Abstract
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.
Highlights
The miniature LS-dual-axis confocal (DAC) microscope developed in this study consists of three major modules (Fig. 1): (1) a main body housing the optics that are unique for the illumination beam and collection beam, the microelectromechanical system (MEMS) scanning mirror and alignment mirrors; (2) a relay objective lens with a lens cap that provides 3x magnification from the focal plane of the microscope within tissue to the back focal plane that is scanned by the main-body optics; and (3) a detector array module
Note that there is vignetting at the edges of the field of view (FOV) due to slight field curvature introduced by the scanning MEMS mirror
Previous miniature point-scanned dual-axis confocal (PS-DAC) microscopes have had modest axial resolutions in the 5 – 10 μm range, limited frame rates of up to 4 frames/sec, and have relied on sophisticated MEMS scanners designed to operate at frequencies in the kHz range [14, 21]
Summary
The most reliable method of diagnosing diseases has been the microscopic visualization of glandular, cellular, and subcellular features from thinly sectioned tissues mounted on glass slides. Histopathology requires the invasive removal of tissues from a patient, which introduces risk of iatrogenic injury and hinders its use for the diagnostic screening of sensitive sites or large areas of tissue In light of these concerns, there has been a growing consensus of the need for miniature in vivo microscopes to enable noninvasive point-of-care pathology. Coupled with the increased pixel dwell times afforded by line scanning, the LS-DAC architecture is capable of sensitive optical-sectioning microscopy of fresh tissues (ex vivo and in vivo) at high frame rates (>16 frames/sec), as exhibited here as well as in a previous study with a large tabletop LS-DAC prototype [34, 35, 39]
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