Igor pro version 6.22a

FLIM experiments were performed on an Olympus FluoView 1000 laser scanning microscope (Olympus, Tokyo, Japan) with a Sepia II and PicoHarp 300 time correlated single photon counting system from PicoQuant (Berlin, Germany). Proteins were tagged with the fluorescent proteins mCitrine (donor) and mCherry (acceptor) to optimize FRET efficiency. All experiments were carried out at 37°C with a 60x water objective. For imaging, samples were simultaneously excited with 488 (10% laser intensity) and 561 nm (31% laser intensity) light using a 405/488/561/633 dichroic mirror. Excitation light was split with a dichroic mirror at 560 nm and detected between 500–550 nm (green channel) and 580–680 nm (red channel). The pinhole was set to 300 μm. FLIM images were acquired using 470 nm excitation (36% intensity) with a pulse frequency of 40 MHz, a 405/470 dichroic mirror, and a 525/15 band path filter. FLIM images were analyzed using IGOR Pro (Version 6.22 A, Wave Metrics, Lake Oswego, OR) with pFLIM3 as previously described (Walther et al., 2011 (link)).

After recovery, raw data were downloaded from the loggers and analyzed using Igor Pro version 6.22A (Wavemetrics Inc.). We used a purpose‐written script to adjust the depth to zero when birds were at the surface between two dives and extract diving parameters from the depth data, including maximum diving depth, dive and postdive duration, and the number of vertical undulations in the bottom phase of the dive for each dive deeper than 1 m, which are generally indicative of prey pursuit (Kato, Ropert‐Coudert, Grémillet, & Cannell, 2006; Ropert‐Coudert, Kato, Wilson, & Cannell, 2006). A bottom phase was defined as the first and last time during a dive that the depth‐change rate became <0.25 m/s (Kato, Ropert‐Coudert, & Chiaradia, 2008). To allow comparison between the different stages, we calculated the mean foraging effort per day as the total diving duration (in minutes) divided by the trip duration (in days). Similarly, we calculated the mean number of dives per day as the total number of dives performed over a trip divided by the trip duration (in days). The prey encounters per unit time used as proxy for foraging efficiency was calculated as the total number of vertical undulations divided by the total diving duration over the trip (Sala, Wilson, & Quintana, 2012).