Visualization of micro-agents and surroundings by real-time multicolor fluorescence microscopy

Abstract Optical microscopy techniques are a popular choice for visualizing micro-agents. They generate images with relatively high spatiotemporal resolution but do not reveal encoded information for distinguishing micro-agents and surroundings. This study presents multicolor fluorescence microscopy for rendering color-coded identification of mobile micro-agents and dynamic surroundings by spectral unmixing. We report multicolor microscopy performance by visualizing the attachment of single and cluster micro-agents to cancer spheroids formed with HeLa cells as a proof-of-concept for targeted drug delivery demonstration. A microfluidic chip is developed to immobilize a single spheroid for the attachment, provide a stable environment for multicolor microscopy, and create a 3D tumor model. In order to confirm that multicolor microscopy is able to visualize micro-agents in vascularized environments, in vitro vasculature network formed with endothelial cells and ex ovo chicken chorioallantoic membrane are employed as experimental models. Full visualization of our models is achieved by sequential excitation of the fluorophores in a round-robin manner and synchronous individual image acquisition from three-different spectrum bands. We experimentally demonstrate that multicolor microscopy spectrally decomposes micro-agents, organic bodies (cancer spheroids and vasculatures), and surrounding media utilizing fluorophores with well-separated spectrum characteristics and allows image acquisition with 1280 $$\times$$ × 1024 pixels up to 15 frames per second. Our results display that real-time multicolor microscopy provides increased understanding by color-coded visualization regarding the tracking of micro-agents, morphology of organic bodies, and clear distinction of surrounding media.