Rapid pseudo-H&E imaging using a fluorescence-inbuilt optical coherence microscopic imaging system

Abstract

A technique using Linnik-based optical coherence microscopy (OCM), with built-in fluorescence microscopy (FM), is demonstrated here to describe cellular-level morphology for fresh porcine and biobank tissue specimens. The proposed method utilizes color-coding to generate digital pseudo-H&E (p-H&E) images. Using the same camera, colocalized FM images are merged with corresponding morphological OCM images using a 24-bit RGB composition process to generate position-matched p-H&E images. From receipt of dissected fresh tissue piece to generation of stitched images, the total processing time is <15 min for a 1-cm² specimen, which is on average two times faster than frozen-section H&E process for fatty or water-rich fresh tissue specimens. This technique was successfully used to scan human and animal fresh tissue pieces, demonstrating its applicability for both biobank and veterinary purposes. We provide an in-depth comparison between p-H&E and human frozen-section H&E images acquired from the same metastatic sentinel lymph node slice (∼10 µm thick), and show the differences, like elastic fibers of a tiny blood vessel and cytoplasm of tumor cells. This optical sectioning technique provides histopathologists with a convenient assessment method that outputs large-field H&E-like images of fresh tissue pieces without requiring any physical embedment.

Fig. 1.

(a) Schematic of the f-OCM experimental setup. BL: broadband light; UVL: ultraviolet light; DM: dichroic mirror; C: container; B: beamsplitter; R: retarder; O: objective; W: water; GP: glass plate; S: specimen; 3D-LS: 3D linear stage; CB: control box; TL: tube lens; CMOS: mono complementary metal-oxide semiconductor camera. (b) Front view of the f-OCM device (AcuOnPath), including the scan machine (left, approximately 40 × 40 × 40 cm³) and the control box (right, approximately 40 × 20 × 40 cm³). (c) Output spectrum and (d) the corresponding interferometric signal of BL.

Fig. 2.

Flowchart of the color-coding process for producing p-H&E images.

Fig. 3.

(a)–(d) Co-localized images of the fresh porcine cerebrum tissue piece: (a) OCM morphological image of the cytoplasmic structure; (b) co-located FM image highlighting the positions of nuclei; (c) red-and-green OCM/FM composite image; and (d) traditional pink-and-blue-violet color scheme with structural features labeled. All images correspond to a tissue depth of 25 μm with the same FOV (908 μm [W] × 736 μm [H]).

Fig. 4.

Two processing methods of fresh tissue samples after dissection by using a surgical blade. Process I shows the material preparation of fresh tissue pieces for producing p-H&E images. Process II shows the material preparation of frozen-sectioned slices for image validation purposes, and comprehensive comparison between p-H&E and H&E-stained images.

Fig. 5.

(a) A large-field p-H&E image (inside large green box of AcuViewer interface) indicated by a navigator (top-right inset figure) with a top-view snapshot of the fresh porcine tissue piece in the container were scanned at a depth of 25 μm. The stitched size of the cropped small green box region is 6.1 × 4.9 mm². (b) and (c) Low- and high-magnified images from red and blue boxes of (a), where the FOVs were 908 μm (W) × 736 μm (H) and 454 μm (W) × 368 μm (H), respectively.

Fig. 6.

P-H&E images of fresh porcine tissue pieces: (a) and (b) lymph node, (c) and (d) liver, and (e) and (f) skeletal muscle. The FOV of images (b), (d), and (f) was 454 μm (W) × 368 μm (H), corresponding to a quarter of the area shown in images (a), (c), and (e), respectively. All images were scanned at a depth of 25 μm.

Fig. 7.

P-H&E images of fresh porcine tissue pieces: (a) and (b) salivary gland, (c) and (d) lung, and (e) and (f) esophagus. The FOV of images (b), (d), and (f) was 454 μm (W) × 368 μm (H), corresponding to a quarter of the area shown in images (a), (c), and (e), respectively. All the images were scanned at a depth of 25 μm.

Fig. 8.

Comparison of p-H&E and frozen-sectioned H&E images using the same fresh tissue pieces prior to warehouse entry of the biobank. Respectively, (a) and (c) show the p-H&E images of the normal and cancerous human thyroid tissue specimens at a scanning depth of 15 μm. (b) and (d) show both the frozen-sectioned H&E images obtained from the original tissue pieces of (a) and (c). Similarly, (e) and (g) separately show the p-H&E images of the normal and cancerous human breast tissue specimens at a scanning depth of 15 μm. (f) and (h) are the frozen-sectioned H&E images produced from the original tissue specimens of (e) and (g). For all figures, the FOV was 908 μm (W) × 736 μm (H).

Fig. 9.

(a) P-H&E and (b) H&E-stained images from the same 10-µm-thick frozen-sectioned slice of fresh IDC-metastatic SLN tissue (each image covers the same 6.5 mm [W] × 6.5 mm [H] area size). Images (c), (e), and (g) are magnified regions from (a), identified by the red, green, and blue boxes, respectively. Similarly, images (d), (f), and (h) are magnified regions from (b), identified by the red, green, and blue dashed boxes, respectively.