Abstract
Cancer is associated with specific cellular morphological changes, such as increased nuclear size and crowding from rapidly proliferating cells. In situ tissue imaging using fluorescent stains may be useful for intraoperative detection of residual cancer in surgical tumor margins. We developed a widefield fluorescence structured illumination microscope (SIM) system with a single-shot FOV of 2.1×1.6 mm (3.4 mm2) and sub-cellular resolution (4.4 µm). The objectives of this work were to measure the relationship between illumination pattern frequency and optical sectioning strength and signal-to-noise ratio in turbid (i.e. thick) samples for selection of the optimum frequency, and to determine feasibility for detecting residual cancer on tumor resection margins, using a genetically engineered primary mouse model of sarcoma. The SIM system was tested in tissue mimicking solid phantoms with various scattering levels to determine impact of both turbidity and illumination frequency on two SIM metrics, optical section thickness and modulation depth. To demonstrate preclinical feasibility, ex vivo 50 µm frozen sections and fresh intact thick tissue samples excised from a primary mouse model of sarcoma were stained with acridine orange, which stains cell nuclei, skeletal muscle, and collagenous stroma. The cell nuclei were segmented using a high-pass filter algorithm, which allowed quantification of nuclear density. The results showed that the optimal illumination frequency was 31.7 µm−1 used in conjunction with a 4×0.1 NA objective ( = 0.165). This yielded an optical section thickness of 128 µm and an 8.9×contrast enhancement over uniform illumination. We successfully demonstrated the ability to resolve cell nuclei in situ achieved via SIM, which allowed segmentation of nuclei from heterogeneous tissues in the presence of considerable background fluorescence. Specifically, we demonstrate that optical sectioning of fresh intact thick tissues performed equivalently in regards to nuclear density quantification, to physical frozen sectioning and standard microscopy.
Highlights
Fluorescence microscopy is an attractive optical technique capable of generating high- resolution images for visualizing tissue microstructure
Structured illumination is an elegant approach to solve the problem of optical sectioning in microscopy, essentially analogous to frequency modulation techniques used to encode electrical signals
Structured illumination is a low-complexity solution for optical sectioning microscopy of thick tissues that has the potential for clinically feasible high throughput microscopy of tumor margins due to its light efficiency and parallel-pixel detection approach
Summary
Fluorescence microscopy is an attractive optical technique capable of generating high- resolution images for visualizing tissue microstructure. One significant morphological change that occurs in cancer is enlargement of nuclear size and increase in nuclear density (i.e. reduction in average inter-nuclear distance) [1]. This is a characteristic, which is commonly exploited by pathologists, to diagnose cancer pathology in hematoxylin and eosin (H&E) stained tissues sections. An appropriate contrast agent could be used with fluorescence microscopy to visualize cell nuclei without requiring extensive sample preparation. This provides an advantage over H&E as the tissue can be imaged intra-operatively in vivo or ex vivo without the need for fixation or H&E staining. Gmitro et al have demonstrated imaging of ovarian tissue and cancer stained with AO using confocal scanning microscopy[6,7,8,9]
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