Sanjeewa Abeytunge

Confocal microscopy with strip mosaicing for rapid imaging over large areas of excised tissue.

Abstract. Confocal mosaicing microscopy is a developing technology platform for imaging tumor margins directly in freshly excised tissue, without the processing required for conventional pathology. Previously, mosaicing on 12-×-12  mm2 of excised skin tissue from Mohs surgery and detection of basal cell carcinoma margins was demonstrated in 9 min. Last year, we reported the feasibility of a faster approach called “strip mosaicing,” which was demonstrated on a 10-×-10  mm2 of tissue in 3 min. Here we describe further advances in instrumentation, software, and speed. A mechanism was also developed to flatten tissue in order to enable consistent and repeatable acquisition of images over large areas. We demonstrate mosaicing on 10-×-10  mm2 of skin tissue with 1-μm lateral resolution in 90 s. A 2.5-×-3.5  cm2 piece of breast tissue was scanned with 0.8-μm lateral resolution in 13 min. Rapid mosaicing of confocal images on large areas of fresh tissue potentially offers a means to perform pathology at the bedside. Imaging of tumor margins with strip mosaicing confocal microscopy may serve as an adjunct to conventional (frozen or fixed) pathology for guiding surgery.

Detection of skin cancer margins in Mohs excisions with high-speed strip mosaicing confocal microscopy: a feasibility study.

Fluorescence confocal mosaicing microscopy is an emerging technology for rapid imaging of nuclear and morphological detail directly in excised tissue, without the need for frozen or fixed section processing. Basal cell carcinomas (BCCs) can be detected with high sensitivity and specificity in Mohs excisions with this approach. For translation to clinical trials and towards potentially routine implementation, a new and faster approach called strip mosaicing confocal microscopy was recently developed.

Performance of full-pupil line-scanning reflectance confocal microscopy in human skin and oral mucosa in vivo

Point-scanning reflectance confocal microscopes continue to be successfully translated for detection of skin cancer. Line-scanning, with the use of a single scanner and a linear-array detector, offers a potentially smaller, simpler and lower cost alternative approach, to accelerate widespread dissemination into the clinic. However, translation will require an understanding of imaging performance deep within scattering and aberrating human tissues. We report the results of an investigation of the performance of a full-pupil line-scanning reflectance confocal microscope in human skin and oral mucosa, in terms of resolution, optical sectioning, contrast, signal-to-noise ratio, imaging and the effect of speckle noise.

Miniature in vivo MEMS-based line-scanned dual-axis confocal microscope for point-of-care pathology.

There is a need for miniature optical-sectioning microscopes to enable in vivo interrogation of tissues as a real-time and noninvasive alternative to gold-standard histopathology. Such devices could have a transformative impact for the early detection of cancer as well as for guiding tumor-resection procedures. Miniature confocal microscopes have been developed by various researchers and corporations to enable optical sectioning of highly scattering tissues, all of which have necessitated various trade-offs in size, speed, depth selectivity, field of view, resolution, image contrast, and sensitivity. In this study, a miniature line-scanned (LS) dual-axis confocal (DAC) microscope, with a 12-mm diameter distal tip, has been developed for clinical point-of-care pathology. The dual-axis architecture has demonstrated an advantage over the conventional single-axis confocal configuration for reducing background noise from out-of-focus and multiply scattered light. The use of line scanning enables fast frame rates (16 frames/sec is demonstrated here, but faster rates are possible), which mitigates motion artifacts of a hand-held device during clinical use. We have developed a method to actively align the illumination and collection beams in a DAC microscope through the use of a pair of rotatable alignment mirrors. Incorporation of a custom objective lens, with a small form factor for in vivo clinical use, enables our device to achieve an optical-sectioning thickness and lateral resolution of 2.0 and 1.1 microns respectively. Validation measurements with reflective targets, as well as in vivo and ex vivo images of tissues, demonstrate the clinical potential of this high-speed optical-sectioning microscopy device.

