Gigapixel surface imaging of radical prostatectomy specimens for comprehensive detection of cancer-positive surgical margins using structured illumination microscopy

Abstract

Achieving cancer-free surgical margins in oncologic surgery is critical to reduce the need for additional adjuvant treatments and minimize tumor recurrence; however, there is a delicate balance between completeness of tumor removal and preservation of adjacent tissues critical for normal post-operative function. We sought to establish the feasibility of video-rate structured illumination microscopy (VR-SIM) of the intact removed tumor surface as a practical and non-destructive alternative to intra-operative frozen section pathology, using prostate cancer as an initial target. We present the first images of the intact human prostate surface obtained with pathologically-relevant contrast and subcellular detail, obtained in 24 radical prostatectomy specimens immediately after excision. We demonstrate that it is feasible to routinely image the full prostate circumference, generating gigapixel panorama images of the surface that are readily interpreted by pathologists. VR-SIM confirmed detection of positive surgical margins in 3 out of 4 prostates with pathology-confirmed adenocarcinoma at the circumferential surgical margin, and furthermore detected extensive residual cancer at the circumferential margin in a case post-operatively classified by histopathology as having negative surgical margins. Our results suggest that the increased surface coverage of VR-SIM could also provide added value for detection and characterization of positive surgical margins over traditional histopathology.

DRAQ5 and Eosin (‘D&E’) as an Analog to Hematoxylin and Eosin for Rapid Fluorescence Histology of Fresh Tissues

“…We have previously demonstrated the ability to diagnose thick tissues using acridine orange and SIM [28, 31]. While the pseudocoloring process can be used with fluorophores other than DRAQ5 and Eosin, DRAQ5’s uniqueness as a DNA-selective, membrane permeant dye with far-red spectral properties provides the potential for it to be paired with many different fluorophores with little spectral interference [32].…”

“…Multiple biopsies may be placed in a cassette and stained concurrently, such that increasing the number of biopsies to be imaged increases the total processing time only by the imaging time per biopsy and not the staining time. By increasing illumination intensity to improve signal, imaging time could be further reduced by a factor of 10 to 30 ms per frame as shown in our prior work using a very bright fluorophore, acridine orange [13, 28]. After imaging, the imaged surface of the biopsy was marked with histological ink and fixed in 10% formalin for a minimum of 48 hours.…”

Partial nephrectomy margin imaging using structured illumination microscopy

“…We have previously developed a video‐rate structured illumination microscope (VR‐SIM) with the area‐throughput and subcellular resolution necessary to evaluate surgical margins intraoperatively . We have demonstrated the utility of VR‐SIM for rapid pathology imaging of small needle‐core renal biopsies , large prostate punch biopsies and radical prostatectomy margins . In this work, we demonstrate the potential of VR‐SIM as a practical and useful alternative to FSA for intraoperative microscopy of PN specimens to detect PSMs.…”

“…Currently, multiple ex vivo microscopy methods are sufficient for imaging small fragments of excised tissue on the order of 1 cm 2 or less , and some have been demonstrated for larger tissue resections on the order of 10 cm 2 while maintaining subcellular lateral resolution . However, surgical tumor resection surface areas in organs such as, for example the breast , prostate and kidney (this paper) can greatly exceed 10 cm 2 . Therefore, very high throughput ex vivo microscopy methods are needed for real‐time PSM detection in these organs with large tumor resection surfaces.…”

Label-free optical imaging technologies for rapid translation and use during intraoperative surgical and tumor margin assessment

“…This method achieved rapid large-area imaging at a modest depth of 50 μm in fresh tissue specimens; however, due to the image detection plane being oriented at 45 deg relative to the specimen surface, the number of pixels per image frame that corresponded to the actual specimen surface was not maximized, compared to en-face approaches. Using en-face structured illumination microscopy, Wang et al 23 imaged the circumferential surface (true surgical margin) of freshly excised radical prostatectomy specimens without any slicing, compression, liquid immersion, or other physical manipulation of the specimens. An imaging throughput rate of 18 megapixels per second of surface image data at 1.3-μm resolution was achieved, with the imaging speed limited by the microscope stage movement and not by the raw imaging throughput, which was capable of >130 megapixels per second.…”