Home › Microscopy & Imaging › Multi-layer Cortical Ca2+ Imaging in Freely Moving Mice with Prism Probes and Miniaturized Fluorescence Microscopy
Microscopy & Imaging JoVE (Open Access) Citable · DOI

Multi-layer Cortical Ca2+ Imaging in Freely Moving Mice with Prism Probes and Miniaturized Fluorescence Microscopy

DOI: 10.3791/55579-v
What you'll learn
  • ✓ Perform surgical implantation of prism probes for multi-layer cortical imaging
  • ✓ Install and align miniaturized fluorescence microscopy equipment on freely moving mice
  • ✓ Acquire and interpret calcium imaging data across multiple cortical layers simultaneously
Protocol

Here, we present a procedure for performing large-scale Ca2+ imaging with cellular-resolution across multiple cortical layers in freely moving mice. Hundreds of active cells can be observed simultaneously using a miniature, head-mounted microscope coupled with an implanted prism probe.

Difficulty
advanced
Total time
~4-6 weeks per mouse (surgery recovery + imaging sessions)
Model organism
Mouse (C57BL/6 or similar)
Biosafety
BSL-1

Steps

1
Perform prism probe implant surgery

Surgically implant the prism probe into target cortical region(s) using stereotactic coordinates and proper anesthesia. Secure the probe and allow adequate recovery time before proceeding to imaging setup.

▶ 01:12
2
Attach baseplate for microscope installation

Fix the baseplate assembly to the mouse skull or implant structure to enable reliable mounting and alignment of the miniaturized fluorescence microscope during imaging sessions.

▶ 04:52
3
Image multiple cortical layers in freely moving mice

Mount the head-mounted miniature microscope and acquire calcium fluorescence data from hundreds of neurons across superficial and deep cortical layers while the mouse moves freely in the behavioral arena.

▶ 07:22
4
Analyze calcium dynamics from imaging data

Process acquired fluorescence images to extract and characterize calcium transient activity patterns from neurons in different cortical layers and correlate with animal behavior.

▶ 08:33
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