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 hours, 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 simultaneous calcium imaging of neurons and behavior 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 bright-field imaging, by tracking tardigrade gait as an example of utility for non-model 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.