Fluorescent beads

Microbial abundance was monitored over the course of the mesozooplankton incubation experiments. Water from the incubations (1.5 mL) was fixed with glutaraldehyde (0.5% final concentration) at room temperature for 10 min. Subsequently, the samples were frozen in liquid nitrogen for 10 min and stored at −80°C. Prior to flow cytometric analysis, the samples were thawed to room temperature and 0.5 mL subsamples were stained with SYBR Green I (1× final concentration) in the dark for 10 min, and 1 μm fluorescent beads (Molecular Probes, 1 × 10⁵ mL⁻¹) were added to the samples as an internal standard. Prokaryotes were enumerated on an Accuri C6 (Becton Dickinson) based on their signature in a plot of green fluorescence vs. side scatter. No significant increase in microbial abundance over time was observed in the incubation experiments (data not shown).

Bacteria and VLPs were enumerated using an Accuri C6 flow cytometer (Becton Dickinson), which was fitted with a 488 nm blue laser and green (530/30 nm), orange (585/40 nm) and red (670 nm) filters. Prior to analysis, triplicate samples were thawed and diluted to 1:10 with 0.2 μm filtered TE buffer (10mM Tris, 1 mM EDTA, pH 8). Samples were then stained with SYBR Green-I solution (1:20000 dilution; Molecular Probes, Eugene, OR) and incubated in the dark at 80°C for 10 min [26 (link)]. As an internal size and fluorescence standard, 1 μm diameter fluorescent beads (Molecular Probes, Eugene, OR) were added to each samples at a final concentration of approximately 10⁵ beads ml^-1 [29 ]. For each sample, forward scatter, side scatter and green (SYBR Green-I) fluorescence were acquired for two minutes. FlowJo (Treestar, Inc.) software was used to analyse data collected from each sample, where differences in cell side scatter and SYBR Green fluorescence were used to discriminate between VLP and bacterial groups [26 (link),30 (link),31 (link)].

Prokaryotes from the collected groundwater were identified and enumerated by flow cytometry using a FACSCanto II flow cytometer (Becton Dickinson). Triplicate samples were thawed and diluted 1:10 with filter TE buffer (10 mM Tris, 1 mM EDTA), stained with SYBR-I Green solution (Molecular Probes) and incubated in the dark for 10 minutes34 (link)36 (link). Fluorescent beads with a diameter of 1 μm (Molecular Probes) were added to each sample for an internal size and concentration standard37 (link). Data for each sample was collected and analysed using FlowJo software (© Treestar) and differences in cell side scatter and SYBR-I Green fluorescence were used to discriminate prokaryotes (Gasol et al.37 (link)).

We used the imaging beam as a reference and aligned the uncaging beam to it via two alignment irises. For fine adjustment, we imaged 1 μm diameter fluorescent beads (Molecular Probes, Inc.) with the 60× objective using both lasers at 750 nm simultaneously but with a blank hologram displayed on the SLM. This double illumination produces two images of the beads if the uncaging beam is not completely aligned to the imaging beam. Fine adjustments in alignment were made by adjusting the direction of the uncaging beam via relay optics just before PBS2 in Fig 1a).
To calibrate the position of the holographic excitation spots, we used mirror images of a bead resulting from scanning a patterned uncaging beam onto the bead sample (without the imaging beam). For instance, imaging a single fluorescent bead with a two-foci uncaging beam results in two mirror images. We imaged fluorescent beads this way while adjusting the x, y and z scaling and rotation factors of the hologram until the mirror images of the beads on the 2P image were centered on the uncaging cross hairs on the GUI (S1b Fig).
We measured the 2P point spread function (PSF) of our system by imaging 100 nm fluorescent beads (Molecular Probes, Inc.) with the 60× and 20× objective lenses, using both imaging and uncaging lasers separately.