Plankton Analysis by Automated Submersible ImaginginFlow Cytometry Transforming

Plankton Analysis by Automated Submersible Imaging-in-Flow Cytometry: Transforming a Specialized Research Instrument into a Broadly Accessible Tool Heidi M. Sosik Robert J. Olson

Long-term goal and strategy • Understand regulation of seasonal to interannual plankton dynamics • Time series observations are key • New sampling and analysis systems must be developed Flow Cytometry in the Lab Flow Cytometry in situ Picophytoplankton Microphytoplankton

2008 NOPP-funded project “Sensors for Measurement of Biological, Bio-Optical or Chemical Properties of the Ocean” Topic Three-way partnership Woods Hole Oceanographic Institution University of Washington Cytopeia, Inc. Two main objectives Ø Transition of Imaging Flow. Cytobot to commercially viable status Promote access for the broader oceanographic and environmental monitoring communities User-tested pre-commercial units Ø Extending Imaging Flow. Cytobot’s target size range Enhance the technology for a next generation instrument Research prototype

Overview • Imaging Flow. Cytobot Ø Description, readiness, motivation for commercialization Ø Why should this community be interested? • Partnerships Ø Cytopeia Ø University of Washington • Proposed work Ø Iterative design optimization strategy Ø Target subsystems • Current Status

Flow. Cytobot Principals from conventional flow cytometry (but automated and submersible) Optimized for “small” cells (~1 -15 mm) Olson et al. 2003 Imaging Flow. Cytobot Derived from Flow. Cytobot design Optimized for large cells (~10 -300 mm) Olson and Sosik 2007 Automated features for extended deployment • Standard analysis • Biofouling control • Realtime humidity sensing & intake valve control 6 -month deployments routine

Imaging Flow. Cytobot Data example Nano/microplankton -Associated images Chl fluoresence (all same scale) Light scattering Individual particle measurements

Remote sensing Martha’s Vineyard Coastal Observatory Air-side observations Sea. PRISM shortwave radiation, winds, etc. In water observations T, S, currents, fluorescence, backscattering, oxygen, flow cytometry and cell imaging Bottle samples chlorophyll, absorption, etc. • • Operational since 2001 24/7 power and data Open to new users Realtime public data access

The Phytoplankton Community at MVCO Flow. Cytobot Picoplankton Imaging Flow. Cytobot Microplankton

Picoplankton to Microplankton event to seasonal to interannual scales Which ones are diatoms? > 130 million images to date Diatoms

Automated image analysis and classification 22 categories (16 phytoplankton genera) 88% overall accuracy Image processing Supervised machine learning algorithm Statistical error correction Sosik and Olson 2007 50 mm

Taxonomic resolution winter / spring 2007 Total for all images January – July Major contributors: 6 Diatom taxa 2007

Seasonality in phytoplankton biomass – Two views Extracted pigment analysis Chl fall / winter peaks diatom blooms … Carbon budget cell image / scattering ↓ cell volume ↓ cell carbon ∑(C cell-1) Flow cytometry

Biomass and community structure How is this C distributed across size classes? Microplankton fall / winter Picoplankton summer Nanoplankton all year

Biomass and community structure HPLC-based (Vidussi et al. approach) Proportion pico v. micro + nano How does this result compare to other methods?

Biomass and community structure HPLC-based (Vidussi et al. approach) Proportion nano … How does this result compare to other methods?

Texas Coast Winter 2008 - First ever DSP event Auto * Manual Port Aransas, TX Imaging Flow. Cytobot 3 – The early warning! Olson, Sosik & Campbell Shellfish recalled & harvest closed within days

Partnerships Cytopeia, Inc. • Influx – high speed cell sorter, open architecture Ø Large bio-medical market ($9 M in 2007 sales) Ø Specialized Influx Mariner for oceanographic users • Contributing engineering and fabrication at no cost Founder Ger van den Engh Experienced R&D team University of Washington • Development of position sensitive detector (PSD)

Commercial Transition Iterative process Build on strengths at WHOI & Cytopeia Leverage MVCO access and existing research prototypes Expand to select outside users Design Optimization WHOI / Cytopeia Evaluation at WHOI / MVCO Evaluation by outside users ~ Commercial units

Design optimization targets • Opto-mechanical system Ø Fixed modules for stability • Fluidics system Ø Custom syringe pump to reduce size and power • Illumination for imaging Ø LEDs to replace Xenon strobe • Signal detection Ø Improved electronics, digital signal processing • Control system Ø Integration • Control software Ø Integration and user-friendliness

Current Status Opto-mechanical components Existing collection of off-the-shelf components to be replaced by Cytopeia’s custom rigid assembly Model 1 Design complete Fabrication complete Under evaluation at WHOI Cytopeia’s rigid fixed assembly detector module Imaging Flow. Cytobot fluidics and optics

Current Status Fluidics system Existing off-the-shelf HPLC syringe pump to be replaced by Custom unit, modified from MBARI design 20% reduction in overall power consumption Model 1 Design acquired Fabrication complete Under evaluation at WHOI custom syringe pump

Imaging Flow. Cytobot – commercial transition Applications Ecological research HAB warning PFT algorithms / validation Cell size class algorithms / validation Carbon budgets Design goals Increase of manufacture & use Reduce size, power consumption Expand dynamic range Adapt analysis methods THANK YOU!