Coolsnap hq2

[Ca²⁺]_i was measured by quantitative fluorescence imaging using the calcium-sensitive dye fura-2. Cells plated on a coverslip were incubated with 4 μM fura-2 acetoxymethyl ester (fura-2 AM; Molecular Probes) for 30 min at 37°C. Fura-2 fluorescence emission was measured at 510 nm in response to alternating excitation at 340 and 380 nm. Images were acquired and stored using a NIKON Eclipse TE300 microscope (with 40x oil immersion objective) and CCD (CoolSNAP HQ₂) camera under computer control (MetaFluor: Molecular Devices). For each coverslip, the background light levels were determined and subtracted from each image before measurement of the fluorescence intensity ratio. [Ca²⁺]_i was determined using the 340 nm/380 nm fluorescence ratio as described previously (Grynkiewicz et al., 1985 (link)). Calibration was performed using cell-free solutions. The perfusion solution used for [Ca²⁺] measurements contained (mM): 118 NaCl, 23 NaHCO₃, 3 KCl, 2 KH₂PO₄, 1 MgCl₂, 1.2 CaCl₂, 11 glucose. The temperature of the perusion solution at the recording chamber was 35°C.

[Ca²⁺]_i was measured by quantitative fluorescence imaging using the calcium-sensitive dye fura-2. Cells plated on the coverslip were incubated with 4 µM fura-2 acetoxymethyl ester (fura-2 AM; Molecular Probes) for 30 min at 37 °C. Fura-2 fluorescence emission was measured at 510 nm in response to alternating excitation at 340 and 380 nm. Images were acquired and stored using a NIKON Eclipse TE300 microscope (with 40x oil immersion objective) and CCD (CoolSNAP HQ₂) camera under computer control (MetaFluor: Molecular Devices). For each coverslip, the background light levels were determined and subtracted from each image before measurement of the fluorescence intensity ratio. [Ca²⁺]_i was determined using the 340 nm/380 nm fluorescence ratio as described previously (Wasicko et al., 1999 (link)). Calibration was performed using cell-free solutions (Grynkiewicz et al., 1985 (link)). The perfusion solution used for [Ca²⁺] measurements contained (mM): 118 NaCl, 23 NaHCO₃, 3 KCl, 1 MgCl₂, 1.2 CaCl₂, 11 glucose.

Chorionated embryos that had been injected with the tbx16 cMO and/or photoactivatable tracers and spot-irradiated at 6 hpf were placed in an agarose template (560-μm × 960-μm wells) filled with E3 medium, oriented with the dorsal shield facing upward. Imaging was performed with a Leica DMI6000B inverted compound microscope equipped with a UVI 6.3x/0.13 objective, GFP and TX2 filters, and a CoolSnap HQ2 monochrome camera controlled by MetaMorph microscopy automation and image analysis software (Molecular Devices). Fluorescent micrographs were acquired at rate of 1 frame per 5 minutes, with exposure times automatically adjusted every five frames to account for progressive photobleaching. ImageJ software (NIH) was used to normalize signal brightness and generate annotated movie montages.