Long-Stokes-Shift mScarlet3 as a Structural Marker for Two-Photon Imaging

Area of Science:

• Neuroscience
• Biophysics
• Genetics

Background:

• Two-photon imaging is crucial for monitoring neurophysiology using genetically encoded sensors.
• Incorporating fluorescent proteins aids in cell-type identification and motion detection.
• Existing red fluorescent proteins often necessitate a separate excitation laser, increasing equipment complexity.

Purpose of the Study:

• To develop a red fluorescent protein for simultaneous imaging with green sensors using a single laser.
• To reduce complexity and cost in two-photon microscopy setups.
• To enable anatomical context for functional neurophysiological data.

Main Methods:

• Creation of transgenic Drosophila expressing a long-Stokes-shift mScarlet variant (LSSmScarlet3).
• Characterization of excitation and emission spectra of LSSmScarlet3.
• In vitro and in vivo assessment of LSSmScarlet3 fluorescence at 920 nm.

Main Results:

• LSSmScarlet3 demonstrates robust fluorescence at 920 nm.
• Minimal channel crosstalk was observed when imaging alongside green sensors.
• Successful in vitro and in vivo imaging was achieved with a single 920-nm laser.

Conclusions:

• LSSmScarlet3 enables simultaneous functional and anatomical imaging in Drosophila.
• This method simplifies two-photon microscopy by reducing the need for multiple lasers.
• The approach integrates neurophysiological data with precise anatomical localization.