There would be clinical value in a miniature optical-sectioning microscope to enable in vivo interrogation of tissues as a real-time and noninvasive alternative to gold-standard histopathology for early disease detection and surgical guidance. To address this need, a reflectance-based handheld line-scanned dual-axis confocal microscope was developed and fully packaged for label-free imaging of human skin and oral mucosa. This device can collect images at >15 frames / s with an optical-sectioning thickness and lateral resolution of 1.7 and 1.1 μm, respectively. Incorporation of a sterile lens cap design enables pressure-sensitive adjustment of the imaging depth by the user during clinical use. In vivo human images and videos are obtained to demonstrate the capabilities of this high-speed optical-sectioning microscopy device.
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.
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.
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.
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.
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.
The line-scanning confocal microscope is simpler than a point-scanning confocal microscope and allows for a smaller
and lower cost footprint, making it attractive for endoscopic clinical use. The optical configuration affects image fidelity.
Here, we present a benchtop version of an endoscopic line-scanning confocal microscope for intraoral imaging, with a
divided pupil and optimal detection configuration (magnification, pixel-to-resolution ratio) to enhance image fidelity.
Improved sectioning performance and reduction of "speckle" noise are demonstrated. A topology for use of a
deformable MEMs mirror-based optical axial focus control for imaging in depth is presented. Preliminary images of
human oral mucosa in vivo demonstrate feasibility for clinical application.
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.
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.
KEYWORDS: Sensors, Clocks, Signal detection, Electronics, Field programmable gate arrays, Microscopes, Control systems, Analog electronics, Confocal microscopy, Scanners
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.
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.
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.
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