensities (GVI = gray value intensities) and contrast (GV SD = gray value standard deviation). Data are presented as mean ± standard deviation, n = 8. *Statistically significant difference between the groups indicated, p < 0.05. Color images available online at www.liebertpub.com/tec