Brain heating induced by near-infrared lasers during multiphoton microscopy

Area of Science:

• Neuroscience
• Biophotonics
• Microscopy

Background:

• Two-photon microscopy and optogenetics require high illumination powers for advanced applications.
• Information on heat generation and tissue damage from high-power illumination in the brain is limited.

Purpose of the Study:

• To quantify temperature changes and identify damage thresholds induced by two-photon microscopy in the mouse neocortex.
• To investigate the influence of wavelength, exposure time, and illumination depth on thermal effects.

Main Methods:

• Utilized thermocouple probes and quantum dot nanothermometers to measure temperature variations in the neocortex of awake and anesthetized mice.
• Characterized heating patterns as a function of illumination parameters and depth.
• Assessed tissue damage using immunohistochemistry for markers like Iba1, GFAP, HSPs, and cleaved caspase-3.

Main Results:

• Heating was most severe hundreds of micrometers below the focal plane due to heat dissipation through the cranial window.
• Continuous illumination (1 mm² area) caused a peak temperature increase of ~1.8°C per 100 mW.
• Powers exceeding 250 mW under continuous illumination induced lasting tissue damage.

Conclusions:

• High-power two-photon microscopy can induce significant thermal effects and neuronal damage in brain tissue.
• Damage thresholds depend on illumination power, duration, and depth.
• Employing limited duty cycles for illumination presents a viable strategy to mitigate damage in high-power microscopy applications.