Miniature in vivo MEMS-based line-scanned dual-axis confocal microscope for point-of-care pathology.
Ontology highlight
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.
Project description:A handheld line-scanned dual-axis confocal (LS-DAC) microscope has been developed for high-speed (16 frames/s) fluorescence imaging of tissues with sub-nuclear resolution. This is the first miniature fluorescence LS-DAC system that has been fully packaged for handheld clinical use on patients. A novel micro-electro-mechanical system scanning mechanism, with synchronized tilting and pistoning, is used to achieve flat-field en face imaging. We show that this facilitates video mosaicking to generate images that sample an extended lateral field of view.
Project description: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.
Project description: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.
Project description:Laser scanning microscopes can be miniaturized for in vivo imaging by substituting optical microelectromechanical system (MEMS) devices in place of larger components. The emergence of multifunctional active optical devices can support further miniaturization beyond direct component replacement because those active devices enable diffraction-limited performance using simpler optical system designs. In this paper, we propose a catadioptric microscope objective lens that features an integrated MEMS device for performing biaxial scanning, axial focus adjustment, and control of spherical aberration. The MEMS-in-the-lens architecture incorporates a reflective MEMS scanner between a low-numerical-aperture back lens group and an aplanatic hyperhemisphere front refractive element to support high-numerical-aperture imaging. We implemented this new optical system using a recently developed hybrid polymer/silicon MEMS three-dimensional scan mirror that features an annular aperture that allows it to be coaxially aligned within the objective lens without the need for a beam splitter. The optical performance of the active catadioptric system is simulated and imaging of hard targets and human cheek cells is demonstrated with a confocal microscope that is based on the new objective lens design.
Project description:This paper describes a handheld laser scanning confocal microscope for skin microscopy. Beam scanning is accomplished with an electromagnetic MEMS bi-axial micromirror developed for pico projector applications, providing 800 x 600 (SVGA) resolution at 56 frames per second. The design uses commercial objective lenses with an optional hemisphere front lens, operating with a range of numerical aperture from NA=0.35 to NA=1.1 and corresponding diagonal field of view ranging from 653 microm to 216 microm. Using NA=1.1 and a laser wavelength of 830 nm we measured the axial response to be 1.14 mum full width at half maximum, with a corresponding 10%-90% lateral edge response of 0.39 mum. Image examples showing both epidermal and dermal features including capillary blood flow are provided. These images represent the highest resolution and frame rate yet achieved for tissue imaging with a MEMS bi-axial scan mirror.
Project description:Advancing molecular therapies for the treatment of skin diseases will require the development of new tools that can reveal spatiotemporal changes in the microanatomy of the skin and associate these changes with the presence of the therapeutic agent. For this purpose, we evaluated a handheld dual-axis confocal (DAC) microscope that is capable of in vivo fluorescence imaging of skin, using both mouse models and human skin. Individual keratinocytes in the epidermis were observed in three-dimensional image stacks after topical administration of near-infrared (NIR) dyes as contrast agents. This suggested that the DAC microscope may have utility in assessing the clinical effects of a small interfering RNA (siRNA)-based therapeutic (TD101) that targets the causative mutation in pachyonychia congenita (PC) patients. The data indicated that (1) formulated indocyanine green (ICG) readily penetrated hyperkeratotic PC skin and normal callused regions compared with nonaffected areas, and (2) TD101-treated PC skin revealed changes in tissue morphology, consistent with reversion to nonaffected skin compared with vehicle-treated skin. In addition, siRNA was conjugated to NIR dye and shown to penetrate through the stratum corneum barrier when topically applied to mouse skin. These results suggest that in vivo confocal microscopy may provide an informative clinical end point to evaluate the efficacy of experimental molecular therapeutics.
Project description:We present software-based methods for automatic phase control and for mosaicing high-speed, Lissajous-scanned images. To achieve imaging speeds fast enough for mosaicing, we first increase the image update rate tenfold from 3 to 30 Hz, then vertically interpolate each sparse image in real-time to eliminate fixed pattern noise. We validate our methods by imaging fluorescent beads and automatically maintaining phase control over the course of one hour. We then image fixed mouse brain tissues at varying update rates and compare the resulting mosaics. Using reconstructed image data as feedback for phase control eliminates the need for phase sensors and feedback controllers, enabling long-term imaging experiments without additional hardware. Mosaicing subsampled images results in video-rate imaging speeds, nearly fully recovered spatial resolution, and millimeter-scale fields of view.
Project description:We present a handheld dual-axes confocal microscope that is based on a two-dimensional microelectromechanical systems (MEMS) scanner. It performs reflectance and fluorescence imaging at 488 nm wavelength, with three-dimensional imaging capability. The fully packaged microscope has a diameter of 10 mm and acquires images at 4 Hz frame rate with a maximum field of view of 400 microm x 260 microm. The transverse and axial resolutions of the handheld probe are 1.7 microm and 5.8 microm, respectively. Capability to perform real time small animal imaging is demonstrated in vivo in transgenic mice.
Project description:Transgenic reporter mice and advances in imaging instrumentation are enabling real-time visualization of cellular mechanisms in living subjects and accelerating the development of novel therapies. Innovative confocal microscope designs are improving their utility for microscopic imaging of fluorescent reporters in living animals. We develop dual-axis confocal (DAC) microscopes for such in vivo studies and create mouse models where fluorescent proteins are expressed in the skin for the purpose of advancing skin therapeutics and transdermal delivery tools. Three-dimensional image volumes, through the different skin compartments of the epidermis and dermis, can be acquired in several seconds with the DAC microscope in living mice, and are comparable to histologic analyses of reporter protein expression patterns in skin sections. Intravital imaging with the DAC microscope further enables visualization of green fluorescent protein (GFP) reporter gene expression in the skin over time, and quantification of transdermal delivery of small interfering RNA (siRNA) and therapeutic efficacy. Visualization of transdermal delivery of nucleic acids will play an important role in the development of innovative strategies for treating skin pathologies.
Project description:A novel method for fabricating lens arrays and other non-rotationally symmetric free-form optics is presented. This is a diamond machining technique using 4 controlled axes of motion - X, Y, Z, and C. As in 3-axis diamond micro-milling, a diamond ball endmill is mounted to the work spindle of a 4-axis ultra-precision computer numerical control (CNC) machine. Unlike 3-axis micro-milling, the C-axis is used to hold the cutting edge of the tool in contact with the lens surface for the entire cut. This allows the feed rates to be doubled compared to the current state of the art of micro-milling while producing an optically smooth surface with very low surface form error and exceptionally low radius error.