Local delivery of fluorescent dye for fiber-optics confocal microscopy of the living heart

Abstract

Fiber-optics confocal microscopy (FCM) is an emerging imaging technology with various applications in basic research and clinical diagnosis. FCM allows for real-time in situ microscopy of tissue at sub-cellular scale. Recently FCM has been investigated for cardiac imaging, in particular, for discrimination of cardiac tissue during pediatric open-heart surgery. FCM relies on fluorescent dyes. The current clinical approach of dye delivery is based on systemic injection, which is associated with high dye consumption, and adverse clinical events. In this study, we investigated approaches for local dye delivery during FCM imaging based on dye carriers attached to the imaging probe. Using three-dimensional confocal microscopy, automated bench tests, and FCM imaging we quantitatively characterized dye release of carriers composed of open-pore foam only and foam loaded with agarose hydrogel. In addition, we compared local dye delivery with a model of systemic dye delivery in the isolated perfused rodent heart. We measured the signal-to-noise ratio (SNR) of images acquired in various regions of the heart. Our evaluations showed that foam-agarose dye carriers exhibited a prolonged dye release vs. foam-only carriers. Foam-agarose dye carriers allowed reliable imaging of 5–9 lines, which is comparable to 4–8 min of continuous dye release. Our study in the living heart revealed that the SNR of FCM images using local and systemic dye delivery is not different. However, we observed differences in the imaged tissue microstructure with the two approaches. Structural features characteristic of microvasculature were solely observed for systemic dye delivery. Our findings suggest that local dye delivery approach for FCM imaging constitutes an important alternative to systemic dye delivery. We suggest that the approach for local dye delivery will facilitate clinical translation of FCM, for instance, for FCM imaging during pediatric heart surgery.

Sensitivity and Specificity of Cardiac Tissue Discrimination Using Fiber-Optics Confocal Microscopy

“…We showed previously that the approach based on sodium fluorescein and dye delivery via carrier is more appropriate for clinical applications of FCM, in particular for translation of FCM for pediatric open heart surgery [ 15 ]. This approach was used to acquire FCM carrier images.…”

“…Our experimental conditions reproduced cardioplegic arrest commonly required for pediatric heart surgery. Two-dimensional image sequences were acquired using a FCM system (FCM 1000; Leica Microsystems GmbH, Wetzlar, Germany) and two methods for local delivery of fluorescent dye [ 14 , 15 ].…”

“…Rodent hearts were also imaged using FCM and a method of local dye delivery based on a dye carrier affixed to the tip of the FCM imaging microprobe. Dye carriers were fabricated according to a recently described method [ 15 ] and loaded for 15 min with fluorescent dye, sodium fluorescein [Fluorescite® (fluorescein injection, USP) 10%; Alcon, Fort Worth, TX, US; 1:1000]. Images from AWM, SAN, and AVN regions of perfused rodent hearts were acquired using this method with a custom fiber-optics microprobe having a numeric aperture of 0.8 (UltraMiniOWD30; Mauna Kea Technologies) at a lateral resolution ( xy ) of 1.4 μm, optical sectioning ( z ) of 7 μm, xy field of view of 186 by 130 μm, frame rate of 12 Hz, and a z-scan range of 26 μm.…”

“…Current clinical applications of FCM include imaging of tissues in gastrointestinal [ 11 ], respiratory [ 12 ] and urinary [ 13 ] system. We recently introduced approaches based on FCM and local delivery of extracellular fluorophores for imaging in the living heart [ 14 , 15 ]. In these studies we demonstrated feasibility of these approaches for cardiac tissue discrimination in the living heart of a rodent model, as well as in the fixed human fetal and pediatric heart.…”