Optical mapping at increased illumination intensities.

Area of Science:

• Biomedical Optics
• Electrophysiology
• Cardiovascular Research

Background:

• Voltage-sensitive fluorescent dyes are crucial for cardiac and neuro-electrophysiology.
• High signal-to-noise ratios necessitate increased illumination, risking photobleaching and phototoxicity.
• Optimal illumination intensities vary by dye and require individual assessment.

Purpose of the Study:

• To evaluate the effects of high illumination intensities on two voltage-sensitive dyes (di-4-ANBDQBS and di-4-ANEPPS) in guinea pig hearts.
• To determine if increased light intensity causes photobleaching or phototoxicity.
• To assess the impact of light absorption-induced tissue heating on electrophysiological parameters.

Main Methods:

• Two voltage-sensitive dyes, di-4-ANBDQBS (660 nm excitation) and di-4-ANEPPS (532 nm excitation), were tested in guinea pig hearts.
• Illumination intensities ranged from 0.1 to 5 mW/mm², exceeding typical literature values.
• Illumination duration was 60 seconds, equivalent to 300 cardiac beats.

Main Results:

• Neither dye exhibited significant photobleaching or phototoxicity within the tested intensity and duration range.
• Higher illumination intensities led to noticeable tissue heating.
• Tissue heating caused a shortening of action potential duration, approximately 30% for 532-nm and 10% for 660-nm excitation.

Conclusions:

• High illumination intensities for optical mapping with di-4-ANBDQBS and di-4-ANEPPS are safe regarding photobleaching and phototoxicity.
• Tissue heating is a significant consequence of high-intensity illumination, affecting electrophysiological measurements.
• These findings are critical for optimizing optical mapping protocols in biomedical applications to account for thermal artifacts.