Deep tissue multiphoton microscopy using longer wavelength excitation.

Area of Science:

• Neuroscience
• Biomedical Optics
• Microscopy

Background:

• Two-photon microscopy (TPM) is crucial for deep tissue imaging in neuroscience.
• Wavelength selection significantly impacts imaging depth due to light scattering and absorption.
• Optimizing excitation wavelength is key to enhancing TPM's penetration in biological tissues.

Purpose of the Study:

• To compare the maximal imaging depth of 775-nm and 1280-nm excitation in two-photon fluorescence microscopy.
• To evaluate the efficacy of longer wavelengths for in vivo and ex vivo imaging of mouse brain vasculature.
• To assess the feasibility of deep-brain blood flow measurements using optimized TPM.

Main Methods:

• In vivo and ex vivo two-photon fluorescence microscopy (TPM) of fluorescently-labeled mouse brain vasculature.
• Comparative analysis of imaging depth achieved with 775-nm and 1280-nm excitation wavelengths.
• Measurement of blood flow speed at depths up to 900 micrometers.

Main Results:

• 1280-nm excitation achieved approximately twice the imaging depth compared to 775-nm excitation.
• High-contrast imaging of blood vessels was obtained at depths up to 1 mm in adult mouse brains.
• Achieved 1 mm imaging depth with approximately 1-nJ pulse energy at the sample surface using 1280-nm excitation.
• Successful blood flow speed measurements were performed at a depth of 900 micrometers.

Conclusions:

• Longer excitation wavelengths, such as 1280 nm, significantly enhance maximal imaging depth in two-photon microscopy.
• 1280-nm TPM offers a viable method for deep-brain in vivo imaging and functional studies, including blood flow.
• This advancement extends the capabilities of TPM for investigating neural circuits and vascular dynamics in intact brains.