Fluorescence Analysis of Phytoplankton Pigment, Group, and Physiological Variability

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Fluorescence Analysis of Phytoplankton Pigment,
Group, and Physiological Variability
Alexander Chekalyuk
NASA Goddard Space Flight Center, Wallops Flight
Facility / Hampton University
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Why Fluorescence?

• High sensitivity spectral discrimination
  between CDOM and pigment fluorescence, Raman and
  elastic scattering
• Enhanced selectivity provided by spectral
  fluorescence excitation
• Potential for assessment of algal pigments,
  physiology, and functional groups
• Variety of platforms and sensors ships,
  drifters, buoys, autonomous vehicles, airplanes
  (LIDAR), satellites (FLAPS)
• Works remotely and can be as accurate as HPLC

Spectral discrimination between CDOM and Pigment
Fluorescence, Raman and Elastic Scattering
CDOM fluorescence
Chlorophyll fluorescence
Chlorophyll a fluorescence
Phycoerythrin fluorescence
Water Raman
Laser, 532 nm
Water Raman
Laser, 473 nm
Selective spectral fluorescence excitation to
assess phytoplankton pigment and taxonomic
composition
Fluorescence induction assessment of
phytoplankton physiology
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Advanced Laser Fluorometer (ALF-1, 2005) for
Measurements in Coastal and Estuarine Waters
ALF system, 5 hours of underway flow-through
operations, up to 33 mi/h
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Advanced Laser Fluorometry Spectral
Deconvolution for Accurate Assessment of Water
Constituents
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ALF Assessment of Chl-a, Phycobiliprotein CDOM
Abundance, Algal Physiology Water Turbidity
Delaware Bay
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ALF Assessment of Chl-a, Phycobiliprotein CDOM
Abundance, Algal Physiology Water Turbidity
Laser elastic scattering
CDOM fluorescence
Phycoerythrin fluorescence
Green Laser Excitation
Chlorophyll-a fluorescence
Raman scattering
Blue Laser Excitation
Raman scattering
Allophycocyanin fluorescence
CDOM fluorescence
Chlorophyll-a fluorescence
Pump-During-Probe (PDP) assessment of algal
physiology
MAB
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A,B Spectral deconvolution (SDC) of
laser-stimulated emissionC Chl-a measurements
vs. HPLC analysis D ALF assessments of water
turbidity
Green laser excitation 11 SDC components
CDOM fluorescence
Raman scattering
Blue laser excitation
A
B
Phycocyanin fluorescence
Phycocyanin Group (phycocyanin, allophycocyanin
and phycocyanoerythrin)
Chlorophyll-a fluorescence
C
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ALF Mappingof red tide bloom in the Pacific
coastal zone of San DiegoCollaborative
study with Dr. Mitchell (SIO), June 2005
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ALF Underway Measurements in the Chesapeake
(upper) and Delaware (lower) Bays
ALF flow-through measurements, up to 33
miles/hour
C
New! (March 31, 2006)
DataFlow measurements
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CDOM
Phycocyanin
Chlorophyll-a
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Fv/Fm
Allophycocyanin
1
2
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Advanced Laser Fluorometry Spectral Analysis of
Phytoplankton Functional Groups
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Chesapeake Bay York River, July 2005
Variability in Water Constituents and
Phytoplankton Composition
Dinoflagellates/diatoms dominance
Relative abundance of cyanobacteria vs.
cryptophytes (zeaxanthin/Chl alloxanthin/Chl)
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ALF Spectral Analysis Assessment of
Dinoflagellates and Diatoms in Coastal Communities
Lab measurements, cultures
Field, mixed populations
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ALF Spectral Analysis Assessment of
Cyanobacteria and Cryptophytes in Coastal Algal
Communities
Lab measurements, cultures
Zea/Alloxanthin vs. PE peak
Field measurements
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Advanced Laser Fluorometry Improved Assessments
of Phytoplankton Photophysiology
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PP LIDAR Airborne Assessment of Phytoplankton
Photo-Physiology
probe beam
150 m
pump laser
pump beam
probe laser
telescope
spectrograph
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PP LIDAR 2D Mapping of Physiology and Pigments
Delaware Bay, March 23, 2002 Duration 3 hours
SeaWiFS Chl-a, March 23, 2002
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Combining Spectral and Variable Fluorescence
Measurements Improved Assessments of
Phytoplankton Photophysiology
ALF, Delaware Bay, March 2006
Chlorophyll-a fluorescence, 680 nm
Phycocyanin fluorescence, 642 nm
Variable fluorescence needs to be corrected for
non-Chl pedestal that may be significant
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Assessments of Chl-a and Phytoplankton
Photophysiology Does the Light History (NPQ)
Really Matter?

