Optical mapping at increased illumination intensities

Abstract

Abstract. Voltage-sensitive fluorescent dyes have become a major tool in cardiac and neuro-electrophysiology. Achieving high signal-to-noise ratios requires increased illumination intensities, which may cause photobleaching and phototoxicity. The optimal range of illumination intensities varies for different dyes and must be evaluated individually. We evaluate two dyes: di-4-ANBDQBS (excitation 660 nm) and di-4-ANEPPS (excitation 532 nm) in the guinea pig heart. The light intensity varies from 0.1 to 5 mW∕mm 2 , with the upper limit at 5 to 10 times above values reported in the literature. The duration of illumination was 60 s, which in guinea pigs corresponds to 300 beats at a normal heart rate. Within the identified duration and intensity range, neither dye shows significant photobleaching or detectable phototoxic effects. However, light absorption at higher intensities causes noticeable tissue heating, which affects the electrophysiological parameters. The most pronounced effect is a shortening of the action potential duration, which, in the case of 532-nm excitation, can reach ∼30%. At 660-nm excitation, the effect is ∼10%. These findings may have important implications for the design of optical mapping protocols in biomedical applications.

Evolution of Action Potential Alternans in Rabbit Heart During Acute Regional Ischemia

“…The optical mapping setup was as described previously [14]. Briefly, fluorescence imaging was carried out using a cooled fast 14-bit EMCCD camera (iXon EM+ DU-860, Andor Technology) equipped with 50 mm focal length objective.…”

Toward microendoscopy-inspired cardiac optogeneticsin vivo: technical overview and perspective

“…High-resolution CCD cameras and EMCCD cameras have been popular for use in optical mapping [5][6][7][30][31][32][33][34][37][38][39][40]127,128,144 and microendoscopy due to their high-spatiotemporal resolution. EMCCD cameras take advantage of avalanche processes to perform the on-chip electron multiplication to enhance SNRs, have higher frame rates, and improved quantum efficiency.…”

“…[12][13][14][15]44,[46][47][48] However, these potentiometric dyes are inherently inefficient. For example, di-4-ANEPPS has a quantum efficiency estimated to be on the order of 0.3 129,130 and is excited at irradiances as low as 0.1 up to 5 mW∕mm 2 before light-induced tissue heating occurs 128 resulting in, at best, a 10% relative change in fluorescence per 100 mV. 5 More recently, new near-infrared dyes, such as di-4-ANBDQPQ and di-4-ANBDQBS, have been used in blood-perfused hearts as their absorption peak is >70 nm higher than that of hemoglobin.…”

“…The two key classes of fast response indicators are voltage (potentiometric) and calcium probes; examples of these indicators can be seen in Table 1. For in vitro voltage sensing, the styryl dyes di-4-ANEPPS, di-8-ANEPPS, and RH-237 undergoing spectral shifts that vary linearly with voltage changes have been used extensively in optical mapping [5][6][7]30,31,35,38,40,127,128 and other cardiac imaging systems. [12][13][14][15]44,[46][47][48] However, these potentiometric dyes are inherently inefficient.…”

“…For example, di-4-ANEPPS and di-4-ANBDQBS (excited by 535 nm and 660 nm, respectively) show little difference in percent of fluorescence change during an action potential for irradiances varied from 0.1 to 5 mW∕mm 2 ; however, at higher irradiances, tissue heating can occur thereby altering the measured action potentials. 128 Other practical considerations include the power consumption of the light source, whether or not cooling units are needed, the size of the device, and how easily the light source can be coupled into the system.…”