Simultaneous measurement and modulation of multiple physiological parameters in the isolated heart using optical techniques

Abstract

Whole-heart multi-parametric optical mapping has provided valuable insight into the interplay of electrophysiological parameters, and this technology will continue to thrive as dyes are improved and technical solutions for imaging become simpler and cheaper. Here, we show the advantage of using improved 2nd-generation voltage dyes, provide a simple solution to panoramic multi-parametric mapping, and illustrate the application of flash photolysis of caged compounds for studies in the whole heart. For proof of principle, we used the isolated rat whole-heart model. After characterising the blue and green isosbestic points of di-4-ANBDQBS and di-4-ANBDQPQ, respectively, two voltage and calcium mapping systems are described. With two newly custom-made multi-band optical filters, (1) di-4-ANBDQBS and fluo-4 and (2) di-4-ANBDQPQ and rhod-2 mapping are demonstrated. Furthermore, we demonstrate three-parameter mapping using di-4-ANBDQPQ, rhod-2 and NADH. Using off-the-shelf optics and the di-4-ANBDQPQ and rhod-2 combination, we demonstrate panoramic multi-parametric mapping, affording a 360° spatiotemporal record of activity. Finally, local optical perturbation of calcium dynamics in the whole heart is demonstrated using the caged compound, o-nitrophenyl ethylene glycol tetraacetic acid (NP-EGTA), with an ultraviolet light-emitting diode (LED). Calcium maps (heart loaded with di-4-ANBDQPQ and rhod-2) demonstrate successful NP-EGTA loading and local flash photolysis. All imaging systems were built using only a single camera. In conclusion, using novel 2nd-generation voltage dyes, we developed scalable techniques for multi-parametric optical mapping of the whole heart from one point of view and panoramically. In addition to these parameter imaging approaches, we show that it is possible to use caged compounds and ultraviolet LEDs to locally perturb electrophysiological parameters in the whole heart.

Fig. 1

Isosbestic points of di-4-ANBDQBS and di-4-ANBDQPQ. a Di-4-ANBDQBS fluorescence in a rat heart (left and right ventricle view; sinus rhythm), excited with red (red LED filtered with F1) and blue (blue LED filtered with F2a) wavelengths. Fluorescence signals (taken from the 4×4-pixel white-square region shown) were collected through the custom-made multi-band filter F3a shown in (c). Illumination with the blue source (Ex2a) yielded no signal during the AP. b Di-4-ANBDQPQ fluorescence in a rat heart (mostly left ventricle view; sinus rhythm), excited with red (red LED filtered with F1) and green (green LED filtered with F2b) wavelengths. Fluorescence signals (taken from the 4×4-pixel white-square region shown) were collected through the custom-made multi-band filter F3b shown in (d). Illumination with the green source (Ex2b) yielded no signal during the AP. c Transmission spectrum of a custom multi-band emission filter that passes V_m (Em1; di-4-ANBDQBS dye) and [Ca²⁺]_i (Em2a; fluo-4 dye) emitted fluorescence signals. F1 and F2a (for di-4-ANBDQBS and fluo-4 excitation, respectively) excitation filter spectra are shown as dashed curves. d Transmission spectrum of a custom multi-band emission filter that passes V_m (Em1; di-4-ANBDQPQ dye) and [Ca²⁺]_i (Em2b; rhod-2 dye) emitted fluorescence signals. F1 and F2b (for di-4-ANBDQPQ and rhod-2 excitation, respectively) excitation filter spectra are shown as dashed curves. Scale bar=5 mm

