alfa instrument introduction-and-applications
DESCRIPTION
Introduction to a novel tool to address the fluorescence complexity of natural waters.TRANSCRIPT
Introduction to a novel tool to address the fluorescence complexity of natural waters
1
What is ALFA? CDOM, Chlorophyll, PBPs, Fv/Fm
Community structure, algal health Provides unprecedented accuracy and versatility
CDOM corrected fluorescence and non‐photochemical quenching corrected CHL
Shipboard flow loop or discrete bottle sampling mode (e.g., rosette casts) Working towards in‐situ version
2
B
0 155 310 465 620Sample Number
0.0
0.1
0.2
0.3
0.4
Fv /
Fm
Fv / FmCDOMChl-a
0
1
2
3
4
5
6
7
8
A.U
.
170 m
How does it work?
3
Sample515 nm Laser 405 nm Laser
PMT
Spectrometer Spectral info
Time response of chlorophyll
Biological Significance
CDOM measurement and correction: Related to community dynamics Overlaps with pigment! Must be corrected to get accurate pigment
and variable fluorescence Raman normalization:
Provides means to compare data to other, benchtop, systems and provides inherent correction for instrument specific artifacts
0 1000 2000 3000 4000 5000 6000 7000 8000 9000 10000 11000
0
20
40
60
80
100
120
140
160
180
200
220
PB0
9_P
DP
\1
Bay_mouth_Pb5MAB_offshoreRiver_St18
0
100
200
300
400
500
600
700
0
100
200
300
400
500
1 2 3 4 50.0
0.1
0.2
0.3
0.4
0.5AU
Selective spectral fluorescence to assess
phytoplankton pigment and taxonomic composition
Fluorescence induction assessment of
phytoplankton physiology
Laser stimulation
Provides enhanced sensitivity over traditional light sources Biological/environmental significance of different lasers CDOM correction (blue laser): 405 nm CHL targeted (green laser): 515 nm
Fast laser pulses permit determination of Fv/Fm
5
Water Raman (R)
PE2
PE3
PE1
Chl-a
6
Spectral Deconvolution: extracting a wealth of information
Collected spectrum (blue) is Raman normalized and component spectra are fitted to derive individual values and correct for CDOM
Simplified spectra: phycoerythrin and chorophyll components
Importance of corrections
7
Chl‐a fluorescence, 1 min vs. 2‐3 h dark adaptation:
Fv/Fm, 1 min vs. 2‐3 h dark adaptation:
Non-photochemical inhibition correction (Fv/Fm) can
provide chlorophyll values equivalent to extraction
and HPLC!
Fv/Fm must be corrected for background
fluorescence!
ALFA provides dark adapted values, regardless of daytime or ambient light!
Operational Modes Bottle vs. sample mode
Designed to plumb directly into shipboard flow loop Simple plumbing Features to integrate
shipboard data (e.g., CTD, GPS)
Special sensor interface to integrate PAR data
Modular pump and click‐connect sampling bottles
8
Bottle Setup
9
Flow Setup
10
Graphical User Interface
11
GUI Breakdown
12
Instrument control Spectra and deconvolution visualization
Relative component
levels
Observation of variable
fluorescence (Fv/Fm)
Transect Mode(Shipboard flow loop)
Example data: Depth profile from bottle samples
13
Example data: Shipboard flow loop transects and satellite data comparison
14
Chlorophyll‐a (Chl‐a) determination comparison
ALF in green, high resolution transect, good comparison to grab samples
Bottle samples red dots, extraction and HPLC
In Dark red MODIS Satellite Chl‐estimates
In pink is ALF FV/FM , Variable Fluorescence: measure of photosystem health (CDOM corrected)
MODIS Satellite Sea Surface Temperature in blue
Orange line is ALF phycoerythrin (PE) a phycobiliprotien, often indicative of cryptophytes
Dark blue is ALF CDOM
Light blue dots are the PE to Chl‐a ratio which give a measure of pigment composition, allowing spatial shifts in community structure to be observed
Gulf of Mexico assemblages
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PE1/Total PE Log Chlorophyll concentration
Normalized PE1 shows change in phycoerythrinpigment composition likely indicative of a change in community structure
Chlorophyll maximums in different areas than phycobiliprotiens showing differences in community pigment structure
Example data: Assessment of Phycobiliprotein‐Containing Photosynthesizing Organisms in Mixed Populations
C
y = 0.41x + 0.001R2 = 0.78
0.000
0.005
0.010
0.015
0.020
0.025
0.00 0.01 0.02 0.03 0.04 0.05
IPE1/Rg
Zea
xant
hin
(mg
m-3
)
A
y = 1.17x + 0.01R2 = 0.77
0.00
0.05
0.10
0.15
0.20
0.00 0.05 0.10 0.15
IPE3/Rg
Allo
xant
hin
(mg
m-3
)
D
y = 0.47x + 0.001R2 = 0.63
0.000
0.005
0.010
0.015
0.020
0.00 0.01 0.02 0.03 0.04
IPE1/Chlag
Zea
xant
hin
Chl
-a -1
B
y = 1.12x + 0.013R2 = 0.63
0.00
0.02
0.04
0.06
0.08
0.10
0.00 0.02 0.04 0.06 0.08
IPE3/Chlag
Allo
xant
hin
Chl
-a -1
Spectral deconvolution of laser‐stimulated emission yields the group‐specific phycoerythrin (PE) spectral indexes:
IPE1/R, IPE2/R, IPE3/R IPE1/Chla, IPE2/Chla, IPE3/Chla
Correlations with HPLC‐retrieved group‐specific pigment biomarkers demonstrate ALF potential for discrimination and quantitative assessment of cryptophytes and cyanobacteria (field data)
Water Raman (R)
PE2
PE3
PE1
Chl-a
ALFA provides vast information on biogeochemical fluorescence properties
ALFA provides highly accurate values for chlorophyll and Fv/Fm
Numerous application spaces: community composition, community health, biological response to
physical forcing, comparison to satellite data, studying pigment degredation, DOM studies, etc.
Future work: In‐situ, potential for advanced lasers (i.e., the science vs. monitoring)
References Chekalyuk, A. and M. Hafez. Advanced laser fluorometry of natural aquatic environments.
2008. Limnol. Oceanogr.: Methods 6, 591‐609. Chekalyuk, A. and M. Hafez. Photo‐physiological variability in phytoplankton chlorophyll
fluorescence and assessment of chlorophyll concentration. 2011. Optics Express 19 (23), 22,643‐22,658.
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ContactsAndrew Barnard Casey MooreVice President, Research & Development [email protected] [email protected]
Cris Orrico Michael TwardowskiSenior Research Associate V.P., Director of [email protected] [email protected]
Corey Koch Jim SullivanRes. Sci./Chemist Res. [email protected] [email protected]
WET Labs, IncPO Box 518
Philomath, OR 97370(541) 929‐5650