A modular multi-color fluorescence microscope for simultaneous tracking of cellular activity and behavior

We present a modular epifluorescence tracking microscope which enables ratiometric imaging of muscles, neurons, and other structures in moving animals. The microscope is assembled entirely from commercial parts within 3 h, making the system broadly accessible. Leveraging the improved brightness and bleaching characteristics of recent genetically encoded indicators and fluorophores, the simple microscope is even suitable for calcium imaging of neurons in behaving animals, as we demonstrate in C. elegans. We also show how muscle dynamics in D. melanogaster larvae can be analyzed and how dual color fluorescence tracking elucidates inter-species interactions by visualizing both predatory nematodes and their prey. Finally, we showcase a configuration for brightfield imaging by tracking tardigrade gait as an example of utility for non-labeled species. The affordability of the hardware and ease of use of the accompanying software make this a suitable tool for education in addition to its use in research.