Fig. 2

V_m and [Ca²⁺]_i optical mapping approach using isosbestic points. a System schematic showing key components (see text for details). Since a single camera (combined with a multi-band filter F3a/b) is used, the V_m and [Ca²⁺]_i optical mapping system requires no challenging optical alignment. b The multi-colour imaging technique: During any camera frame exposure, either the V_m or the [Ca²⁺]_i (Em1 or Em2a/b) signal is acquired by illuminating the dual-dye-loaded tissue with excitation source Ex1 or Ex2a/b, respectively. At sufficiently high camera frame rates and with interpolation, this technique provides a straightforward method of multi-parametric optical mapping. c V_m (red) and [Ca²⁺]_i (blue) fluorescence signals (camera signals on a 16-bit scale) taken from the 4×4-pixel white-square region shown from a rat heart dual-loaded with di-4-ANBDQBS and fluo-4. d V_m (red) and [Ca²⁺]_i (green) fluorescence signals (camera signals on a 16-bit scale) taken from the 4×4-pixel white-square region shown from a rat heart dual-loaded with di-4-ANBDQPQ and rhod-2. Scale bar=5 mm

Fig. 3

V_m, [Ca²⁺]_i and ‘ischemia’ optical mapping. a Transmission spectrum of an off-the-shelf triple-band emission filter that passes V_m (di-4-ANBDQPQ), [Ca²⁺]_i (rhod-2) and NADH (endogenous) fluorescence, due to excitation with sources Ex1, Ex2 and Ex3, respectively (see text for details). Ex1 and Ex2 excitation filter spectra are shown as dashed curves. b Oscilloscope traces of camera frame-exposure (top trace; 5 V=exposure on) and photodiode signals (bottom trace) recorded from LED excitation sources Ex1, Ex2 and Ex3 (triggered by the frame-exposure signal via a microcontroller). These traces show the non-overlapping on/off cycles of the three excitation sources, and the output stability of the LEDs. c Normalised fluorescence intensity maps (colourbar shown) at progressive time points during sinus rhythm (left and right ventricle view). The delay of the CaT relative to the AP peak (~15 ms delay) is clearly visible. d The change in the normalised fluorescence intensity maps (colourbar shown) of NADH during the course of 15 min before and after left anterior descending artery proximal occlusion (tie-off point marked by a red circle in the bottom panel). Scale bar=5 mm

Fig. 4

V_m and [Ca²⁺]_i panoramic optical mapping using a single camera. a System schematic showing key components (see text for details). A heart-sized model made from Blu-Tak (top right and bottom right) was used to guide positioning of the two back mirrors, lenses and camera. Transmission spectra for filters F1, F2b and F3b are shown in Fig. 1d. Sides S1, S2 and S3 represent projections from three angles, 120° apart, where S1 represents the primary view and S2 and S3 the secondary mirror image views. b V_m (red) and [Ca²⁺]_i (green) fluorescence signals (camera signals on a 16-bit scale) taken from the 4×4-pixel white-square region shown from a rat heart, in sinus rhythm, dual-loaded with di-4-ANBDQPQ and rhod-2. Top, a grayscale fluorescence image (V_m) of sides S1, S2 and S3 (S1 is mostly a view of the left ventricle). The red circle indicates the location of electrical point stimulation. c Normalised fluorescence intensity maps (colourbar shown) at progressive time points during 5 Hz local electrical pacing. The delay of the CaT relative to the AP peak (~21 ms delay) is clearly visible. Scale bar=5 mm (for primary view S1)

Fig. 5

Loading and local flash photolysis of NP-EGTA in the whole heart. a Left, a grayscale fluorescence image (V_m) of the rat heart (mostly right ventricle view). The remaining panels are normalised [Ca²⁺]_i fluorescence intensity maps (colourbar shown) at progressive time points during sinus rhythm, after NP-EGTA loading. b [Ca²⁺]_i (blue, green and red) fluorescence signals (camera signals on a 16-bit scale) taken from 8×8-pixel regions (left panel in (a)) from the heart, in sinus rhythm. Left, shows CaT after loading of NP-EGTA. Middle, altered CaT at site 1 (and unaltered CaT at sites 2 and 3) after region 1 local flash photolysis. Right, altered CaT at sites 1 and 2 (and unaltered CaT at site 3) after region 2 local flash photolysis. c Normalised [Ca²⁺]_i fluorescence intensity maps (colourbar shown) at progressive corresponding (to a) time points, highlighting CaT widening at site 1 (left, two panels) and sites 1 and 2 (right, two panels), during sinus rhythm. Scale bar=5 mm