Zet642 20x

Fluorescence imaging was carried out on an inverted microscope (Nikon Instruments, Eclipse Ti2) with the Perfect Focus System, applying an objective-type TIRF configuration with an oil-immersion objective (Nikon Instruments, Apo SR TIRF 100×, NA 1.49, Oil). A 561 nm and 640 nm (MPB Communications Inc, 2W, DPSS-system) laser were used for excitation. The laser beam was passed through clean-up filters (Chroma Technology, ZET561/10, ZET642/20x) and coupled into the microscope objective using a beam splitter (Chroma Technology, ZT561rdc, ZT647rdc). Fluorescence light was spectrally filtered with an emission filter (Chroma Technology, ET600/50m and ET575lp, ET705/72m and ET665lp) and imaged on a sCMOS camera (Andor, Zyla 4.2 Plus) without further magnification, resulting in an effective pixel size of 130 nm (after 2 × 2 binning).

For imaging the voltage dye di-4-ANBDQPQ, two red LEDs (part #: CBT-90 Red; Luminus Devices Inc., Woburn, MA) were used to excite dye-loaded tissue (Fig. 9a,b). The excitation light from each LED was passed through a plano-convex lens (part #: LA1951; Thorlabs Inc., Newton, NJ) and excitation filter (part #: ZET642/20X; Chroma Technology Corp, Bellows Falls, VT). Fluorescence emission light from the tissue was passed through a custom-made emission filter (part #: ET700LP; Chroma Technology Corp) and then collected with a camera lens (part #: DO-1795; Navitar Inc., Rochester, NY). The same emission filter and camera lens were used for both the CMOS and EMCCD camera. For imaging voltage dye RH237 and calcium dye rhod-2AM, the same configuration as Optical Mapping System 2 was used.