The Development of Lateral Microscopy as an Imaging System to Characterize Cell Morphology.
Walz, Jenna.
2019
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The development of optical microscopy and its advances over time have led to significant and continual breakthroughs in biological research. However, as innovations to light microscopes increase, the complexity and cost of these systems also increase, which, in turn, decreases their accessibility to researchers and clinicians. To address this limitation, we fabricated a lateral optical microscope ... read moreentirely from affordable commercialized components to enable brightfield imaging of biological processes in an unconventional field of view. Specifically, the lateral microscope allows direct observation of cell-substrate interactions in real-time on any substrate—transparent, opaque, or coated—without requiring labels or specialized optical features. As a result, lateral microscopy is ideally suited to improve our understanding of cell morphology, especially during early adhesion events. In Chapter 1, we describe prominent optical microscopy techniques that have emerged throughout history, and in Chapter 2, we acknowledge limitations of these approaches to highlight the utility of lateral microscopy. Furthermore, we demonstrate the capabilities of the lateral microscope by quantifying dynamic changes in cell morphology during the first 90 min of adhesion to various materials. We determined the rates of change in contact angle of HeLa, 3T3, HEK293, and MDA-MB-231 cells on five different substrates: glass, collagen-coated glass, Nylon, PTFE, and collagen-alginate hydrogels. We used these rates of change to compare the adhesion of different cell lines on each surface, and to rank the adhesion-promoting capacities of the five surfaces for each cell line. For HeLa, 3T3, and HEK293 cells, we observed maximal rates of change in contact angle (0.058 min-1) on collagen-coated glass substrates. All five cell lines exhibited minimal rates of change (0.003 min-1) on PTFE. Lateral microscopy also revealed a unique morphology among MDA-MB-231 breast cancer cells during initial adhesion, which we quantified using measurements of changes in cell height. These results suggest that the lateral microscope will not only enable more comprehensive, quantitative studies of cell adhesion to inform the development of biomaterials, but it will ultimately assist in advancing our understanding of many important biological processes and discovering new behaviors related to cell adhesion. In Chapter 3, we implemented lateral microscopy as a means for immunophenotyping. Immunophenotyping is typically achieved using flow cytometry, but any influence a biomarker may have on adhesion or surface recognition cannot be determined concurrently. With the lateral microscope, we were able to correlate cell surface biomarker expression levels with quantitative descriptions of cell morphology. We observed single cells from two T cell lines and two B cell lines adhere to antibody-coated substrates and quantified this adhesion using contact angle measurements. We found that SUP-T1 and CEM CD4+ cells, both of which express similar levels of CD4, experienced average changes in contact angle that were not statistically different from one another on surfaces coated in anti-CD4. However, MAVER-1 and BJAB K20 cells, both of which express different levels of CD20, underwent average changes in contact angle that were significantly different from one another on surfaces coated in anti-CD20. Our results indicate that changes in cell contact angle on antibody-coated substrates reflect the expression levels of corresponding antigens on the surfaces of cells as determined by flow cytometry. Our lateral microscopy approach offers a more reproducible and quantitative alternative to evaluate adhesion compared to commonly used wash assays and can be extended to many additional immunophenotyping applications to identify cells of interest within heterogeneous populations. Finally, in Chapter 4, we used lateral microscopy to develop an assay to rapidly predict chondrocyte phenotype during long-term culture, which has the potential to transform cartilage tissue engineering efforts. Maintaining the chondrogenic phenotype, which is marked by rounded cell morphologies, low proliferation rates, and the synthesis of cartilage extracellular matrix proteins, has posed many challenges for researchers, particularly due to the properties of scaffold materials. Depending on their composition, these materials can be thick, uneven, or opaque, which precludes real-time analyses of chondrocyte phenotype with traditional microscopy approaches. As a result, endpoint analyses are typically performed to obtain phenotypical information, which wastes valuable time and money. Using the lateral microscope, we determined the average contact angle of a population of chondrocytes on a particular substrate after 90 min of adhesion to predict whether or not the substrate promotes the chondrogenic phenotype long-term. We performed this assay with primary bovine chondrocytes on a panel of substrates with varying elastic moduli. We determined that after 90 min of adhesion to stiff substrates (i.e., titanium, glass, and polystyrene), chondrocytes adopted flattened morphologies corresponding to average contact angles less than 90 degrees, which did not correlate with the chondrogenic phenotype as determined by gene expression analyses after 9 days of culture on the same substrates. On the softest substrate investigated (i.e., PDMS), chondrocytes maintained rounded morphologies corresponding to an average contact angle greater than 90 degrees, which indicated that these cells would uphold the chondrogenic phenotype over extended time periods. Thus, lateral microscopy serves as a promising approach for predicting cell-biomaterial interactions, eliminating expensive, late-stage analyses, especially for cartilage tissue engineering applications.
Thesis (Ph.D.)--Tufts University, 2019.
Submitted to the Dept. of Chemistry.
Advisor: Charles Mace.
Committee: Charles Sykes, Joshua Kritzer, and Amy Peterson.
Keyword: Analytical chemistry.read less - ID:
- 1c18dv39f
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