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Microscopy & Imaging JoVE (Open Access) Citable · DOI

Detection of Microregional Hypoxia in Mouse Cerebral Cortex by Two-photon Imaging of Endogenous NADH Fluorescence

DOI: 10.3791/3466-v
What you'll learn
  • ✓ Perform two-photon imaging of NADH fluorescence to detect tissue hypoxia
  • ✓ Prepare mouse cranial window for in vivo cortical imaging
  • ✓ Monitor microcirculation and blood oxygenation during imaging
  • ✓ Interpret microregional oxygen supply patterns in cerebral cortex
Protocol

Here we describe a method to directly visualize microregional tissue hypoxia in the mouse cortex in vivo. It is based on concurrent two-photon imaging of nicotinamide adenine dinucleotide (NADH) and the cortical microcirculation. This method is useful for high resolution analysis of tissue oxygen supply.

Difficulty
advanced
Total time
~3–4 hours per mouse
Model organism
Mouse (strain not specified, assumed C57BL/6 or similar)
Biosafety
BSL-1

Steps

1
Prepare animal for two-photon imaging

Anesthetize and position the mouse for cranial access. This step establishes proper surgical anesthesia and immobilization required for stable imaging.

▶ 01:42
2
Create open skull cranial window

Perform surgical exposure of the cortex via small craniotomy and install a cover glass. This provides optical access to the cerebral cortex for microscopy.

▶ 02:56
3
Inject dye and monitor blood oxygenation

Administer intravenous contrast agent and measure arterial oxygen saturation throughout the imaging session. This enables simultaneous visualization of microcirculation.

▶ 06:40
4
Acquire two-photon images of NADH

Perform concurrent two-photon microscopy to image endogenous NADH fluorescence and detect microregional hypoxia in cortical tissue.

▶ 07:58
5
Analyze hypoxia measurement results

Interpret NADH fluorescence patterns and correlate with microcirculatory data to identify and quantify tissue hypoxia regions.

▶ 09:30
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