Deep two-photon microscopic imaging through brain tissue using the second singlet state from fluorescent agent...

Area of Science:

• Biomedical Optics
• Microscopy
• Plant Science

Background:

• Deep tissue imaging is limited by light scattering and absorption.
• Two-photon (2P) microscopy offers improved resolution and reduced phototoxicity.
• Excitation of higher electronic states can alter fluorescence properties.

Purpose of the Study:

• To investigate two-photon (2P) excitation of the second singlet (S₂) state for deep optical microscopic imaging.
• To assess the feasibility of imaging chlorophyll α (Chl α) fluorescence through biological tissue.
• To determine the maximum imaging depth achievable using the 2P S₂ state technique.

Main Methods:

• Utilized two-photon (2P) excitation of the second singlet (S₂) state of chlorophyll α (Chl α).
• Employed 800 nm excitation and observed 685 nm emission, within the tissue optical window (650-950 nm).
• Combined 2P microscopy with a spinach leaf positioned beneath freshly sliced rat brain tissue.

Main Results:

• Achieved strong Chl α fluorescence emission at 685 nm via 2P S₂ state excitation.
• Successfully imaged Chl α fluorescence through a thick layer of rat brain tissue.
• Demonstrated an imaging depth of up to 450 μm.

Conclusions:

• Two-photon (2P) excitation of the S₂ state is a viable method for deep optical microscopic imaging.
• This technique allows for non-invasive visualization of biological samples, like Chl α in plants, through scattering media such as brain tissue.
• The 2P S₂ state approach significantly enhances imaging depth in biological tissues.