Slides were imaged using an Olympus Microscope DP72 Camera on an Olympus BX51 fluorescence microscope. Slides were calibrated from pixels to microns for both 0.8 µm and 8 µm to determine what length of pixels equates to microns. Our data includes conversion ratios (0.0012 for 0.8 µm and 0.0072 for 8 µm), which should be applied when determining biomass or abundance, along with the volume filtered. These ratios account for the area of the images taken and represent the proportion of cells actually counted for the 0.8 µm and 8 µm samples, and they have already been incorporated into the dataset presented.

Twenty 8-µm images (20x objective lens) and thirty 0.8-µm images (60x objective lens) were captured using filter sets that were suited for proflavine (green fluorescence with 482 nm excitation, 536 nm emission filters), auto-chlorophyll fluorescence (red fluorescence with 450-490 nm excitation, 660-680 nm emission) and DAPI (blue fluorescence with 350 nm excitation, 465 nm emission) to capture the fluorescence of cell proteins, chlorophyll and DNA. The images were subsequently analyzed to identify phototrophs and heterotrophs, with cells marked based on the presence of both fluorescence signals (indicating phototrophs) or only proflavine fluorescence (indicating heterotrophs).ImageJ image analysis software (v 1.52a or 1.53c) was utilized for processing images, involving manually outlining cells in order to calculate feret length, area and width. Cells that were >50% out of frame or broken were not included in analyses. Images were calibrated to microns and a conversion factor was applied to include the number of images, area of the slide and area imaged with the specific camera/lens combination. 

To determine the true filtration diameter, a light microscope was used to examine a 25 mm glass fiber filter (GF/F) filter that had a small amount of dyed water filtered through. It was discovered that the filter funnel blocks roughly 12% of the 25-mm filter and the filtered region had a diameter of 22 mm. Equations 1-5 show the equations used to calculate cell width, biovolume, ESD (equivalent spherical diameter) and biomass, where * implies we assumed that cell height = cell width/2. ESD was used as a consistent measure of mean cell size since many plankton have an irregular shape. The height of a cell was assumed to be roughly equivalent to half of the cell width since cells are often flattened during filtration (Taylor et al., 2011) with the exception of diatoms. The biomass of diatoms (which were the only taxon we could conclusively identify) was estimated allometrically using equation 5 while all other cell biomass (non-diatoms) was estimated allometrically using equation 4. (Menden-Deuer and Lessard, 2000). 

Equation 1: Cell width = (4/π) x (Area of the cell/Feret length of the cell)

Equation 2: Biovolume = (4/3)(π) x (Feret Length/2) x (Cell Width/2) x (Cell Height*/2)

Equation 3: ESD = 2 x (3 x Biovolume/4π)^(1/3)

Equation 4: Biomass (non-diatoms) = 0.216 x Biovolume^0.939

Equation 5: Biomass (diatoms) = 0.288 x Biovolume^0.811