Flow 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. 

Groups of Microbes 
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.

Challenges of 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, cytometers such as the Attune® Acoustic Focusing Cytometer use ultrasonic waves to focus particles and require 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 syringe-driven sample handling with excitation of divinyl-chlorophylls with the 405 nm laser allows for direct enumeration of Prochlorococcus spp. in SYBR® Green I–stained samples.  

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