Multicolor fluorescence microscopy for tracking magnetic micro-agents

The field of microrobotics opens the door to new therapeutic interventions by providing access to hard-to-reach regions of the human body. The envisioned interventions are based on the wireless movement of the micro-agents, part of the microrobotics systems designed to interact with the environments, using external stimuli (e.g., magnetic, sound, light, chemical, temperature). Although a wide variety of micro-agents have been designed, fabricated, and actuated, sensor integration remains a challenge due to size limitations. In order to understand and verify the functionality of the micro-agents, imaging modalities are utilized as external sensors and considered the only feedback source. Since experimental testbeds are fabricated using optically transparent materials, microscopy techniques have been extensively used in preliminary tests and lab-on-a-chip applications. Single-band fluorescence and bright-field microscopy are commonly used to visualize the micro-agents with spatiotemporal resolution. However, single-band fluorescence microscopy renders visualization of only micro-agents and does not provide information about physical surroundings. Clear visualization of surroundings, as well as the micro-agents, is a requirement to reach the desired location. Bright-field microscopy enables visualization of micro-agents and surroundings, but acquired images do not reveal encoded information about identification and distinction. In this thesis, multicolor fluorescence microscopy is first applied for visualizing magnetically-actuated micro-agents and dynamic surroundings by relatively fast spectrum band change (multiplexing). The main difference of multicolor microscopy is that a sample containing micro-agents is fully resolved in different spectrum bands, and color-coded visualization is acquired. Since color is a visual cue for detection, acquired images enable an increased understanding of the micro-agent functionality and clear distinction of the surrounding media. This thesis aims to fill the gap in the literature on performing real-time multicolor microscopy for micro-agents and surroundings with three parts divided into six chapters.