Second and third harmonic generation microscopy visualizes key structural components in fresh unprocessed healthy human breast tissue

Abstract

Real‐time assessment of excised tissue may help to improve surgical results in breast tumor surgeries. Here, as a step towards this purpose, the potential of second and third harmonic generation (SHG, THG) microscopy is explored. SHG and THG are nonlinear optical microscopic techniques that do not require labeling of tissue to generate 3D images with intrinsic depth‐sectioning at sub‐cellular resolution. Until now, this technique had been applied on fixated breast tissue or to visualize the stroma only, whereas most tumors start in the lobules and ducts. Here, SHG/THG images of freshly excised unprocessed healthy human tissue are shown to reveal key breast components—lobules, ducts, fat tissue, connective tissue and blood vessels, in good agreement with hematoxylin and eosin histology. DNA staining of fresh unprocessed mouse breast tissue was performed to aid in the identification of cell nuclei in label‐free THG images. Furthermore, 2‐ and 3‐photon excited auto‐fluorescence images of mouse and human tissue are collected for comparison. The SHG/THG imaging modalities generate high quality images of freshly excised tissue in less than a minute with an information content comparable to that of the gold standard, histopathology. Therefore, SHG/THG microscopy is a promising tool for real‐time assessment of excised tissue during surgery.

Recent advances in nonlinear optics for bio-imaging applications

“…Huizen et al applied the THG combined SHG microscopy to imaging of fresh unprocessed healthy human tissue. They compared the images with the gold standard hematoxylin and eosin histological images and identify the key structural components in the tissue 143 .…”

Epithelium segmentation and automated Gleason grading of prostate cancer via deep learning in label‐free multiphoton microscopic images

“…MPM has advantages of increasing the imaging depth, reducing phototoxicity and photobleaching , and the ability to generate pathology‐like images based on the natural intrinsic fluorescence properties of tissues. It has been widely used in cancer research and diagnosis, such as brain cancer , breast cancer , and esophageal cancer . In 2011, Tewari et al employed MPM to identify relevant prostatic and periprostatic structures in prostate cancer surgery.…”

Structural Assessment of Scars Using Optical Techniques

“…Collagen fibers induce a bright SHG signal, whereas intrinsic fluorophores in the cytoplasm of the dermal cells outline the cells. In vitro images were acquired using a previously described setup [87]. Scale bar: 500 μm Simultaneous recording allows a redox ratio to be calculated.…”

“…19.2 Combined 2PEF (green) and SHG (red) image of elastin and collagen fibers, respectively. Unprocessed ex vivo human skin, specifically the reticular dermis, was imaged using the setup described in [87]. A stack of 35 images was acquired in the Z-axis.…”