Browsing by Author "Brown, J Quincy"

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    Optimization of a widefield structured illumination microscope for non-destructive assessment and quantification of nuclear features in tumor margins of a primary mouse model of sarcoma.
    (PloS one, 2013-01) Fu, Henry L; Mueller, Jenna L; Javid, Melodi P; Mito, Jeffrey K; Kirsch, David G; Ramanujam, Nimmi; Brown, J Quincy
    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 mm(2)) 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 (v=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.
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    Wavelength optimization for quantitative spectral imaging of breast tumor margins.
    (PloS one, 2013-01) Lo, Justin Y; Brown, J Quincy; Dhar, Sulochana; Yu, Bing; Palmer, Gregory M; Jokerst, Nan M; Ramanujam, Nirmala
    A wavelength selection method that combines an inverse Monte Carlo model of reflectance and a genetic algorithm for global optimization was developed for the application of spectral imaging of breast tumor margins. The selection of wavelengths impacts system design in cost, size, and accuracy of tissue quantitation. The minimum number of wavelengths required for the accurate quantitation of tissue optical properties is 8, with diminishing gains for additional wavelengths. The resulting wavelength choices for the specific probe geometry used for the breast tumor margin spectral imaging application were tested in an independent pathology-confirmed ex vivo breast tissue data set and in tissue-mimicking phantoms. In breast tissue, the optical endpoints (hemoglobin, β-carotene, and scattering) that provide the contrast between normal and malignant tissue specimens are extracted with the optimized 8-wavelength set with <9% error compared to the full spectrum (450-600 nm). A multi-absorber liquid phantom study was also performed to show the improved extraction accuracy with optimization and without optimization. This technique for selecting wavelengths can be used for designing spectral imaging systems for other clinical applications.