Visualizing astrocytes in the deep mouse brain in vivo.

Area of Science:

• Neuroscience
• Biomedical Imaging
• Cell Biology

Background:

• Astrocytes are crucial glial cells in the central nervous system, but their visualization in deep brain regions in vivo has been challenging.
• Existing imaging techniques, such as two-photon fluorescence microscopy, have limitations in achieving sufficient depth for studying deep brain structures.
• The development of advanced imaging methods is essential for understanding astrocyte function in complex neural circuits.

Purpose of the Study:

• To demonstrate the efficacy of three-photon fluorescence imaging for visualizing astrocytes in the deep brain in vivo.
• To optimize imaging parameters by measuring the wavelength-dependent three-photon action cross-section of sulforhodamine 101 (SR101).
• To achieve unprecedented imaging depths for astrocyte visualization compared to existing methods.

Main Methods:

• Utilized three-photon fluorescence imaging with sulforhodamine 101 (SR101) labeling for astrocytes and vasculature in live mouse brains.
• Performed wavelength-dependent measurements of the three-photon action cross-section (ησ ₃ ) of SR101 to guide excitation wavelength selection.
• Quantitatively compared signal differences between labeled blood vessels and astrocytes to assess imaging challenges.

Main Results:

• Achieved an imaging depth of 1340 μm into the mouse brain, demonstrating the technique's deep imaging capability.
• Successfully visualized astrocytes at depths of 910 μm, significantly deeper (30%) than achievable with two-photon fluorescence microscopy.
• Confirmed that imaging depth is not limited by signal-to-background ratio, unlike in two-photon imaging.

Conclusions:

• Three-photon fluorescence imaging of SR101-labeled astrocytes is a powerful technique for in vivo deep brain visualization.
• This method overcomes the depth limitations of two-photon microscopy, enabling deeper exploration of astrocyte function.
• Further research is needed to address challenges in visualizing astrocytes below white matter tracts.