High-Resolution Rapid Diagnostic Imaging of Whole Prostate Biopsies Using Video-Rate Fluorescence Structured Illumination Microscopy

Abstract

Rapid assessment of prostate core biopsy pathology at the point-of-procedure could provide benefit in a variety of clinical situations. Even with advanced transrectal ultrasound guidance and saturation biopsy protocols, prostate cancer can be missed in up to half of all initial biopsy procedures. In addition, collection of tumor specimens for downstream histologic, molecular, and genetic analysis is hindered by low tumor yield due to inability to identify prostate cancer grossly. However, current point-of-procedure pathology protocols, such as frozen section analysis (FSA), are destructive and too time- and labor-intensive to be practical or economical. Ex vivo microscopy of the excised specimens, stained with fast-acting fluorescent histology dyes, could be an attractive nondestructive alternative to FSA. In this work, we report the first demonstration of video-rate structured illumination microscopy (VR-SIM) for rapid high-resolution diagnostic imaging of prostate biopsies in realistic point-of-procedure timeframes. Large mosaic images of prostate biopsies stained with acridine orange are rendered in seconds and contain excellent contrast and detail, exhibiting close correlation with corresponding hematoxylin and eosin histology. A clinically relevant review of VR-SIM images of 34 unfixed and uncut prostate core biopsies by two independent pathologists resulted in an area under the receiver operative curve (AUC) of 0.82-0.88, with a sensitivity ranging from 63% to 88% and a specificity ranging from 78% to 89%. When biopsies contained more than 5% tumor content, the sensitivity improved to 75% to 92%. The image quality, speed, minimal complexity, and ease of use of VR-SIM could prove to be features in favor of adoption as an alternative to destructive pathology at the point-of-procedure.

Figure 1

VR-SIM images and subsequent H&E slide images of Biopsy A1 confirmed as malignant. A) Wide-field (i.e., without SIM) image of the entire biopsy, B) VR-SIM mosaic image of the entire biopsy, comprising 205.5 megapixels. C) Digital image of the corresponding H&E section. D) VR-SIM and E) H&E zoom images of the regions of interest marked by the correspondingly labeled boxes in B and C, depicting an area of normal skeletal muscle and fibrous stroma. F) VR-SIM and G) H&E zoom images of the regions of interest marked by the correspondingly labeled boxes in B and C, depicting an area of malignant glands.

Figure 2

VR-SIM images and subsequent H&E slide images of biopsy A19 confirmed as malignant. A) VR-SIM mosaic image of the entire biopsy, comprising 205.5 megapixels, B) Digital image of the corresponding H&E section. Zooms of the dashed yellow boxes in A and B are shown in C and D, respectively. Corresponding areas of interest between the VR-SIM image and the H&E image are denoted by numbered circles (1 = Gleason grade 3 cancer, 2 = Gleason grade 4 cancer, and 3 = benign glands).

Figure 3

VR-SIM images and subsequent H&E slide images of biopsy B18 classified as non-malignant, but containing high-grade prostatic intraepithelial neoplasia (HGPIN). A) VR-SIM mosaic image of the entire biopsy, comprising 205.5 megapixels, and B) Digital image of the corresponding H&E section. Zooms of the dashed yellow boxes in A and B are shown in C and D, respectively. C) A close-up of a gland containing PIN (yellow arrow) in the VR-SIM image, with D) the corresponding H&E image.

Figure 4

Examples of commonly observed features in prostate biopsies (A, C, E), along with the corresponding H&E section histology (B, D, F). A & C: Corpora amylacea, C & D: Perineural invasion, E & F: Nerve ganglion.