Live cell ultraviolet microscopy: a comparison between two- and three-photon excitation

Area of Science:

• Biophotonics
• Neuroscience
• Spectroscopy

Background:

• Serotonin, a crucial neurotransmitter, is often imaged using fluorescence microscopy.
• Ultraviolet fluorophores like serotonin present challenges for conventional imaging techniques.
• Multiphoton microscopy offers advantages for deep tissue and live-cell imaging.

Purpose of the Study:

• To compare the efficacy of two-photon excitation (2PE) using visible light versus three-photon excitation (3PE) using infrared light for serotonin imaging.
• To evaluate the performance of both excitation schemes in solution and in live cells.
• To investigate the underlying reasons for observed differences in imaging performance.

Main Methods:

• Utilized a visible laser for 2PE and an infrared laser for 3PE to excite the ultraviolet fluorophore serotonin.
• Quantified signal levels and detectivity of serotonin in buffer solutions under varying laser powers.
• Performed live-cell imaging of vesicular serotonin in serotonergic cells using both 2PE and 3PE techniques.

Main Results:

• 3PE required higher power (33 mW at 740 nm) than 2PE (5 mW at 550 nm) to achieve a comparable signal level.
• 3PE demonstrated significantly higher detectivity (220 µM) compared to 2PE (12 µM) for serotonin in solution.
• Surprisingly, 3PE yielded superior image contrast for live-cell imaging of vesicular serotonin, attributed to a concentration-dependent spectral shift.

Conclusions:

• Three-photon excitation provides superior detectivity for serotonin compared to two-photon excitation in solution.
• The concentration-dependent spectral shift of serotonin emission is a critical factor influencing image contrast in live-cell imaging.
• Three-photon excitation emerges as a promising technique for high-contrast imaging of serotonin in complex biological systems.