Sms 598 application of remote and in situ ocean optical measurements to ocean biogeochemistry
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SMS 598: Application of Remote and In-situ Ocean Optical Measurements to Ocean Biogeochemistry. Fluorescence. Mary Jane Perry 6 July 2007.

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SMS 598: Application of Remote and In-situ Ocean Optical Measurements to Ocean Biogeochemistry

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Sms 598 application of remote and in situ ocean optical measurements to ocean biogeochemistry

SMS 598: Application of Remote and In-situ Ocean Optical Measurements to OceanBiogeochemistry

Fluorescence

Mary Jane Perry6 July 2007


Sms 598 application of remote and in situ ocean optical measurements to ocean biogeochemistry

1. What is fluorescence?2. What fluoresces in the ocean?3. Fluorescence as a proxy4. Types of fluorescence5. Instrumentation issues6. Examples7. Today’s labs


Sms 598 application of remote and in situ ocean optical measurements to ocean biogeochemistry

Fluorescence: Re-emission of energy as a photon as an electron relaxes from a electronic excited stateFraction of energy absorbed atshorter wavelengths (higher frequency, higher energy) is re-emitted as a photon at longer wavelengths (lower frequency, lower energy).E = h = hc/Property of some molecules (not all)


Sms 598 application of remote and in situ ocean optical measurements to ocean biogeochemistry

Collin’s lecture Tuesday:Absorption of a photon occurs if AND ONLY IFthe energy of the photon (E = h = hc/) is equal to the energy difference of an electron in the ground state (S0) and higher electronic states (Sn).Absorption is an “electronic transition”; (O(10-15 s))


Sms 598 application of remote and in situ ocean optical measurements to ocean biogeochemistry

Collin’s lecture Tuesday:Absorption of a photon occurs if AND ONLY IFthe energy of the photon (E = h = hc/) is equal to the energy difference of an electron in the ground state (S0) and higher electronic states (Sn).Absorption is an “electronic transition”; (O(10-15 s))

Vibrational states w/in electronic states

Primary mechanism of energy loss to permit an electron to relax or return to S0 is by loss of heat (IR radiation); so-called radiationless decay; (O(10-12 s)).


Sms 598 application of remote and in situ ocean optical measurements to ocean biogeochemistry

http://www.micro.magnet.fsu.edu/primer/java/fluorescence/exciteemit/index.html

http://micro.magnet.fsu.edu/primer/java/jablonski/jabintro/index.html


Sms 598 application of remote and in situ ocean optical measurements to ocean biogeochemistry

Chlorophyll absorption (direct or via accessory pigs)

Chlorophyll excited electron:

Photochemistry (charge separation)

Heat (many pathways)

Fluorescence at 686 nm (O(10-9 s))


Sms 598 application of remote and in situ ocean optical measurements to ocean biogeochemistry

Fluorescence emission1. always from lowest vibrational state of Sn2. red shifted – Stokes shift (higher , lower E)3. mirror image of absorption


Sms 598 application of remote and in situ ocean optical measurements to ocean biogeochemistry

F = E() . conc .fF = E() . a() .fwhereE() is excitation lamp energyconc is concentration a is () is absorption fis quantum yield of fluorescence = moles photons fluoresced moles photons absorbedif fwere constant, F ~ conc or a


Sms 598 application of remote and in situ ocean optical measurements to ocean biogeochemistry

Collin, Station 2 of today’s labfluorescence excitation/emission* match of wavelengths E() and a() * energy transfer of chlorophyll ain vivo (living cell) vs. in vitro (out of cell, solvent)


Sms 598 application of remote and in situ ocean optical measurements to ocean biogeochemistry

What fluoresces in the ocean?Chlorophyll a– red(note, chlorophyll b only fluoresces in solvent –in vitro– so tightly coupled to chlorophyll a in membrane)PE– phycoerythrin (orange)CDOM – broad excitation, with some peaksGreen fluorescent protein (used in molecular staining) –from coral, jellyfish and some protozoans


Sms 598 application of remote and in situ ocean optical measurements to ocean biogeochemistry

CDOM F – proxy for aCDOMfor radiative transfer

aCDOM– proxy for DOM for carbon cycling

PE – specific taxa

Chl F – proxy for Chl – proxy for phytoplankton

and input to productivity and carbon models


Sms 598 application of remote and in situ ocean optical measurements to ocean biogeochemistry

Our ability to use proxies in any quantitative sense depends on this relationship:F = E() . conc .ff depends on temperature and environment(pH, ionic strength, interaction with other molecules for dissipation of energy, etc.)

chlorophyll a fluorescence in vitro (solvent, acetone)

f ~ 0.33

chlorophyll a fluorescence in vivo (living cell)

f ~ <0.05 – 0.03


Sms 598 application of remote and in situ ocean optical measurements to ocean biogeochemistry

