Sanjee Abeytunge

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.

Line-scanning reflectance confocal microscopy of human skin: comparison of full-pupil and divided-pupil configurations.

Line-scanning, with pupil engineering and the use of linear array detectors, may enable simple, small, and low-cost confocal microscopes for clinical imaging of human epithelial tissues. However, a fundamental understanding of line-scanning performance within the highly scattering and aberrating conditions of human tissue is necessary, to translate from benchtop instrumentation to clinical implementation. The results of a preliminary investigation for reflectance imaging in skin are reported.

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.

Large area mapping of excised breast tissue by fluorescence confocal strip scanning: a preliminary feasibility study

Lumpectomy, in conjunction with radiation and chemotherapy drugs, together comprise breast-conserving treatment as an alternative to total mastectomy for patients with breast tumors. The tumor is removed in surgery and sent for pathology processing to assess the margins, a process that takes at minimum several hours, and generally days. If the margins are not clear of tumor, the patient must undergo a second surgery to remove residual tumor. This re-excision rate varies by institution, but can be as high as 60%. Currently, no intraoperative microscopic technique is used routinely to examine tumor margins in breast tissue. A new technique for rapidly scanning large areas of tissue has been developed, called confocal strip scanning, which provides high resolution and seamless mosaics over large areas of intact tissue, with nuclear and cellular resolution and optical sectioning of about 2 microns. Up to 3.5 x 3.5 cm2 of tissue is imaged in 13 minutes at current stage speeds. This technique is demonstrated in freshly excised breast tissue, using a mobile confocal microscope stationed in our pathology laboratory. Twenty-five lumpectomy and mastectomy cases were used as a testing ground for reflectance and fluorescence contrast modes, resolution requirements and tissue fixturing configurations. It was concluded that fluorescent imaging provides the needed contrast to distinguish ducts and lobules from surrounding stromal tissue. Therefore the system was configured with 488 nm illumination, with acridine orange fluorescent dye for nuclear contrast, with the aim of building an image library of malignant and benign breast pathologies.

FPGA-based electronics for confocal line scanners with linear detector arrays

One-dimensional linear detector arrays have been used in the development of microscopes. Our confocal line scanning microscope electronics incorporate two printed circuit boards: control board and detector board. This architecture separates control electronics from detection electronics allowing us to minimize the footprint at microscope detector head. The Field Programmable Gate array (FPGA) on the control board generates timing and synchronization signals to three systems: detector board, frame grabber and galvanometric mirror scanner. The detector is kept away from its control electronics, and the clock and control signals are sent over a differential twisted-pair cable. These differential signals are translated to single ended signals and forwarded to the detector at the microscope detector head. The synchronization signals for the frame grabber are sent over a shielded cable. The control board also generates a saw tooth analog ramp to drive the galvanometric mirror scanner. The analog video output of the detector is fed into an operational amplifier where the white and the black levels are adjusted. Finally the analog video is send to the frame grabber via a shielded cable. FPGA-based electronics offer an inexpensive convenient means to control and synchronize simple line-scanning confocal microscopes.

Full-Pupil Line-Scanning Confocal Microscope for Imaging Weakly Scattering Tissues: Comparison to Divided-Pupil

Confocal reflectance full-pupil and divided-pupil line-scanning microscopes provide optical sectioning of 1-2?m and image nuclear detail in skin. Line-scanning with linear detectors is a simpler alternative to point-scanning for imaging weakly scattering epithelial tissues.

A handheld MEMS-based line-scanned dual-axis confocal microscope for early cancer detection and surgical guidance (Conference Presentation)

Considerable efforts have been recently undertaken to develop miniature optical-sectioning microscopes for in vivo microendoscopy and point-of-care pathology. These devices enable in vivo interrogation of disease as a real-time and noninvasive alternative to gold-standard histopathology, and therefore could have a transformative impact for the early detection of cancer as well as for guiding tumor-resection procedures. Regardless of the specific modality, various trade-offs in size, speed, field of view, resolution, contrast, and sensitivity are necessary to optimize a device for a particular application. Here, a miniature MEMS-based line-scanned dual-axis confocal (LS-DAC) microscope, with a 12-mm diameter distal tip, has been developed for point-of-care pathology. The dual-axis architecture has demonstrated superior rejection of out-of-focus and multiply scattered photons compared to a conventional single-axis confocal configuration. The use of line scanning enables fast frame rates (≥15 frames/sec), which mitigates motion artifacts of a handheld device during clinical use. We have developed a method to actively align the illumination and collection beams in this miniature LS-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 application, enables the device to achieve an axial 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 that this high-speed LS-DAC microscope can achieve high-contrast imaging of fluorescently labeled tissues with sufficient sensitivity for applications such as oral cancer detection and guiding brain-tumor resections.