• Airborne LIDAR in situ measurements
• Left 2-fold daytime drop in Chl-a
  fluorescence yield
• Right 10-fold daytime decline in variable
  fluorescence, (Fm-Fo)/Fm
• Presumed mechanisms
• Instant photoregulation by ambient light
  (photochemical quenching)
• Long-term (1 h recovery) photoinhibition
  (light history)

Night
Day
Chl-a fluorescence, 1 min vs. 2-3 h dark
adaptation
Fv/Fm, 1 min vs. 2-3 h dark adaptation
Field ALF flow-through dark-chamber (no ambient
light) daytime measurements Horizontal quick
(1 min) dark adaptation Vertical long (gt1
hour) dark adaptation Conclusion no long-term
photoinhibition of photosynthesis in the upper
water layer exposed to intense (noon) ambient
light (no light history?) Note
Contradicts FRRF field/lab data Consequences
Accurate flow-through fluorescence assessments of
Chl-a concentration and phytoplankton
photophysiology regardless ambient light
Shipboard (horizontal) vs. laboratory (vertical)
flow-through ALF measurements of Chl-a
fluorescence (left) and variable fluorescence
(right) both slopes 1 ! No NPQ?
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Coming Soon New Instruments and Platforms
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New Instruments-AOL4 Green and
Wavelength-Tunable Excitation (OPO)
Hyperspectral Signal Detection (ICCD spectrometer)
telescope
generator
AOL YAG laser, 100 mJ
GN
GPS
OPO 1 mJ, 400-670 nm
PC
PC
computer
beam expander
OS2

Gater
SP
SP
PMT
ICCD
Hyper-spectral detector, 400800 nm
water surface
150 m
spectrograph
OPO beam
AOL YAG laser beam
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New Platforms Unmanned Airborne Vehicle (UAV)
Compact hyperspectral radiometer Aerosonde
UAV for low-cost meso- and synoptic-scale Ocean
color measurements (GSFC/WFF)

• Test Flight
• July 2006

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Feasibility of Space-borne Measurements
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Can We Do That From Space? (PhyLM vs. FLAPS)

• New Technology Needs
• Powerful lasers
• Large-aperture telescope
• Hyperspectral sensor

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17 Fluorescence Lidar Active-Passive Suite
(FLAPS)
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Can we do it with natural fluorescence?
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Avenue to Explore Regional Assessments of Algal
Physiology Productivity from Natural
Fluorescence
View from Moon 361979 km above 2714'S 8232'W

• Measurements of natural fluorescence vs. PAR
  under low-light regime may provide regional
  mapping of phytoplankton physiology
• A platform Geostationary satellite?

FP
CFE
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A Question Why Do We Not Measure Natural
Fluorescence of Phycobiliprotein Pigments?
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Phycocyanin fluorescence, 640-650 nm
Phycoerythrin fluorescence, 575-590 nm
?
Chlorophyll-a fluorescence, 5 mg/l
Phycocyanin fluorescence, 1 mg/l
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Summary Fluorescence Analysis of Phytoplankton
Pigment, Group and Physiological Variability

• Conclusion
• The key solutions for fluorescence analysis in
  optically complex coastal and estuarine waters
  are
• Broadband hyperspectral measurements of water
  emission stimulated with several laser excitation
  wavelengths
• Spectral deconvolution of the emission patterns
  to retrieve the constituent bands
• Combining spectral and variable fluorescence
  measurements
• This provides potential for
• o      Improved assessments of Chl-a
  concentration and phytoplankton photophysiology
• o      Detection, characterization and
  quantitative assessments of phycobiliprotein
  pigments
• o      Detection, discrimination and quantitative
  assessments of phytoplankton functional groups
• (e.g., dinoflagellates vs. diatoms cyanobacteria
  vs. cryptophytes)
• Future directions
• Methods (i) Fluorescence assessment of
  non-fluorescence accessory pigments, and (ii)
  pigment, group and physiological assessments from
  natural fluorescence
• Instruments (I) new generation NASA Airborne
  Oceanographic LIDAR (AOL-4), (ii) Ocean color
  from UAV platforms and (iii) feasibility of
  space-borne Fluorescence LIDAR Active-Passive
  Suite (FLAPS)
• Further evaluation of the new methods and
  instruments in a range of environments, including
  coastal, estuarine and blue oceanic waters, and
  spatial/temporal scales