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.
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.
For the removal of superficial and nodular basal cell carcinomas (BCCs), laser ablation provides certain advantages relative to other treatment modalities. However, efficacy and reliability tend to be variable because tissue is vaporized such that none is available for subsequent histopathological examination for residual BCC (and to confirm complete removal of tumor). Intra-operative reflectance confocal microscopy (RCM) may provide a means to detect residual tumor directly on the patient and guide ablation. However, optimization of ablation parameters will be necessary to control collateral thermal damage and preserve sufficient viability in the underlying layer of tissue, so as to subsequently allow labeling of nuclear morphology with a contrast agent and imaging of residual BCC. We report the results of a preliminary study of two key parameters (fluence, number of passes) vis-à-vis the feasibility of labeling and RCM imaging in human skin ex vivo, following ablation with an erbium:yttrium aluminum garnet laser.
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.
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.
Laser ablation may be a promising method for removal of skin lesions, with the potential for better cosmetic outcomes and reduced scarring and infection. An obstacle to implementing laser ablation is that the treatment leaves no tissue for histopathological analysis. Pre-operative and intra-operative mapping of BCCs using confocal microscopy may guide the ablation of the tumor until all tumor is removed. We demonstrate preliminary feasibility of confocal microscopy to guide laser ablation of BCCs in freshly excised tissue from Mohs surgery. A 2940 nm Er:YAG laser provides efficient ablation of tumor with reduced thermal damage to the surrounding tissue.
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 mm 2 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 mm 2 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 mm 2 of skin tissue with 1-μm lateral resolution in 90 s. A 2.5-×-3.5 cm 2 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.
Confocal mosaicing microscopy is a developing technology platform for imaging tumor
margins directly in fresh tissue, without the processing that is required for conventional
pathology. Previously, basal cell carcinoma margins were detected by mosaicing of
confocal images of 12 x 12 mm2 of excised tissue from Mohs surgery. This mosaicing
took 9 minutes. Recently we reported the initial feasibility of a faster approach called
"strip mosaicing" on 10 x 10 mm2 of tissue that was demonstrated in 3 minutes. In this
paper we report further advances in instrumentation and software. Rapid mosaicing of
confocal images on large areas of fresh tissue potentially offers a means to perform
pathology at the bedside. Thus, strip mosaicing confocal microscopy may serve as an
adjunct to pathology for imaging tumor margins to guide surgery.
Confocal point-scanning microscopy has been showing promise in the detection, diagnosing and mapping of skin lesions
in clinical settings. The noninvasive technique allows provides optical sectioning and cellular resolution for in vivo
diagnosis of melanoma and basal cell carcinoma and pre-operative and intra-operative mapping of margins. The imaging
has also enabled more accurate "guided" biopsies while minimizing the otherwise large number of "blind" biopsies.
Despite these translational advances, however, point-scanning technology remains relatively complex and expensive.
Line-scanning technology may offer an alternative approach to accelerate translation to the clinic. Line-scanning, using
fewer optical components, inexpensive linear-array detectors and custom electronics, may enable smaller, simpler and
lower-cost confocal microscopes. A line is formed using a cylindrical lens and scanned through the back focal plane of
the objective with a galvanometric scanner. A linear CCD is used for detection. Two pupil configurations were
compared for performance in imaging human tissue. In the full-pupil configuration, illumination and detection is made
through the full objective pupil. In the divided pupil approach, half the pupil is illuminated and the other half is used for
detection. The divided pupil configuration loses spatial and axial resolution due to a diminished NA, but the sectioning
capability and rejection of background is improved. Imaging in skin and oral mucosa illustrate the performance of the
two configurations.
Imaging large areas of tissue rapidly and with high resolution may enable rapid pathology at the bedside. The limited field of view of high-resolution microscopes requires the merging of multiple images that are taken sequentially to cover a large area. This merging or mosaicing of images requires long acquisition and processing times, and produces artifacts. To reduce both time and artifacts, we developed a mosaicing method on a confocal microscope that images morphology in large areas of excised tissue with sub-cellular detail. By acquiring image strips with aspect ratios of 10:1 and higher (instead of the standard ∼1:1) and "stitching" them in software, our method images 10×10 mm2 area of tissue in about 3 min. This method, which we call "strip mosaicing," is currently three times as fast as our previous method.
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.
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