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Description
Single cell phenotypic heterogeneity studies reveal more information about the pathogenesis process than conventional bulk methods. Furthermore, investigation of the individual cellular response mechanism during rapid environmental changes can only be achieved at single cell level. By enabling the study of cellular morphology, a single cell three-dimensional (3D) imaging system can be used to diagnose fatal diseases, such as cancer, at an early stage. One proven method, CellCT, accomplishes 3D imaging by rotating a single cell around a fixed axis. However, some existing cell rotating mechanisms require either intricate microfabrication, and some fail to provide a suitable environment for living cells. This thesis develops a microvorterx chamber that allows living cells to be rotated by hydrodynamic alone while facilitating imaging access. In this thesis work, 1) the new chamber design was developed through numerical simulation. Simulations revealed that in order to form a microvortex in the side chamber, the ratio of the chamber opening to the channel width must be smaller than one. After comparing different chamber designs, the trapezoidal side chamber was selected because it demonstrated controllable circulation and met the imaging requirements. Microvortex properties were not sensitive to the chambers with interface angles ranging from 0.32 to 0.64. A similar trend was observed when chamber heights were larger than chamber opening. 2) Micro-particle image velocimetry was used to characterize microvortices and validate simulation results. Agreement between experimentation and simulation confirmed that numerical simulation was an effective method for chamber design. 3) Finally, cell rotation experiments were performed in the trapezoidal side chamber. The experimental results demonstrated cell rotational rates ranging from 12 to 29 rpm for regular cells. With a volumetric flow rate of 0.5 µL/s, an irregular cell rotated at a mean rate of 97 ± 3 rpm. Rotational rates can be changed by altering inlet flow rates.
ContributorsZhang, Wenjie (Author) / Frakes, David (Thesis advisor) / Meldrum, Deirdre (Thesis advisor) / Chao, Shih-hui (Committee member) / Wang, Xiao (Committee member) / Arizona State University (Publisher)
Created2011
Description
A single cell is the very fundamental element in an organism; however, it contains the most complicated and stochastic information, such as DNA, RNA, and protein expression. Thus, it is a necessity to study stochastic gene expression in order to discover the biosignatures at the single-cell level. The heterogeneous gene expression of single cells from an isogenic cell population has already been studied for years. Yet to date, single-cell studies have been confined in a fashion of analyzing isolated single cells or a dilution of cells from the bulk-cell populations. These techniques or devices are limited by either the mechanism of cell lysis or the difficulties to target specific cells without harming neighboring cells.
This dissertation presents the development of a laser lysis chip combined with a two-photon laser system to perform single-cell lysis of single cells in situ from three-dimensional (3D) cell spheroids followed by analysis of the cell lysate with two-step reverse transcription-quantitative polymerase chain reaction (RT-qPCR). The 3D spheroids were trapped in a well in the custom-designed laser lysis chip. Next, each single cell of interest in the 3D spheroid was identified and lysed one at a time utilizing a two-photon excited laser. After each cell lysis, the contents inside the target cell were released to the surrounding media and carried out to the lysate collector. Finally, the gene expression of each individual cell was measured by two-step RT-qPCR then spatially mapped back to its original location in the spheroids to construct a 3D gene expression map.
This novel technology and approach enables multiple gene expression measurements in single cells of multicellular organisms as well as cell-to-cell heterogeneous responses to the environment with spatial recognition. Furthermore, this method can be applied to study precancerous tissues for a better understanding of cancer progression and for identifying early tumor development.
This dissertation presents the development of a laser lysis chip combined with a two-photon laser system to perform single-cell lysis of single cells in situ from three-dimensional (3D) cell spheroids followed by analysis of the cell lysate with two-step reverse transcription-quantitative polymerase chain reaction (RT-qPCR). The 3D spheroids were trapped in a well in the custom-designed laser lysis chip. Next, each single cell of interest in the 3D spheroid was identified and lysed one at a time utilizing a two-photon excited laser. After each cell lysis, the contents inside the target cell were released to the surrounding media and carried out to the lysate collector. Finally, the gene expression of each individual cell was measured by two-step RT-qPCR then spatially mapped back to its original location in the spheroids to construct a 3D gene expression map.
This novel technology and approach enables multiple gene expression measurements in single cells of multicellular organisms as well as cell-to-cell heterogeneous responses to the environment with spatial recognition. Furthermore, this method can be applied to study precancerous tissues for a better understanding of cancer progression and for identifying early tumor development.
ContributorsWang, Guozhen (Author) / Meldrum, Deirdre R (Thesis advisor) / Chao, Shih-hui (Committee member) / Wang, Hong (Committee member) / Goryll, Michael (Committee member) / Arizona State University (Publisher)
Created2016