Using the Attune Acoustic Focusing Cytometer to Detect Marine Photosynthetic PicoplanktonFlow cytometry is a powerful tool for studying the biology, ecology, and biogeochemistry of marine photosynthetic picoplankton. These organisms are intrinsically fluorescent due to their photopigment content, and differences in photopigment composition can be used to distinguish the various groups. Prochlorococcus and Synechococcus are the two major groups of microbes that comprise photosynthetic picoplankton; they have been extensively studied for their principal role in primary production. Prochlorococcus are the smallest and most abundant photosynthetic organisms known and, along with Synechococcus, have a large impact on the global carbon cycle. Prochlorococcus are approximately 0.6 μm in size and contain the red-fluorescent divinyl chlorophylls a and b. At 1 μm, cells of Synechococcus are larger and contain orange-fluorescent phycoerythrin in addition to red-fluorescent chlorophyll. These differences allow for the discrimination of natural populations of Prochlorococcus and Synechococcus in environmental samples [1–5].
Typical Photosynthetic Picoplankton Population Analysis
Analysis of marine photosynthetic picoplankton is routinely performed using flow cytometry, although this testing has presented some challenges. The excitation of the intrinsically fluorescent photosynthetic picoplankton has conventionally been performed using a 488 nm laser, a wavelength that is not optimal for the divinyl chlorophyll–containing Prochlorococcus. Flow cytometers that utilize high velocity or high-volume sheath fluid to focus cells (hydrodynamic focusing) for laser interrogation are typically employed for this analysis. These flow cytometers are usually pressure-driven, making direct cell counting of discrete populations require either weighing samples pre- and post-analysis or adding counting beads to samples. In addition, the use of 488 nm–excitable nucleic acid–binding dyes for determining cell counts of the heterotrophic population (Bacteria and Archaea) obscures the intrinsic fluorescence of the picophytoplankton populations. To compensate multiple samples must be analyzed to assess the entire microbial population.
Simplifying Photosynthetic Picoplankton Detection
Conventional cytometers employ large sheath-to-sample flow rates to hydrodynamically focus particles. In contrast, the Attune Acoustic Focusing Cytometer uses ultrasonic waves to focus particles and requires significantly lower sheath fluid flow rates. The Sensitive mode on the Attune cytometer further reduces the instrument sheath flow rate, thereby slowing the particle velocity. By slowing the particle velocity, researchers can increase the laser interrogation and photon collection times for dim, low-background populations (e.g., the inherently dimly fluorescent Prochlorococcus from oligotrophic surface water samples). In addition, the 405 nm laser enables better excitation of divinyl chlorophylls from Prochlorococcus and enhances separation of distinct picophytoplankton populations from background signal. Syringe-driven sample fluidics permit the direct counting of cells in a given population. Combining this with excitation of divinyl chlorophylls using the 405 nm laser helps enable direct enumeration of Prochlorococcus in SYBR Green I–stained samples.
Figure 1 demonstrates the utility of combining a slow particle flow rate, excitation with the 405 nm laser, and analysis of a defined sample volume. Prochlorococcus could be accurately discriminated and directly counted in samples from the very oligotrophic Station ALOHA surface water, where these marine picophytoplankton contain low amounts of divinyl chlorophylls (Figure 2).
Figure 1. Station ALOHA oligotrophic surface water sample analyzed using Sensitive and Standard modes. Sensitive mode (25 μL/min) (left) allows for better separation of the inherently dim red-fluorescent (y-axis) Prochlorococcus (Pro) population from the remaining SYBR Green I–stained cells, as compared to Standard mode (25 μL/min) (right). Slowing the particle flow rate can increase the laser interrogation and photon collection times from dim, low-background populations.
Figure 2. Analysis of aquatic samples using 488 nm and 405 nm excitation. Few commercial cytometers are capable of resolving the Prochlorococcus population from this very oligotrophic part of the ocean, and fewer still will resolve this picophytoplankton population and provide a direct cell count. (Left) Red fluorescence from 488 nm excitation (x-axis) vs. divinyl chlorophyll fluorescence from 405 nm excitation (y-axis), showing separation of populations of picophytoplankton from an unstained Station ALOHA surface water sample. The concentration of Prochlorococcus (Pro) (27,000 events) observed in the Station ALOHA oligotrophic surface water sample was 216,000 cells/mL. The Synechococcus (Syn) cell count from this sample was 2,100 cells/mL. The picoeukaryote (Euk) cell count for this sample was 1,100 cells/mL. FluoSpheres 1.0 μm yellow-green fluorescent microspheres were added as an internal reference. (Right) Red fluorescence from 488 nm excitation (x-axis) vs. divinyl chlorophyll fluorescence from 405 nm excitation (y-axis), showing separation of populations of picophytoplankton from an unstained Station ALOHA Deep Chlorophyll Maximum (DCM) water sample. The direct cell count for Prochlorococcus observed for the Station ALOHA site DCM sample was 113,000 cells/mL. The Synechococcus cell count calculated from the DCM sample was 300 cells/mL. The picoeukaryote (Euk) cell count for this sample was 2,400 cells/mL. FluoSpheres 1.0 μm yellow-green fluorescent microspheres were added as an internal reference.
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