Development of three-dimensional single-cell optomechanical techniques for investigating immune therapy mechanisms

Abstract

this is particularly true when imaging cells in three dimensions (3D). To address the rapid, long-term dynamics of the adaptive immune response, developing a high-speed, volumetric, and label-free microscopy tool is required. In this thesis, we propose and experimentally demonstrate the development of the 3D single-cell optomechanical platform for a volumetric, long-term and high-speed assessment of immune dynamics. Our platform mainly exploits optical diffraction tomography, a label-free tomographic microscopy technique which enables quantitative spatiotemporal analyses of intracellular protein density distributions. The proposed system provides the high-throughput imaging capability at 140 nm lateral, 400 nm axial, and 2 seconds temporal resolution. We integrated fluorescence-based structured illumination microscopy to enable 3D quantitative structural analysis of the immunological synapse by chimeric antigen receptor T cells. Additionally, we simultaneously measure and transduce 3D traction force of the immunological synapse by a cytotoxic T cell by combining a a method of holographic optical tweezers and traction force microscopy. The demonstrated method enables a high-throughput, label-free, and 3D measurement of immune-cell mechanics.

Immunotherapy represents a new paradigm to obtain remarkable potency and durability in cancer treatment—by exploiting the immune system, not the tumor itself. Ongoing research mostly focuses on identifying, targeting, and modulating critical immune signaling pathways to improve efficacy physiologically. Recently, biophysical experiments have revealed that cellular forces have biophysically critical roles in activating the adaptive immune response. In this regard, we hypothesize that understanding the mechanobiology of immune dynamics is important for advancing immunotherapy. Optical microscopy is indispensable for assessing the mechanobiology of live immune cells. Conventional imaging methods rely on fluorescent markers. However, they have limitations concerning spatiotemporal resolution, photobleaching, and phototoxicity