Two-photon microscopy: imaging in scattering samples and three-dimensionally resolved flash photolysis

Area of Science:

• Biophysics
• Cell Biology
• Microscopy

Background:

• Conventional confocal fluorescence microscopy has limitations in scattering samples and precise photolysis.
• Two-photon microscopy offers advantages in depth penetration and spatially resolved flash photolysis.

Purpose of the Study:

• To evaluate the benefits of non-descanned fluorescence detection in two-photon microscopy.
• To investigate the spatio-temporal properties of two-photon excited flash photolysis (TPEFP) for studying cellular calcium dynamics.

Main Methods:

• Incorporation of non-descanned fluorescence detection into a two-photon microscope system.
• Two-photon excited flash photolysis (TPEFP) using DM-nitrophen in test solutions and cardiac myocytes.
• Examination of calcium release events and diffusion properties using EGTA and low-affinity calcium indicators.

Main Results:

• Non-descanned detection improved signal strength by over an order of magnitude at depths >40 microm in scattering samples.
• Low-power TPEFP (approx. 4 mW, <50 ms) successfully generated repeatable calcium release events in cardiac myocytes, mimicking natural calcium sparks.
• Calcium diffusion in myocytes appeared similar to Fluo-3 diffusion in solution.
• EGTA improved temporal resolution but not spatial spread of calcium signals during TPEFP.

Conclusions:

• Non-descanned detection is crucial for deep imaging in scattering biological samples with two-photon microscopy.
• TPEFP provides a powerful tool for studying cellular calcium signaling with high spatial and temporal control.
• The study offers insights into calcium diffusion dynamics within cardiac myocytes.