Scanning darkfield high-resolution microendoscope for label-free microvascular imaging

Abstract

Characterization of microvascular changes during neoplastic progression has the potential to assist in discriminating precancer and early cancer from benign lesions. Here, we introduce a novel high-resolution microendoscope that leverages scanning darkfield reflectance imaging to characterize angiogenesis without exogenous contrast agents. Scanning darkfield imaging is achieved by coupling programmable illumination with a complementary metal-oxide semiconductor (CMOS) camera rolling shutter, eliminating the need for complex optomechanical components and making the system portable, low-cost (<$5,500) and simple to use. Imaging depth is extended by placing a gradient-index (GRIN) lens at the distal end of the imaging fiber to resolve subepithelial microvasculature. We validated the capability of the scanning darkfield microendoscope to visualize microvasculature at different anatomic sites in vivo by imaging the oral cavity of healthy volunteers. Images of cervical specimens resected for suspected neoplasia reveal distinct microvascular patterns in columnar and squamous epithelium with different grades of precancer, indicating the potential of scanning darkfield microendoscopy to aid in efforts to prevent cervical cancer through early diagnosis.

Fig. 1.

(A) Block diagram and (B) photograph of the portable DF-HRME imaging system. The distal tip of a thin, flexible fiber optic bundle is enclosed in a 3D-printed probe holder and placed in contact with the tissue epithelium. The probe relays the image to the portable optical system at the proximal end of the bundle. The system is controlled via a GUI on a laptop; high resolution video of microvasculature is displayed in real time without the need for an exogenous contrast agent. (C) Optical diagram of the DF-HRME. Scanning darkfield illumination is used to enable reflectance imaging of microvasculature through the fiber bundle. A DLP is used to project a scanning structured illumination pattern at the proximal face of the fiber bundle; synchronized detection is performed using a CMOS camera. An offset is introduced between the illumination and detection apertures to reduce internal reflection. Arrows indicate the directions of scanning at the probe surfaces, DLP and CMOS camera. DF-HRME: scanning darkfield high-resolution microendoscope; DLP: digital light projector; CMOS: complementary metal-oxide semiconductor camera; GUI: graphical user interface.

Fig. 2.

Scanning darkfield imaging is implemented to suppress background signal due to strong internal reflection from fiber bundle surfaces. (A) Schematic of the spatiotemporal coordination of illumination and detection aperture sequences for non-scanning widefield imaging and scanning darkfield imaging. In widefield imaging, the entire field of view is illuminated by the DLP as the detection aperture (width D and shutter time T) of the CMOS camera scans across the entire sensor to capture image information. In scanning darkfield imaging, a sequence of illumination line pairs (width 4D and spaced by 8D from center to center) is scanned across the field of view synchronized with but spatially offset from the detection aperture sequence. The corresponding background images captured with no sample are shown below; scanning darkfield imaging suppresses internal reflection. (B) Normalized background signal for the DF-HRME system in widefield and scanning darkfield modes. (Scale bar: 100 µm)

Fig. 3.

Design and characterization of sub-surface imaging probes. (A) Schematic of the imaging probe inserted in a probe holder. Inset shows detailed design of the distal end of the imaging probe. A GRIN lens is epoxied to the distal tip of the bundle to extend the focusing depth. (B) Experimental measurements demonstrate the relationship between probe imaging depth (d₂) and the distance between the fiber bundle and the GRIN lens (d₁) for all imaging depths (blue dashed line), and for three imaging probes designed to image at depths of 70, 170 and 270 µm beneath the tissue surface (orange dashed line). (C) Photograph of imaging probe before (left) and after (right) insertion into a 3D-printed probe holder to facilitate in vivo imaging.

Fig. 4.

Scanning darkfield images consistently shows improved image contrast compared to widefield images. (A) Comparison of representative images acquired using widefield imaging and scanning darkfield imaging at different anatomic sites in the oral cavity of a healthy volunteer. Images were acquired using a probe with a 70 µm imaging depth. Corresponding intensity profiles along the red dashed lines are plotted for each image pair. Red arrows indicate the microvascular features that are clearly resolved in darkfield images but not distinguishable in widefield images. Black brackets denote vessels resolved in darkfield images. (B) Relationship between widefield and darkfield image contrast from 25 randomly selected microvessels. Each point indicates the contrast of one microvessel. WF: widefield imaging; DF: scanning darkfield imaging. (Scale bar: 100 µm)

Fig. 5.

Representative DF-HRME images acquired using probes with different imaging depths (0 µm, 70 µm, 170 µm and 270 µm) at different anatomic sites in the oral cavity of healthy volunteers. Intensity line profiles of representative microvessels (red arrows) are shown at the bottom right of each panel. Vasculature is in focus and clearly resolved in images acquired by probes with imaging depths of 70 µm and 170 µm. Focused large branching vessels (blue arrows) and hairpin capillary loops (green arrows) are highlighted. (Scale bar: 100 µm)

Fig. 6.

Representative DF-HRME images acquired at different imaging depths from 10 cervical LEEP specimens show various microvascular patterns at different anatomic sites and with different pathologies. In cervical tissue of all types, vasculature is better resolved at imaging depths of 0 and 70 µm compared to depths of 170 and 270 µm. DF-HRME: scanning darkfield high-resolution microendoscope; LEEP: loop electrosurgical excision procedure; SCJ: squamocolumnar junction; HSIL: high-grade squamous intraepithelial lesion. (Scale bar: 100 µm)

Fig. 7.

DF-HRME of cervical LEEP specimens clearly reveals distinct microvascular features with different anatomic locations and histopathologic results. DF-HRME images at imaging depths of 0 and 70 µm are shown along with corresponding histopathology images. (A) At a representative site of columnar epithelium, DF-HRME images show branching vessels with a network structure at the superficial surface. The histopathology image confirms the presence of several vessels at the superficial columnar epithelium (arrow). (B) At a representative SCJ site, DF-HRME images reveal the transition of vascular features from branching vessels to individual hairpin capillary loops. Arrows in the histopathology image indicate the depth change of vessels at SCJ. (C) At a representative site of the normal squamous epithelium, hairpin capillaries with small loops that are relatively straight and parallel to each other can be observed at both 0 µm and 70 µm imaging depths in DF-HRME images. The histopathology image shows the presence of a few microvessels in the subepithelial region. (D) At a representative site of squamous epithelium with ulceration, elongated vessels with multiple branches are observed in DF-HRME images. Black arrows in the corresponding histopathology image indicate the presence of dense vasculature in the ulceration area. At a HSIL lesion, the DF-HRME images show tortuous capillary loops. The histopathology image indicates increased vasculature in HSIL. DF-HRME: scanning darkfield high-resolution microendoscope; LEEP: loop electrosurgical excision procedure; SCJ: squamocolumnar junction; HSIL: high-grade squamous intraepithelial lesion. (Scale bars for DF-HRME and histopathology images: 100 µm)