Facing the future with flow cytometry

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Facing the future with flow cytometry
SCOR 115 Survey Analysis of Plankton

• Peter Burkill
• National Oceanography Centre, Southampton

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What do we want?

• Holy grail optimal plankton sampling system

No one system can sample the plankton Dr Sonia
Batten 19th May
First, we need to define what plankton we want to
sample
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Quantifying the oceans plankton
Size (µm)
102
104
100
10-2
Virioplankton
WP2 CPR nets
Bacterioplankton
Phytoplankton
Protozooplankton
Mesozooplankton
Morphological complexity
Light microscopy
Other approaches
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Other considerations
Conc (no/mL)
1012
Detrital particles
109
106
103
Living particles
100
10-3
10-2
100
102
104
Size (µm)
Problems increase
Problems decrease
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Single cell analysis (AFC) to the rescue
Flow cell
Optical filters
Sample
Laser beam
F4
P4
F3
Output from flow cell is optically filtered (F),
measured (P) and stored in computer
F2
F1
P3
P2
P1
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Protozooplankton State of the art

• Normal microscopic analysis (3 / day)
• AFC SYBR Green DNA dye (30 / h)

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Comparison of phototrophic picoplankton (PP)
counts made using the stained protist protocol
versus unstained picophytoplankton protocol
slope 1.006 ? 0.006, r2 0.98, n 423, P lt
0.0001
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On line analyses with 100m resolution
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If we can do flow AT sea, can we do it IN the
sea?
designed by marine scientists for marine science
in 2002

• Capabilities
• Abundance
• Particle sizing
• Better particle discrimination using pulse
  shapes
• Sampling
• Wider range 1-1000 µm
• Wider detection 1 - 104 per ml
• Larger sample volume 4 ml
• Sampling depth 0 - 200 m
• Sampling frequency every 10 minutes (in
  autonomous mode)
• Sampling resolution with Autosub approx. 500 m
• Data Acquisition modes
• Manual via simple button (lab. use and off a
  wire)
• Interactively via PC and serial link (lab. use
  and off a wire)
• Pre-programmed for Autosub missions and on-line
  analysis
• Long distance via Inmarsat Std C satellite -
  potential

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In situ fine-scale mapping state of the art
enormous practical advantages
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Stepping into the future microflow
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Evolution of AFC
1996 BD FACSort plug n play
2002 Dako MoFlo hi-speed sorter
1986 Coulter EPICS V seatrials
2003 Cytobuoy Cytosub
The future MicroFlow
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Quantifying the oceans plankton
Size (µm)
102
104
100
10-2
Virioplankton
WP2 CPR nets
Bacterioplankton
Phytoplankton
Protozooplankton
Mesozooplankton
Generalised Instrument for Sampling More of the
planktOn (GISMO)
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Summary

• Flow cytometers are hugely powerful generic
  technology
• Traditionally used in lab and onboard ships
• Phytoplankton, bacterioplankton, protozooplankton
  
• Cytobuoy/Cytosub are the first true marine
  cytometers
• The huge breakthrough is Microflow cheap, rugged,
  disposable
• This is the basis for sampling smaller plankton
  in GISMO