HiCCE-128: An open hardware FMC module for High-Channel Count Electrophysiology

Electrophysiology has recently evolved into an interactive, high-throughput endeavor. Recording from dozens to hundreds of electrodes is today routine; novel means of manipulating the system in real time, through electrical stimulation, optogenetics or sensory manipulation are allowing us to decipher neural circuit function at an unparalleled rate. To contribute to the wide dissemination of such techniques, we present an open hardware project, High-Channel Count Electrophysiology (HiCCE), aiming to produce low-cost, high-channel count (≥128 channels) electrophysiology instrumentation capable of fast data acquisition rates, real-time processing and feedback capabilities. Our design is centered on an open standard, FPGA Mezzanine Card (FMC), which permits a varied choice of FPGA carrier architectures suited to different laboratory experimental needs. The HiCCE-128, a low-cost highperformance 128-channel data acquisition board for small voltage signals, is being introduced. It is a FMC module that can be operated from any FPGA carrier conforming to the FMC/VITA57 standard. This specialized board with a low input referred noise of about 3 μV is capable of acquiring 128 channels simultaneously at 31.25 kS/s per channel with 16 effective bits of resolution. We present the global architecture and some preliminary measurement to illustrate its potential for electrophysiological and medical applications.

Mobile large area confocal scanner for imaging tumor margins: initial testing in the pathology department

Surgical oncology is guided by examining pathology that is prepared during or after surgery. The preparation time for Mohs surgery in skin is 20-45 minutes, for head-and-neck and breast cancer surgery is hours to days. Often this results in incomplete tumor removal such that positive margins remain. However, high resolution images of excised tissue taken within few minutes can provide a way to assess the margins for residual tumor. Current high resolution imaging methods such as confocal microscopy are limited to small fields of view and require assembling a mosaic of images in two dimensions (2D) to cover a large area, which requires long acquisition times and produces artifacts. To overcome this limitation we developed a confocal microscope that scans strips of images with high aspect ratios and stitches the acquired strip-images in one dimension (1D). Our “Strip Scanner” can image a 10 x 10 mm2 area of excised tissue with sub-cellular detail in about one minute. The strip scanner was tested on 17 Mohs excisions and the mosaics were read by a Mohs surgeon blinded to the pathology. After this initial trial, we built a mobile strip scanner that can be moved into different surgical settings. A tissue fixture capable of scanning up to 6 x 6 cm2 of tissue was also built. Freshly excised breast and head-and-neck tissues were imaged in the pathology lab. The strip-images were registered and displayed simultaneously with image acquisition resulting in large, high-resolution confocal mosaics of fresh surgical tissue in a clinical setting.

Full pupil line-scanning confocal microscope for imaging weakly scattering tissues: comparison to divided pupil

Confocal reflectance full-pupil and divided-pupil line-scanning microscopes provide optical sectioning and image nuclear detail in skin. Line-scanning with linear detectors is a simpler alternative to point-scanning for imaging weakly scattering epidermis and the oral epithelium. With illumination of 830 nm, a water immersion lens of numerical aperture 0.9 and slit width three times smaller than the diffraction-limited line width, the instrumental full width at half maximum (FWHM) optical sectioning (linespread function) for the full-pupil design is 1.4 +/- 0.07 μm, which degrades through fullthickness human epidermis to 2.8 +/- 0.78 μm. The lateral resolution is 0.7±0.10 μm, which degrades to 1.6±0.28 μm through human epidermis. The divided-pupil design demonstrates instrumental optical sectioning of 1.7 μm, which degrades to 7.6 μm through human epidermis. The lateral resolution is 1.0 μm, which degrades to 1.7 μm. Heavy scattering in the dermis decreases contrast. Images of skin in-vivo show nuclear detail as expected with the predicted and experimentally verified sectioning. However, pixel crosstalk and speckle artifact degrade image quality in strongly scattering and aberrating tissues. The sources of degradation (aberration and scattering) are evaluated for the two design to assess the feasibility of these techniques for in vivo imaging.