Intraoperative assessment of breast surgical margins will be of value for reducing the rate of re-excision surgeries for lumpectomy patients. While frozen-section histology is used for intraoperative guidance of certain cancers, it provides limited sampling of the margin surface (typically <1 % of the margin) and is inferior to gold-standard histology, especially for fatty tissues that do not freeze well, such as breast specimens. Microscopy with ultraviolet surface excitation (MUSE) is a nondestructive superficial optical-sectioning technique that has the potential to enable rapid, high-resolution examination of excised margin surfaces. Here, a MUSE system is developed with fully automated sample translation to image fresh tissue surfaces over large areas and at multiple levels of defocus, at a rate of ∼5 min / cm2. Surface extraction is used to improve the comprehensiveness of surface imaging, and 3-D deconvolution is used to improve resolution and contrast. In addition, an improved fluorescent analog of conventional H&E staining is developed to label fresh tissues within ∼5 min for MUSE imaging. We compare the image quality of our MUSE system with both frozen-section and conventional H&E histology, demonstrating the feasibility to provide microscopic visualization of breast margin surfaces at speeds that are relevant for intraoperative use.
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.
Systemic delivery of 5-aminolevulinic acid leads to enhanced fluorescence image contrast in many tumors due to the increased accumulation of protoporphyrin IX (PpIX), a fluorescent porphyrin that is associated with tumor burden and proliferation. The value of PpIX-guided resection of malignant gliomas has been demonstrated in prospective randomized clinical studies in which a twofold greater extent of resection and improved progression-free survival have been observed. In low-grade gliomas and at the diffuse infiltrative margins of all gliomas, PpIX fluorescence is often too weak to be detected with current low-resolution surgical microscopes that are used in operating rooms. However, it has been demonstrated that high-resolution optical-sectioning microscopes are capable of detecting the sparse and punctate accumulations of PpIX that are undetectable via conventional low-power surgical fluorescence microscopes. To standardize the performance of high-resolution optical-sectioning devices for future clinical use, we have developed an imaging phantom and methods to ensure that the imaging of PpIX-expressing brain tissues can be performed reproducibly. Ex vivo imaging studies with a dual-axis confocal microscope demonstrate that these methods enable the acquisition of images from unsectioned human brain tissues that quantitatively and consistently correlate with images of histologically processed tissue sections.
Video-rate optical-sectioning microscopy of living organisms would allow for the investigation of dynamic biological processes and would also reduce motion artifacts, especially for in vivo imaging applications. Previous feasibility studies, with a slow stage-scanned line-scanned dual-axis confocal (LS-DAC) microscope, have demonstrated that LS-DAC microscopy is capable of imaging tissues with subcellular resolution and high contrast at moderate depths of up to several hundred microns. However, the sensitivity and performance of a video-rate LS-DAC imaging system, with low-numerical aperture optics, have yet to be demonstrated. Here, we report on the construction and validation of a video-rate LS-DAC system that possesses sufficient sensitivity to visualize fluorescent contrast agents that are topically applied or systemically delivered in animal and human tissues. We present images of murine oral mucosa that are topically stained with methylene blue, and images of protoporphyrin IX-expressing brain tumor from glioma patients that have been administered 5-aminolevulinic acid prior to surgery. In addition, we demonstrate in vivo fluorescence imaging of red blood cells trafficking within the capillaries of a mouse ear, at frame rates of up to 30 fps. These results can serve as a benchmark for miniature in vivo microscopy devices under development.
We have developed a line-scanned dual-axis confocal (LS-DAC) microscope with
subcellular resolution suitable for real time diagnostic imaging at shallow depths. This design
serves as a benchtop prototype for a handheld version of the LS-DAC intended for rapid point-of-care
pathology. We have assessed the performance trade-offs between the LS-DAC and a point-scanned
dual-axis confocal (PS-DAC) microscope via diffraction-theory analysis, Monte-Carlo
simulations, and characterization experiments with phantoms and fresh tissues. In addition, we are
exploring the use of a sCMOS detector array and rapid 3D deconvolution to improve the
sensitivity and resolution of our LS-DAC microscope.
A strategy is presented to enable optical-sectioning microscopy with improved contrast and
imaging depth using low-power (0.5 mW) diode laser illumination. While the DAC architecture’s
intersecting illumination and collection beams significantly improves the spatial-filtering and opticalsectioning
performance of confocal microscopy, we propose that modulating the spatial alignment of the
dual-axis beams at a frequency f, such the focal volume signal of the microscope is modulated at 2f, further
provides nearly an order-of-magnitude improvement in optical-sectioning contrast. Lock-in detection is
used to remove the unmodulated background light, thereby enhancing our ability to image deeply within
highly scattering tissues.
We are developing a miniature fiber-optic spectral-detection device and topical-staining protocol
to rapidly detect multiplexed surface-enhanced Raman scattering (SERS) nanoparticles (NPs) targeted to
cell-surface biomarkers in fresh tissues. Ex vivo and in vivo experiments were performed to optimize our
strategy for the rapid detection of multiple cell-surface biomarkers following a brief (5 min) topical
application of SERS NPs on tissues. The simultaneous detection and ratiometric quantification of targeted
and nontargeted NPs allows for an unambiguous assessment of molecular expression that is insensitive to
nonspecific variations in NP concentrations, potentially enabling point-of-care surgical guidance or early
disease detection.
Dual-axis confocal (DAC) microscopy has been found to exhibit superior rejection of out-of-focus and multiply scattered background light compared to conventional single-axis confocal microscopy. DAC microscopes rely on the use of separated illumination and collection beam paths that focus and intersect at a single focal volume (voxel) within tissue. While it is generally recognized that the resolution and contrast of a DAC microscope depends on both the crossing angle of the DAC beams, 2θ , and the focusing numerical aperture of the individual beams, α , a detailed study to investigate these dependencies has not been performed. Contrast and resolution are considered as two main criteria to assess the performance of a point-scanned DAC microscope (DAC-PS) and a line-scanned DAC microscope (DAC-LS) as a function of θ and α . The contrast and resolution of these designs are evaluated by Monte-Carlo scattering simulations and diffraction theory calculations, respectively. These results can be used for guiding the optimal designs of DAC-PS and DAC-LS microscopes.
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