Point-of-care pathology with miniature microscopes

Advances in optical designs are enabling the development of miniature microscopes that can examine tissue in situ for early anatomic and molecular indicators of disease, in real time, and at cellular resolution. These new devices will lead to major changes in how diseases are detected and managed, driving a shift from today's diagnostic paradigm of biopsy followed by histopathology and recommended therapy, to non-invasive point-of-care diagnosis with possible same-session definitive treatment. This shift may have major implications for the training requirements of future physicians to enable them to interpret real-time in vivo microscopic data, and will also shape the emerging fields of telepathology and telemedicine. Implementation of new technologies into clinical practice is a complex process that requires bridging gaps between clinicians, engineers and scientists. This article provides a forward-looking discussion of these issues, with a focus on malignant and pre-malignant lesions, by first highlighting some of the clinical areas where point-of-care in vivo microscopy could address unmet needs, and then by reviewing the technological challenges that are being addressed, or need to be addressed, for in vivo microscopy to become a standard clinical tool

Micromirror-scanned dual-axis confocal microscope utilizing a gradient-index relay lens for image guidance during brain surgery

A fluorescence confocal microscope incorporating a 1.8-mm-diam gradient-index relay lens is developed for in vivo histological guidance during resection of brain tumors. The microscope utilizes a dual-axis confocal architecture to efficiently reject out-of-focus light for high-contrast optical sectioning. A biaxial microelectromechanical system (MEMS) scanning mirror is actuated at resonance along each axis to achieve a large field of view with low-voltage waveforms. The unstable Lissajous scan, which results from actuating the orthogonal axes of the MEMS mirror at highly disparate resonance frequencies, is optimized to fully sample 500×500 pixels at two frames per second. Optically sectioned fluorescence images of brain tissues are obtained in living mice to demonstrate the utility of this microscope for image-guided resections

Quantifying Cell-Surface Biomarker Expression in Thick Tissues with Ratiometric Three-Dimensional Microscopy

The burgeoning fields of in vivo three-dimensional (3D) microscopy and endomicroscopy, as well as ex vivo tissue cytometry have introduced new challenges for tissue preparation and staining with exogenous molecular contrast agents. These challenges include effective delivery of the agents, and once delivered, distinguishing between bound verses unbound molecular probes. If applied topically, there are additional issues with rinsing off unbound probe, which can be nonuniform and inefficient in thick tissues, thus leading to ambiguous contrast and a large nonspecific background that may obscure molecule-specific staining. Therefore, we have developed a ratiometric 3D microscopy scheme that not only reduces the effects of nonspecific sources of contrast, but also enables quantification of the relative binding affinity of imaging probes to their biomarker targets. Here we demonstrate this ratiometric approach by simultaneously imaging a HER2/neu (erbB2)-targeted monoclonal antibody labeled with one fluorophore and an isotype-matched negative control antibody labeled with another fluorophore. By taking a pixel-by-pixel calibrated ratio between the signals from each fluorescent image channel, accurate quantification of specific versus nonspecific binding affinity is achieved with cultured cells, yielding data that are in agreement with analyses via flow cytometry. We also demonstrate quantitative 3D microscopic imaging of biomarker expression in tissue models and in thick human biopsy samples of normal, HER2-negative, and HER2-positive breast tumors. This strategy enables rapid, quantitative, and unambiguous volumetric microscopy of biomarker expression in thick tissues, including whole biopsies, and will enable real-time optical assessment of disease markers in the living body

A Real-Time Clinical Endoscopic System for Intraluminal, Multiplexed Imaging of Surface-Enhanced Raman Scattering Nanoparticles

<div><p>The detection of biomarker-targeting surface-enhanced Raman scattering (SERS) nanoparticles (NPs) in the human gastrointestinal tract has the potential to improve early cancer detection; however, a clinically relevant device with rapid Raman-imaging capability has not been described. Here we report the design and <i>in vivo</i> demonstration of a miniature, non-contact, opto-electro-mechanical Raman device as an accessory to clinical endoscopes that can provide multiplexed molecular data via a panel of SERS NPs. This device enables rapid circumferential scanning of topologically complex luminal surfaces of hollow organs (e.g., colon and esophagus) and produces quantitative images of the relative concentrations of SERS NPs that are present. Human and swine studies have demonstrated the speed and simplicity of this technique. This approach also offers unparalleled multiplexing capabilities by simultaneously detecting the unique spectral fingerprints of multiple SERS NPs. Therefore, this new screening strategy has the potential to improve diagnosis and to guide therapy by enabling sensitive quantitative molecular detection of small and otherwise hard-to-detect lesions in the context of white-light endoscopy.</p></div

Imaging device and system.

<p>(a) Photographs of the components of the distal end of the device adjacent to a quarter (diameter = 24 mm) for scale. All the components shown, less the motor, were custom designed and fabricated for this device. (b) Close-up photograph of the distal end of the fully functional device. The fiber bundle was enclosed and sealed within a flexible extrusion sheath. The window was placed between the scan mirror and the tissue in order to seal the inner mechanisms of the device from fluids in the surrounding environment. The use of a toroidal mirror compensates for beam distortion from the curvature of the glass window in order to maintain a collimated beam. Utilization of a 50-degree inclination angle of the toroidal mirror effectively eliminates back reflections from the window into the fiber bundle detector. (c) System overview. A continuous wave (CW) laser at 785 nm was used and the Raman-scattered light is collected through the multi-mode fibers of the fiber bundle. At the proximal end of the fiber bundle, the multimode fibers are arranged into a vertical array for efficient coupling to the spectrometer. A long-pass filter at the entrance of the spectrometer filters out the illumination light. A function generator signal controls a motor control board to finely tune the rotational speed of the motor to the desired speed of 1 rev/s. (d) Photograph of the completed fully functional system.</p

Schematic of Raman-imaging system being used in parallel with white-light endoscopy.

<p>(a) The device is designed such that it can be inserted through the accessory channel of a clinical endoscope. As the endoscope is being retracted in the GI tract, the device simultaneously scans the lumen. The collected Raman-scattered light is analyzed, and an image is displayed to the user (also see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0123185#pone.0123185.s001" target="_blank">S1 Video</a>). (b) Expanded schematic of the distal end of the device. The schematic illustrates the position of the device relative to the end of the endoscope. A brushless DC motor that rotates a mirror causing the collimated beam to sweep 360 degrees, enabling luminal imaging of the colon wall. The device is not required to be in contact with the tissue, which is enabled through the use of the collimated illumination beam. A custom, miniature, concentrically segmented, air-spaced doublet lens having a non-reciprocal optical path consists of a plano-convex lens and an adjacent plano-concave lens with a central hole. The doublet lens increases collection efficiency at longer, clinically relevant working distances.</p