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.

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 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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Published Version (Please cite this version)

10.1371/journal.pone.0068868

Publication Info

Fu, Henry L, Jenna L Mueller, Melodi P Javid, Jeffrey K Mito, David G Kirsch, Nimmi Ramanujam and J Quincy Brown (2013). 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, 8(7). p. e68868. 10.1371/journal.pone.0068868 Retrieved from https://hdl.handle.net/10161/26067.

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Scholars@Duke

Whitley

Melodi Javid Whitley

Assistant Professor of Dermatology

Melodi Javid Whitley, MD, PhD
Assistant Professor of Dermatology
Assistant Program Director for Trainee Research
Director of Transplant Dermatology

I am a physician scientist focused on the dermatologic care of solid organ transplant recipients.  Clinically, I manage the the complex dermatologic side effects of immunosuppression with a focus on high-risk skin cancer.  My research focuses on understanding the drivers of cutaneous malignancy in this population using translational approaches.

Kirsch

David Guy Kirsch

Adjunct Professor in the Department of Radiation Oncology

My clinical interests are the multi-modality care of patients with bone and soft tissue sarcomas and developing new sarcoma therapies. My laboratory interests include utilizing mouse models of cancer to study cancer and radiation biology in order to develop new cancer therapies in the pre-clinical setting.

Ramanujam

Nimmi Ramanujam

Robert W. Carr, Jr., Distinguished Professor of Biomedical Engineering

Ramanujam obtained her Ph.D. degree at the University of Texas at Austin. She progressed through the ranks as an academic researcher; the first five years as a research scientist and postdoctoral fellow at the University of Pennsylvania, the next five as an assistant professor at the University of Wisconsin, Madison, and the following five as an associate professor in the Department of Biomedical Engineering at Duke University. In 2011 she was promoted to full professor.  Ramanujam is internationally recognized for her contributions in innovation, education and entrepreneurship and received numerous awards most notably, the IEEE  Biomedical Engineering Award Technical Field Award and the Social Impact Abie award . She is a Fulbright scholar, a member of the National Academy of Inventors, and a fellow of international professional societies in her field. She has also been invited for speaking engagements at the United Nations, as a TEDx speaker, and been invited to give plenary talks on her work all over the world.  

Ramanujam addresses pressing challenges in women’s cancers, specifically, cervical and breast cancer. Ramanujam creates technologies that transform complex diagnostic instruments and therapies into accessible, affordable, and appropriate solutions.  Several of these products are now being used in several countries in the U.S., Latin America, and Africa. She has developed a network of partners including academic institutions and hospitals, non-governmental organizations, ministries of health, and commercial partners to implement these technologies in diverse healthcare settings globally.

She has used her expertise in imaging and human-centered design to develop the Pocket colposcope which is on the WHO list of devices for cervical cancer imaging. A sister device, the Callascope is a self-use speculum-free imaging device, which allows women to screen themselves privately without the need for an intrusive pelvic exam. She has developed a translational microscope called the CapCell Scope to identify biomarkers of metabolism that reflect tumor behavior, including growth, proliferation, and treatment resistance, aimed at informing drug selection for breast cancer treatment. She has developed an ultra-low-cost injectable liquid ablation therapy that disrupts tumors locally as well as elicits an anti-tumor immune response to address an important gap – the lack of access to surgery to the world’s most vulnerable populations. 

Ramanujam has also created several global initiatives that strive to achieve enduring impact in health and education. Her innovations have a common wellspring - they are all connected and come from a place of wanting to create and make something that doesn’t exist.

The most prominent is a consortium called Women Inspired Strategies for Health (WISH) to improve cervical cancer prevention in low-resource settings globally.  She is working with partners worldwide to ensure that technologies and strategies for addressing cervical cancer are adopted by cancer control programs in geographically and economically diverse healthcare settings. These partnerships have resulted in see-and-treat cancer control strategies in the least resourced settings that are in clinical deserts. WISH has been recognized by the MacArthur Foundation as one of the top 100 most transformative and impactful solutions, a testament to its significance in redesigning the health system.

Ramanujam has launched an arts and storytelling initiative, The Invisible Organ to raise awareness of sexual and reproductive health inequities. An educational documentary with a similar name was created and has been screened at conferences and by multiple artists and students across the U.S. This film was officially selected for the Women at the Center Film Festival at the International Papillomavirus Conference in 2020. She also co-led the curation of an art exhibit to bring together a collection of visual arts, medical photography, sculptures, and installations, both a physical exhibit and a digital moving gallery to express the stigma and shame associated with female anatomy.

She has also created a global education program that intersects design thinking, STEM concepts, and the U.N. Sustainable Development Goals to promote social justice awareness: Ignite. The participatory learning curricula have been implemented in more than four countries with broad-ranging impact. For example, students living around the contaminated Lake Atitlan, in Guatemala learned how to design engineering solutions for clean water. Similarly,for personal use during frequent power outages.


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