Single-Molecule Imaging in Living Drosophila Embryos with Reflected Light-Sheet Microscopy

Area of Science:

• Biophysics
• Developmental Biology
• Microscopy

Background:

• Single-fluorophore imaging in multicellular organisms is challenging due to high background noise.
• Achieving a sufficient signal-to-noise ratio for single-molecule imaging requires specialized techniques.

Purpose of the Study:

• To adapt reflected light-sheet microscopy (RLSM) for single-molecule imaging in opaque, late-stage Drosophila embryos.
• To quantify molecular diffusion dynamics within the complex environment of living embryos.

Main Methods:

• Modified RLSM with microprisms for improved alignment in opaque samples.
• Imaging of septate-junction complex proteins to delineate epidermal structures.
• Single-particle tracking of fluorescently labeled Dextran molecules within embryos.

Main Results:

• Successfully imaged single 10 kDa Dextran molecules within living Drosophila embryos.
• Quantified Dextran diffusion in free space (∼6.4 μm²/s) and directional movement in epidermal tissue (∼0.1 μm²/s).
• Corroborated findings with imaging of Rab5-GFP and existing data on morphogen/vesicle transport.

Conclusions:

• Adapted RLSM significantly enhances signal-to-noise ratio for single-molecule imaging in opaque biological samples.
• RLSM provides insights into molecular diffusion dynamics in complex multicellular environments.
• This technique holds promise for studying single molecules and complexes in vivo.