1. 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

2. Flow Cytometry in the Lab Flow Cytometry in situ Picophytoplankton Microphytoplankton 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

3. 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  Transition of Imaging FlowCytobot to commercially viable status Promote access for the broader oceanographic and environmental monitoring communities  User-tested pre-commercial units  Extending Imaging FlowCytobot’s target size range Enhance the technology for a next generation instrument  Research prototype

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

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

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

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

8. The Phytoplankton Community at MVCO FlowCytobot Picoplankton Imaging FlowCytobot Microplankton

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

10. 50  m 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

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

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

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

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

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

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

18. Partnerships Cytopeia, Inc. Influx – high speed cell sorter, open architecture  Large bio-medical market ($9M in 2007 sales)  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)

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

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

21. Imaging FlowCytobot fluidics and optics Cytopeia’s rigid fixed assembly detector module 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 Current Status

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

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