Dual-color deep-tissue three-photon microscopy with a multiband infrared laser

Area of Science:

• Biophotonics
• Microscopy
• Neuroscience

Background:

• Multiphoton microscopy and fluorescent indicators are vital biological tools.
• Three-photon (3P) microscopy using short-wavelength infrared (SWIR) light offers deep-tissue imaging potential.
• Current methods require novel strategies for in-depth multicolor fluorescence imaging.

Purpose of the Study:

• To develop a novel multiband SWIR source for advanced 3P microscopy.
• To enable simultaneous deep-tissue multicolor fluorescence imaging.
• To demonstrate the utility of this new source for biological research.

Main Methods:

• Developed a novel multiband SWIR laser source emitting ultrashort pulses at 1.3 and 1.7 µm.
• Optimized laser characteristics for 3P microscopy (sub-70 fs, 1.25 MHz, µJ pulse energy).
• Achieved simultaneous 3P excitation of green and red fluorescent proteins and third-harmonic generation.

Main Results:

• Demonstrated simultaneous 3P excitation of GFP and red fluorescent proteins (mRFP, mCherry, tdTomato).
• Successfully performed in-depth dual-color 3P imaging in fixed mouse brain, chick embryo spinal cord, and live zebrafish brain.
• Observed improved signal-to-background ratio compared to multicolor two-photon imaging.

Conclusions:

• The novel multiband SWIR source facilitates advanced deep-tissue, multicolor 3P imaging.
• This technology enhances imaging capabilities in scattering biological tissues.
• Opens new avenues for multiparametric imaging deep within living organisms.