Reflectance confocal microscope for imaging oral tissues in vivo, potentially with line scanning as a low-cost approach for clinical use

Reflectance confocal microscopy with a line scanning approach potentially offers a smaller, simpler and less expensive approach than traditional methods of point scanning for imaging in living tissues. With one moving mechanical element (galvanometric scanner), a linear array detector and off-the-shelf optics, we designed a compact (102x102x76mm) line scanning confocal reflectance microscope (LSCRM) for imaging human tissues in vivo in a clinical setting. Custom-designed electronics, based on field programmable gate array (FPGA) logic has been developed. With 405 nm illumination and a custom objective lens of numerical aperture 0.5, lateral resolution was measured to be 0.8 um (calculated 0.64 um). The calculated optical sectioning is 3.2 um. Preliminary imaging shows nuclear and cellular detail in human skin and oral epithelium in vivo. Blood flow is also visualized in the deeper connective tissue (lamina propria) in oral mucosa. Since a line is confocal only in one dimension (parallel) but not in the other, the detection is more sensitive to multiply scattered out of focus background noise than in the traditional point scanning configuration. Based on the results of our translational studies thus far, a simpler, smaller and lower-cost approach based on a LSCRM appears to be promising for clinical imaging.

Assessment of fresh breast tissue specimens with confocal strip-mosaicking microscopy in an emulated pathology setting (Conference Presentation)

Confocal microscopy is in clinical use to diagnose skin cancers in the United States and in Europe. Potentially, this technology may provide bed-side pathology in breast cancer surgery during tumor removal. Initial studies have described major findings of invasive breast cancers as seen on fluorescence confocal microscopy. In many of these studies the region of interest (ROI) used in the analysis was user-selected and small (typically 15 square-mm). Although these important findings open exploration into rapid pathology, further development and implementation in a surgical setting will require examination of large specimens in a blinded fashion that will address the needs of typical surgical settings. In post surgery pathology viewing, pathologists inspect the entire pathology section with a low (2X) magnification objective lens initially and then zoomed in to ROIs with higher magnification lenses (10X to 40X) magnifications to further investigate suspected regions. In this study we explore the possibility of implementation in a typical surgical setting with a new microscope, termed confocal strip-mosaicking microscope (CSM microscope), which images an area of 400 square-mm (2 cm x 2 cm) of tissue with cellular level resolution in 10 minutes. CSM images of 34 human breast tissue specimens from 18 patients were blindly analyzed by a board-certified pathologist and correlated with the corresponding standard fixed histopathology. Invasive tumors and benign tissue were clearly identified in CSM images. Thirty specimens were concordant for images-to-histopathology correlation while four were discordant. Preliminary results from on-going work to molecularly target tumor margin will also be presented.

A handheld optical-sectioning device for early detection and surgical guidance

Miniature dual-axis confocal (DAC) microscopes are being developed for the early detection of oral cancers and for guiding brain tumor resection. The design of MEMS-scanned DAC microscopes, and their analysis and optimization are described.

Abstract P2-03-03: Feasibility of evaluation of breast tissue using confocal microscopy with strip mosaicing

