4D Evaluation Approach for Cell Signaling Activity in Multicellular Tumor Spheroids

Two-photon microscopy is a fluorescence microscopic approach with excellent depth reach that allows for the minimally invasive visualization of molecules deep within living organisms and tissues. In recent years, its application in cancer research has become increasingly popular. However, observing drug responses in individual cells in three-dimensional tumor tissues has remained a challenge in cancer research for long. Our research group has established a method to evaluate the activity of intracellular signaling molecules in four dimensions (XYZ + T) using sophisticated two-photon microscopic, FRET-based functional fluorescent probing, and automatic deep learning-based cell detection and tracking methods. In this chapter, we introduce our approach in detail.