Three types of fluorescence:1) active – artificial light source for E()– static: use for profiles of chl fluorescence; moorings; mobile platforms– time resolved (true tr is ~ femo/pico s for chemistry, like whole burning in CDOM; could consider pump & probe, variable F) 2) passive – sun is light source for E()


Sms 598 application of remote and in situ ocean optical measurements to ocean biogeochemistry

Instrumentation issues (a few):Sensors – trend toward smaller, lighter, low power, robust, more sensitive, smaller sensing volume; biofouling issuesE() varies among instrumentsaps() will vary among cells, based on accessory pigments; does E() match aps()? Manufacturer change in LEDs to 470 nm. Different (), different accessory pigmentsCalibrationsensor side: dark reading, temperature response of electronics and optics, stability and driftfluorophore side: (CDOM, Chl): temperature response (-1–2%/ºC), behavior of fAttenuation of signal – turbidity (nonlinear response)


Sms 598 application of remote and in situ ocean optical measurements to ocean biogeochemistry

Example of passive or solar-stimulated fluorescence from Babin and Huot (recall Curt’s lecture, Hydrolight output)


Sms 598 application of remote and in situ ocean optical measurements to ocean biogeochemistry

Other issues:1) satellite images only available on clear days; bias of high light/quenching; what is f? 2) how to interpret, E(), a (), depth resolution

from Babin and Huot;they caution its use in

turbid waters (not F)


Sms 598 application of remote and in situ ocean optical measurements to ocean biogeochemistry

F = E() . conc .f

Note: important temperature effect on f

(watch out if room temp changes)

~ - 1–2% F / ºC

F

Not just chl a, also degradation

pigments (pheophytin a).

Fo reading = chl a + pheo; add H+;

Fa reading = new pheo + old pheo

(2 readings, 2 equations, 2 unknowns)

BUT: also chlorophyll b and its

degradation products. Filter set.

concentration

Example of active /static benchtop application (Ststion 1):fluorescence of solvent-extracted chlorophyll a


Sms 598 application of remote and in situ ocean optical measurements to ocean biogeochemistry

Chlorophyll mg/m3

PAR

GoMOOS Buoy E (Roesler)

From Falkowski and Raven 1997

Chlorophyll fluorescence and extracted concentration of chlorophyll early AM vs. noon.

Example of active /static in situ application for living cells (Station 3) two types: flush-face and flow-through.Used on ship-based profiling systems, moorings, floats and gliders.

Glider fluorescence, Wash. Coast


Example of mid day fluorescence quenching

Mid-day fluorescence quenching

Example of mid-day fluorescence quenching

0

  • Quenching observed to 11m

  • Fluoresence quenched up to 80% at surface

Depth (m)

40

112.48

Year Day

118.2

-- Mixed Layer Depth (MLD)

So maybe for biomass, should we concentrate on night-time measurements in vivo fluorescence measurements?

Sackmann et al, MS.


Mid day fluorescence quenching

Mid-day fluorescence quenching

  • MORNING

  • MID-DAY

  • AFTERNOON

Sackmann et al., unpub.


Mid day fluorescence quenching1

Mid-day fluorescence quenching

  • MORNING

  • MID-DAY

  • AFTERNOON

Sackmann et al., unpub.


Mid day fluorescence quenching2

Mid-day fluorescence quenching

  • MORNING

  • MID-DAY

  • AFTERNOON

Sackmann et al., unpub.


Sms 598 application of remote and in situ ocean optical measurements to ocean biogeochemistry

PAR

Fluorescence

Time

Figure 2: Damariscotta River in situ chlorophyll a fluorescence and PAR (μmol photons/s/m2)vs. time.


Sms 598 application of remote and in situ ocean optical measurements to ocean biogeochemistry

PAR

Fv/Fm

Time

Figure 4:Normalized variable fluorescence (Fv/Fm) and PAR (μmol photons/s/m2) vs. time.


Sms 598 application of remote and in situ ocean optical measurements to ocean biogeochemistry

Fluorescence induction curve (issues of timing and of initial state)

Fv = Fm - Fo

Fluorescence induction curve: rapid rise and slow decline


Fluorescence induction curves for dark adapted cells

Fluorescence induction curves, for dark-adapted cells

Fast rise (< second); #1 – low light;

#2 – high light adapted; #3 DCMU

Slow rise (< minute)

photoreduction of QA to QA- and

connectivity among Reaction Centers

photochemical, thermal

and other quenching


Sms 598 application of remote and in situ ocean optical measurements to ocean biogeochemistry

Saturday experiment* automoatic: continuous measurement of PAR, F, and bb* student teams; hourly sampling of chlorophyll a (extract) and Fv/Fm with FIRe (Station 4)Questions:How does incident light affect in situ F of phytoplankton in the DRE (tides, mixing, variable PAR)?What is the relationship between quenching or photoinhibition of in situ F and Fv/Fm? And could Fv/Fm help to interpret F?Lightening ––––– don’t sample!


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