Gary Peterson

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.

Quantitative molecular phenotyping with topically applied SERS nanoparticles for intraoperative guidance of breast cancer lumpectomy

There is a need to image excised tissues during tumor-resection procedures in order to identify residual tumors at the margins and to guide their complete removal. The imaging of dysregulated cell-surface receptors is a potential means of identifying the presence of diseases with high sensitivity and specificity. However, due to heterogeneities in the expression of protein biomarkers in tumors, molecular-imaging technologies should ideally be capable of visualizing a multiplexed panel of cancer biomarkers. Here, we demonstrate that the topical application and quantification of a multiplexed cocktail of receptor-targeted surface-enhanced Raman scattering (SERS) nanoparticles (NPs) enables rapid quantitative molecular phenotyping (QMP) of the surface of freshly excised tissues to determine the presence of disease. In order to mitigate the ambiguity due to nonspecific sources of contrast such as off-target binding or uneven delivery, a ratiometric method is employed to quantify the specific vs. nonspecific binding of the multiplexed NPs. Validation experiments with human tumor cell lines, fresh human tumor xenografts in mice, and fresh human breast specimens demonstrate that QMP imaging of excised tissues agrees with flow cytometry and immunohistochemistry, and that this technique may be achieved in less than 15 minutes for potential intraoperative use in guiding breast-conserving surgeries.

Combined reflectance confocal microscopy-optical coherence tomography for delineation of basal cell carcinoma margins: an ex vivo study

Abstract. We present a combined reflectance confocal microscopy (RCM) and optical coherence tomography (OCT) approach, integrated within a single optical layout, for diagnosis of basal cell carcinomas (BCCs) and delineation of margins. While RCM imaging detects BCC presence (diagnoses) and its lateral spreading (margins) with measured resolution of ∼1  μm, OCT imaging delineates BCC depth spreading (margins) with resolution of ∼7  μm. When delineating margins in 20 specimens of superficial and nodular BCCs, depth could be reliably determined down to ∼600  μm, and agreement with histology was within about ±50  μm.

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.

Evaluation of breast tissue with confocal strip-mosaicking microscopy: a test approach emulating pathology-like examination

Abstract. Confocal microscopy is an emerging technology for rapid imaging of freshly excised tissue without the need for frozen- or fixed-section processing. Initial studies have described imaging of breast tissue using fluorescence confocal microscopy with small regions of interest, typically 750×750  μm2. We present exploration with a microscope, termed confocal strip-mosaicking microscope (CSM microscope), which images an area of 2×2  cm2 of tissue with cellular-level resolution in 10 min of excision. Using the CSM microscope, we imaged 34 fresh, human, large breast tissue specimens from 18 patients, blindly analyzed by a board-certified pathologist and subsequently correlated with the corresponding standard fixed histopathology. Invasive tumors and benign tissue were clearly identified in CSM strip-mosaic images. Thirty specimens were concordant for image-to-histopathology correlation while four were discordant.

A BRDF model for scratches and digs

Scratch-dig is an acknowledged cosmetic specification that is often misused as a specification to limit scattered light. In spite of its shortcomings, the stray light analyst is often called upon to write this specification, or at least approve it. An analytic model that relates a bidirectional reflectance distribution function (BRDF) to a scratch-dig specification is proposed. Applying scalar diffraction theory and some idealizations for the shape, orientation, and number of scratches and digs; the magnitude and functional form of the BRDF is derived. The effective BRDF associated with the scratch dig specification is compared with the magnitude and functional form of the BRDF from surface roughness and contamination.

Stray light test station for measuring point source transmission and thermal background of visible and infrared sensors

Breault Research Organization has designed and built a stray light test station. The station measures the point source transmission and background thermal irradiance of visible and infrared sensors. Two beam expanders, including a large 0.89 meter spherical mirror, expand and collimate light from laser sources at 0.658 and 10.6 µm. The large mirror is mounted on a gimbal to illuminate sensors at off-axis angles from 0° to 10°, and azimuths from 0° to 180°. Sensors with apertures as large as 0.3 meters can be tested with the existing facility. The large mirror is placed within a vacuum chamber so cryogenic infrared sensors can be tested in a vacuum environment. A dark cryogenic cold plate can be translated into the field of view of a sensor to measure its background thermal irradiance.

Wide-field imaging combined with confocal microscopy using a miniature f/5 camera integrated within a high NA objective lens

Wide-field (WF) imaging paired with reflectance confocal microscopy can noninvasively detect skin cancer with high accuracy. However, two separate devices are required to perform each imaging procedure. We describe a new concept that integrates the two into one device: a miniature WF color camera within the objective lens used for confocal microscopy, providing simultaneous sub-surface cellular imaging and WF surface morphologic imaging. The camera, inserted between a hyperhemisphere front lens and a back lens group of the objective, commands a field of view of 4.0 mm, with a resolution better than 30 μm, while confocal optical sectioning is preserved at sharper than 2.5 μm.

In vivo intraoral reflectance confocal microscopy of an amalgam tattoo

The majority of oral pigmentations are benign lesions such as nevi, melanotic macules, melanoacanthomas or amalgam tattoos. Conversely, mucosal melanomas are rare but often lethal; therefore, excluding oral melanomas in this setting is crucial. Reflectance confocal microscopy is a non-invasive, in vivo imaging system with cellular resolution that has been used to distinguish benign from malignant pigmented lesions in the skin, and more recently in the mucosa. However, lesions located posteriorly in the oral cavity are difficult to assess visually and difficult to biopsy due to their location. Herein we present a patient with previous multiple melanomas presenting with an oral amalgam tattoo in the buccal mucosa, which was imaged using an intraoral telescopic probe attached to a commercially available handheld RCM. In this case report we describe this novel probe, the first RCM description of an amalgam tattoo and we discuss its differences with the findings described in oral melanomas.

Progress in reflectance confocal microscopy for imaging oral tissues in vivo

We report progress in development and feasibility testing of reflectance confocal microscopy (RCM) for imaging in the oral cavity of humans. We adapted a small rigid relay telescope (120mm long x 14mm diameter) and a small water immersion objective lens (12mm diameter, NA 0.7) to a commercial handheld RCM scanner (Vivascope 3000, Caliber ID, Rochester NY). This scanner is designed for imaging skin but we adapted the front end (the objective lens and the stepper motor that axially translates) for intra-oral use. This adaption required a new approach to address the loss of the automated stepper motor for acquisition of images in depth. A helical spring-like cap (with a coverslip to contact tissue) was designed for approximately 150 um of travel. Additionally other methods for focusing optics were designed and evaluated. The relay telescope optics is being tested in a clinical setting. With the capture of video and “video-mosaicing”, extended areas can be imaged. The feasibility of imaging oral tissues was initially investigated in volunteers. RCM imaging in buccal mucosa in vivo shows nuclear and cellular detail in the epithelium and epithelial junction, and connective tissue and blood flow in the underlying lamina propria. Similar detail, including filiform and fungiform papillae, can be seen on the tongue in vivo. Clinical testing during head and neck surgery is now in progress and patients are being imaged for both normal tissue and cancerous margins in lip and tongue mucosa.