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

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

2008 NOPP-funded project“Sensors for Measurement of Biological, Bio-Optical or Chemical Properties of the Ocean” Topic

Three-way partnershipWoods Hole Oceanographic InstitutionUniversity of WashingtonCytopeia, 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

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

Automated features for extended deployment

• Standard analysis

• Biofouling control

• Realtime humidity sensing & intake valve control

Imaging FlowCytobotDerived from FlowCytobot designOptimized for large cells (~10-300 m)

Olson and Sosik 2007

FlowCytobotPrincipals from conventional flow cytometry

(but automated and submersible)Optimized for “small” cells (~1-15 m)

Olson et al. 2003

6-month deployments routine

Imaging FlowCytobotData example

Nano/microplankton

-Associated images (all same scale)

Chl

flu

ores

ence

Light scattering

Individual particle measurements

Air-side observationsSeaPRISM

shortwave radiation, winds, etc.

In water observationsT,S, currents, fluorescence,

backscattering, oxygen,

flow cytometry and cell imaging

Martha’s VineyardCoastal

Observatory

Bottle sampleschlorophyll, absorption, etc.

Remote sensing

• Operational since 2001

• 24/7 power and data

• Open to new users

• Realtime public data access

The Phytoplankton Community at MVCO

FlowCytobot Picoplankton

ImagingFlowCytobot

Microplankton

Picoplankton to Microplankton event to seasonal to interannual

scales

> 130 million images to date

Which ones are diatoms?

Jan Apr Jul Oct Jan0

2

4

6

8x 10

6B

iovo

lum

e ( m

3 m

l-1)

2006200720082009

Diatoms

Jan Apr Jul Oct Jan

102

104

106

Synechococcus

Cel

l con

cent

ratio

n (m

l -1 )

2006200720082009

50 m

Automated image analysis and classification

22 categories (16 phytoplankton genera)

88% overall accuracy

Image processing

Supervised machine

learningalgorithm

Statisticalerrorcorrection

Sosik and Olson 2007

Taxonomic resolution winter / spring 2007

Total for all images

January – July

Major contributors: 6 Diatom taxa

2007

Jan Feb Mar Apr May Jun Jul0

2

4

6x 10

6

Bio

volu

me

( m

3 ml-1

)

Jan Feb Mar Apr May Jun Jul0

2

4

6x 10

6

Bio

volu

me

( m

3 ml-1

)

Jan Feb Mar Apr May Jun Jul0

5

10

15x 10

5

Bio

volu

me

( m

3 ml-1

)

Chaetoceros spp.

Jan Feb Mar Apr May Jun Jul0

5

10

15x 10

5

Bio

volu

me

( m

3 ml-1

)

Guinardia spp.

Jan Feb Mar Apr May Jun Jul0

2

4

6

8x 10

5

Bio

volu

me

( m

3 ml-1

)

Thalassiosira spp.

Jan Feb Mar Apr May Jun Jul0

5

10x 10

5

Bio

volu

me

( m

3 ml-1

)

Leptocylindrus spp.

Jan Feb Mar Apr May Jun Jul0

0.5

1

1.5

2x 10

6

Bio

volu

me

( m

3 ml-1

)

Thalassionema spp.

Jan Feb Mar Apr May Jun Jul0

2

4

6x 10

5

Bio

volu

me

( m

3 ml-1

)

Guinardia flaccida

Jan Feb Mar Apr May Jun Jul0

2

4

6x 10

6

Bio

volu

me

( m

3 ml-1

)

Seasonality in phytoplankton biomass – Two views

J F M A M J J A S O N D J0

50

100

150

Phy

topl

ankt

on C

arbo

n (

g L -

1)

2003 2004 2005 2006 2007 2008 2009 20100

2

4

6

8

10

12C

hlor

ophy

ll (

g

L-1)

Carbon budgetcell image / scattering

↓cell volume

↓

cell carbon

∑(C cell-1)

Chl

fall / winter peaks

diatom blooms…

Extracted pigment analysis

Flow cytometry

Biomass and community structure

J F M A M J J A S O N D J0

50

100

150

Phy

topl

ankt

on C

arbo

n (

g L -

1)

J F M A M J J A S O N D J0

0.2

0.4

0.6

0.8

1

Phy

topl

ankt

on c

arbo

n fr

actio

n

picoplankton (< 2 m)

nanoplankton (2-20 m)microplankton (20-200 m)

How is this C distributed

across size classes?

Microplanktonfall / winter

Picoplanktonsummer

Nanoplanktonall year

J F M A M J J A S O N D J0

50

100

150

Phy

topl

ankt

on C

arbo

n (

g L -

1)

J F M A M J J A S O N D J0

0.2

0.4

0.6

0.8

1

Pro

port

ion

of p

hyto

plan

kton

FCM pico CFCM nano+micro CHPLC picoHPLC nano+micro

Biomass and community structure

J F M A M J J A S O N D J0

0.2

0.4

0.6

0.8

1

Phy

topl

ankt

on c

arbo

n fr

actio

n

picoplankton (< 2 m)

nanoplankton (2-20 m)microplankton (20-200 m)

HPLC-based(Vidussi et al. approach)

Proportion

pico v.

micro + nano

How does this result compare to other methods?

J F M A M J J A S O N D J0

0.2

0.4

0.6

0.8

1

Pro

port

ion

of p

hyto

plan

kton

FCM nano CHPLC nano

Biomass and community structure

J F M A M J J A S O N D J0

0.2

0.4

0.6

0.8

1

Phy

topl

ankt

on c

arbo

n fr

actio

n

picoplankton (< 2 m)

nanoplankton (2-20 m)microplankton (20-200 m)

HPLC-based(Vidussi et al. approach)

Proportion

nano…

How does this result compare to other methods?

Jan Feb Mar Apr0

50

100

150

Din

ophy

sis

(cel

ls m

l-1)

Texas CoastWinter 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

PartnershipsCytopeia, 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)

Design OptimizationWHOI / 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

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

Imaging FlowCytobot fluidics and optics

Cytopeia’s rigid fixed assemblydetector module

Opto-mechanicalcomponents

Existing collection of off-the-shelf

components

to be replaced by

Cytopeia’s custom rigid assembly

Model 1Design completeFabrication complete

Under evaluation at WHOI

Current Status

Current Status

Fluidics system

Existing off-the-shelf HPLC syringe

pump

to be replaced by

Custom unit, modified from MBARI design

Model 1Design acquiredFabrication completeUnder evaluation at

WHOI

20% reduction in overall power consumption

custom syringe pump

Imaging FlowCytobot – commercial transition

ApplicationsEcological researchHAB warningPFT algorithms / validationCell size class algorithms / validationCarbon budgets

Design goals

Increase ease of manufacture & use Reduce size, power consumption

Expand dynamic range

Adapt analysis methods

THANK YOU!