Background: Confocal microscopic strip mosaicing (CSM) provides noninvasive optical sectioning and high resolution, which allows for imaging of nuclear and morphological detail in freshly excised tissue. CSM can image large areas of tissue at micron-level resolution in minutes, which may offer an advantage over standard histology that requires days. We have conducted a preliminary investigation of the feasibility of this technology for the evaluation of breast tissue from surgical excision specimens. Design: In a prospective study, 80 fresh human breast tissue samples from surgical excision specimens of 24 patients were imaged using a prototype confocal strip scanner. Fresh tissue specimens were immersed in Acridine Orange (AO) for 45 seconds to stain the nuclei, then pressed against the glass imaging window and imaged with a 30X, 0.75 numerical aperture (measured) objective lens and a 488 nm laser. Images were acquired in two modes of contrast: in fluorescence (with AO), showing nuclear morphology, and in reflectance (endogenous), showing stroma. The use of fluorescence for nuclear staining mimics the use of hematoxylin in pathology, and the use of reflectance eosin. Use of two contrast modes allows the fluorescence image to be colorized purple and the reflectance image pink, producing confocal strip mosaics that mimic H&E histology in appearance. Specimens were subsequently fixed in formalin and routinely processed to obtain H&E stained sections. H&E and confocal images were compared by the study pathologist (M.M.) Results: Freshly excised breast tissue samples as large as 2 cm x 2 cm were imaged in less than five minutes, with 1-micron resolution and measured optical sectioning of 6 microns. We compared the CSM images against standard histopathology images. In our series we evaluated the following histologies: 12 invasive carcinoma (11 ductal, 1 lobular), 3 ductal carcinoma in-situ, 3 lobular carcinoma in situ, 1 atypical lobular hyperplasia, 1 atypical ductal hyperplasia and various benign lesions such as fat necrosis, fibrocystic changes, and ductal hyperplasia. In confocal images invasive and in situ carcinoma as well as benign ducts and lobules were distinguished from surrounding stromal tissue. Limitations that are typically encountered in standard histology, such as distinguishing low grade ductal carcinoma in situ (DCIS) from lobular carcinoma in situ (LCIS) or atypical proliferations were encountered in the grayscale confocal images as well. Conclusion: In this initial feasibility study, CSM produced images that could be diagnosed as benign or neoplastic by the study pathologist. Further study is needed to build an image library of breast histology and compare reproducibility of histologic diagnoses between CSM (grayscale and colorized images) and traditional optical microscopy, and assess interobserver reproducibility in diagnosis. CSM potentially provides rapid and noninvasive evaluation of breast parenchyma, and has a potential application for intraoperative margin assessment of resected breast specimens. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P2-03-03.

Performance of line-scanning confocal microscopy in human skin: investigation of potential for clinical translation

Line-scanning, using 8-10 optical components, linear-array detectors and custom-FPGA electronics, may enable smaller, simpler and lower-cost confocal microscopes to accelerate translation to the clinic. The adaptability of commercially available low-cost array detectors for confocal microscopy is being investigated. Measurements of optical sectioning and lateral resolution showed good agreement with theory, and are comparable to that of point-scanning systems. LSFs through full thickness of human epidermis show a two-fold degradation in sectioning performance. Imaging of human epidermis in vivo demonstrates nuclear and cellular detail down to the basal layer with a bench top setup and also a compact clinical prototype. Blood flow in oral mucosa can be imaged using the clinical prototype. However, speckle and background noise degrade contrast and resolution of the image.

Full-pupil versus divided-pupil confocal line-scanners for reflectance imaging of human skin in vivo

A full-pupil confocal line-scanning microscope is under development for imaging human skin in vivo in reflectance. The new design potentially offers an alternative to current point- and line-scanners that may simplify the optics, electronics and mechanics, and lead to simpler and smaller confocal microscopes. With a combination of a cylindrical lens and an objective lens, the line-scanner creates a focused line of laser light in the object plane within tissue. An oscillating galvanometric mirror scans the focused line transverse to its axis. The backscattered light from the tissue is de-scanned and focused onto a linear CMOS detector array. Preliminary measurements of the axial line-spread function, with a 30x, 0.9-NA water immersion objective lens and illumination wavelength of 633 nm, determined the optical sectioning to be 10 μm. The new design is simple, requiring only eight optical components. However, the disadvantage is non-confocality in one dimension that results in 20% weaker sectioning than with a point-scanner, and reduced contrast in scattering tissue. The images of standard reflective targets such as a mirror and grating as well as dermis-like scattering target such as paper offer a preliminary glimpse into the performance of the line-scanner. A similar alternative design is the divided-pupil (theta) line-scanner, which provides 50% weaker sectioning than with a point scanner, but better contrast and less speckle due to the theta configuration. Such line scanners may prove useful for routine imaging of humans in clinical settings.