Fig. 2.

Optical configuration results in negligible crosstalk between fluorescent probes. (a) Passbands of all the filters used in the optical configuration. (b) Two sides of a single, large camera sensor display a duplicated ruler image from each emission band with negligible focal shift. (c) Illustration of misaligned (right) and proper alignment (left) of images. (d) i: 800 × 256 pixel image of a back illuminated ruler is shown before splitting into two 384 × 256 pixel images; left: long wavelength path (710 + nm), middle: black boundary between images (16 pixels), right: short wavelength path (585 ± 20 nm). (ii) 800 × 256 image when the a plate is inserted to block the light path on the left (710 + nm). (iii) 800 × 256 image when the path on the right is blocked (585 ± 20 nm). (iiii) Profile traces corresponding to the full image (black), left path blocked (blue, 710 + nm) and right path blocked (red, 585 ± 20 nm) demonstrate a lack of crosstalk along the light paths. Arrow denotes location of the boundary between images without crosstalk. (e) Left: Detected fluorescence on the epicardial surface after single probe administration (Rhod2). Center: High quality calcium transients through the 585 nm emission filter with no detectable signal through the 710 nm longpass filter. Right: Maximum counts from each channel after Rhod2 (only) administration. (f) Left: Detected fluorescence on the epicardial surface after single probe administration (RH237). Center: High quality optical action potentials through the 710 nm emission filter, with minimal signal through the 585 nm emission. Right: Maximum counts from each channel after RH237 (only) administration. Note: Bar plots (E, F) on the right collected from different rat hearts, each loaded with a single fluorescent probe (n = 3 each plot). Scale bar = 1 cm