Subcellular-resolution molecular imaging within living tissue by fiber microendoscopy

Abstract

Conventional histopathology involves sampling, sectioning and staining of tissue specimens prior to microscopic evaluation, and provides diagnostic information at a single location and point in time. In vivo microscopy and molecular-targeted optical labeling are two rapidly developing fields, which together have the potential to provide anatomical and functional indications of disease by staining and imaging tissue in situ. To address the need for high-resolution imaging instrumentation, we have developed a compact, robust, and inexpensive fiber-optic microendoscopy system based around wide-field LED illumination, a flexible 1 mm diameter fiber-optic bundle, and a color CCD camera. We demonstrate the sub-cellular resolution imaging capabilities of the system through a series of experiments, beginning with simultaneous imaging of three different cancer cell lines in culture, each targeted with a distinct fluorescent label. We used the narrow diameter probe to access subcutaneous tumors in an in vivo murine model, allowing direct comparison of microendoscopy images with macroscopic images and histopathology. A surgically resected tissue specimen from the human oral cavity was imaged across the clinical margin, demonstrating qualitative and quantitative distinction between normal and cancerous tissue based on sub-cellular image features. Finally, the fiber-optic microendoscope was used on topically-stained normal human oral mucosa in vivo, resolving epithelial cell nuclei and membranes in real-time fluorescence images. Our results demonstrate that this imaging system can potentially complement conventional diagnostic techniques, and support efforts to translate emerging molecular-diagnostic and therapeutic agents into clinical use.

Fig. 1. Fiber-optic microendoscopy system

(a) Schematic diagram. (b) Photograph of fiber-optic bundles on a US penny. Bundles 1 and 2 have outer diameters of 1.0 mm and 0.5 mm respectively.

Fig. 2. System resolution and multiplexed cell culture imaging

(a) Image of a standard US Air Force resolution target, demonstrating resolution of the 4.4 μm-wide bars in group 6, element 6 (circled). (b) False-color composite image of a 3-D collagen construct containing 1483 oral squamous cell carcinoma cells (red), SK-BR-3 breast cancer cells (green), and SiHa cervical cancer cells (blue), with each cell type stained with a spectrally-distinct fluorophore. (c) Single frame image of the same tissue construct acquired in real-time with a single long-pass emission filter. 1483 cells appear red, SK-BR-3 cells yellow, and SiHa cells appear light green. All scale bars represent 50 μm.

Fig. 3. Murine tumor model imaging

(a) Photograph of an athymic nude mouse showing the fiber microendoscope and subcutaneous tumor. (b) Macroscopic fluorescence image (CRI Maestro) acquired following direct injection of fluorescent contrast agent at the tumor site. (c) Image acquired by the fiber microendoscope in the living mouse, as the fiber is advanced through the tumor within the lumen of a 16-gauge needle (frame from Video 1, 2.87 MB movie). Tumor cell nuclei appear as bright green dots, with connective tissue within the tumor mass. (d) Corresponding histology section following tumor excision. Scale bars represent 100 μm.

Fig. 4. Surgical specimen imaging

(a) Normal epithelium. Left; photograph of fiber probe in contact with excised tumor tissue. Yellow border represents the clinically-abnormal region identified by the surgeon (AG). Center; microendoscopy image of tissue with probe at location indicated in photograph. Right; corresponding transverse histopathology section from the imaged region. (b) Tumor region. Microendoscopy image (center) and histology (right) demonstrate squamous carcinoma throughout the entire epithelium. Scale bars represent 100 μm. (c) Graph of calculated nuclear to cytoplasmic ratio for microendoscopy images in (a, b). The dashed line represents an N/C ratio of 0.08.

Fig. 5. In vivo human tissue imaging

(a) Fiber microendoscopy imaging of normal human oral mucosa in vivo, following topical application of fluorescent acriflavine neutral (0.05% in saline). This image was acquired with a scientific-grade CCD camera. (b) Image of the same tissue acquired with a standard CCD camera (frame from Video 2, 3.10 MB movie). In both images, cell nuclei appear bright surrounded by dark cytoplasm. Scale bars represent 100 μm.