Development and validation of a low-cost tandem-lens mesoscope for wide field fluorescence imaging of neural activity

Suwanee, GA

Advancements in mesoscopic imaging have enabled large field-of-view (FOV) fluorescence microscopy, facilitating the study of neural activity across cortical networks. Traditional two-photon and confocal microscopy techniques provide high spatial resolution but are limited in their ability to capture large-scale neuronal interactions due to their restricted FOV. Mesoscopic imaging bridges this gap by allowing simultaneous observation of widespread neural circuits, making it a powerful tool for studying distributed activity patterns, such as those involved in sensorimotor processing. This approach is particularly useful for investigating the cerebello-thalamo-cortical pathway, where coordinated activity across multiple brain regions is critical. Following the design outlined by Jose et al., a low-cost tandem-lens mesoscope was constructed. This study focuses on the assembly, calibration, and initial validation of the mesoscope using fluorescence microscopy techniques. The mesoscope was assembled using commercially available optical components in a tandem-lens configuration. The system consists of a Thorlabs CS505MU CMOS camera, MVL75M23 objective lens, and MVL50M23 imaging lens, allowing for two distinct FOVs: 12.6 × 10.5 mm (large FOV, 1.5 cm WD) and 6 × 5 mm (small FOV, 1.0 cm WD). Excitation was provided by a 470 nm LED (Thorlabs SOLIS 470C), with emission captured through a DMLP490L dichroic mirror and Chroma fluorescence filters optimized for GCaMP imaging. The dichroic mirror was positioned at a 45-degree angle to direct excitation light toward the sample while allowing fluorescence emission to pass through to the camera. The system was mounted on a 60 mm optical cage, incorporating a dovetail translation stage for fine z-axis adjustments. To validate imaging capability, a Snellen chart was placed underneath the scope. Images were acquired using ThorCam software and analyzed for spatial resolution and signal uniformity. The working distance (WD) was adjusted using the translational stage to optimize focus. Preliminary imaging of the Snellen chart demonstrated the ability to visualize structures with high contrast across both FOV configurations. The system effectively captured fine-scale details, confirming its suitability for widefield imaging. In future experiments, validation of the mesoscope will be tested using the natural fluorescence of pollen grains. Also, application of the mesoscope for in vivo imaging of mice brains during a reaching task will be performed, capturing neuronal activity in the thalamus using GCaMP-expressing mice. This will enable real-time investigation of the cerebello-thalamo-cortical pathway during skilled motor behavior, contributing to a better understanding of neural circuits involved in movement control.