Dataset: 1_copepod gut fluorescence

A Nikon model E400 epifluorescence microscope at 20X magnification, outfitted with a Nikon B-2A longpass filter cube (470EX/515LP, Nikon) and a custom bandpass filter cube (430EX/680EM, Omega Optical) was used. The longpass filter pair provided a brighter overall image than the bandpass filter pair and also fluoresced non-chlorophyll structures (e.g., the copepod exoskeleton fluoresces green with blue excitation). The bandpass filter cube showed fluorescence from only chlorophyll and phaeopigments. Photographs of the copepods were taken at each filter setting using a Canon® Digital model T3i single lens reflex (SLR) camera (ISO 6400, F 1/15) remotely controlled with the Canon Electro-Optical System (EOS) Utility software to produce clear images of the epifluorescence.

Images were processed with Photoshop CS6 (Adobe®). Total gut area was manually digitized using the lasso and measurement tools on the longpass-filtered images. The mean gut area of 12 individuals for each copepod species and stage was used for calculations. The threshold tool was used to select areas of the longpass-filter images containing pigment (gut pigment area) that exceeded baseline fluorescence. Signal intensity was not used in the calculation of the gut pigment index because pigment content and composition differed among phytoplankton taxa. Pixels of the bandpass-filtered images exceeding the threshold value were selected and their area estimated using the measurement tool. The gut-pigment area was then divided by the total gut area yielding a relative GPI for each copepod. The same procedure was used to process images of copepods sampled from the control bottles. Copepods with <5% GPI were assumed not to have fed, as the GPI values in those samples were within the range of indices estimated from the controls.