SMS 598: Application of Remote and In-situ Ocean Optical Measurements to Ocean Biogeochemistry Mary Jane Perry 6 July 2007

1. What is fluorescence? 2. What fluoresces in the ocean? 3. Fluorescence as a proxy 4. Types of fluorescence 5. Instrumentation issues 6. Examples 7. Today’s labs

Fluorescence: Re-emission of energy as a photon as an electron relaxes from a electronic excited state Fraction of energy absorbed at shorter 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)

Collin’s lecture Tuesday: Absorption of a photon occurs if AND ONLY IF the energy of the photon (E = h = hc/  ) is equal to the energy difference of an electron in the ground state (S 0 ) and higher electronic states (S n ). Absorption is an “electronic transition”; (O( s))

Primary mechanism of energy loss to permit an electron to relax or return to S 0 is by loss of heat (IR radiation); so-called radiationless decay; (O( s)). Vibrational states w/in electronic states

Chlorophyll absorption (direct or via accessory pigs) Chlorophyll excited electron: 1.Photochemistry (charge separation) 2.Heat (many pathways) 3.Fluorescence at 686 nm (O(10 -9 s))

Fluorescence emission 1. always from lowest vibrational state of S n 2. red shifted – Stokes shift (higher, lower E) 3. mirror image of absorption

F = E( ). conc.  f F = E( ). a( ).  f where  E( ) is excitation lamp energy conc is concentration a is ( ) is absorption  f is quantum yield of fluorescence = moles photons fluoresced moles photons absorbed if  f were constant, F ~ conc or a

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

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 peaks Green fluorescent protein (used in molecular staining) – from coral, jellyfish and some protozoans

CDOM F – proxy for a CDOM for radiative transfer a CDOM – proxy for DOM for carbon cycling Chl F – proxy for Chl – proxy for phytoplankton and input to productivity and carbon models PE – specific taxa

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

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( )

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

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

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)

Example of active /static benchtop application (Ststion 1): fluorescence of solvent-extracted chlorophyll a F concentration 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. F = E( ). conc.  f Note: important temperature effect on  f (watch out if room temp changes) ~ - 1–2% F / ºC

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. From Falkowski and Raven 1997 Chlorophyll fluorescence and extracted concentration of chlorophyll early AM vs. noon. Chlorophyll mg/m 3 PAR GoMOOS Buoy E (Roesler) Glider fluorescence, Wash. Coast

Example of mid-day fluorescence quenching Quenching observed to 11m Fluoresence quenched up to 80% at surface Year Day Depth (m) Mixed Layer Depth (MLD) Mid-day fluorescence quenching Sackmann et al, MS. So maybe for biomass, should we concentrate on night-time measurements in vivo fluorescence measurements?

Mid-day fluorescence quenching MORNING MID-DAY AFTERNOON Sackmann et al., unpub.

Mid-day fluorescence quenching MORNING MID-DAY AFTERNOON Sackmann et al., unpub.

Mid-day fluorescence quenching MORNING MID-DAY AFTERNOON Sackmann et al., unpub.

Figure 2: Damariscotta River in situ chlorophyll a fluorescence and PAR ( μ mol photons/s/m 2 ) vs. time. Time PAR Fluorescence

Figure 4:Normalized variable fluorescence (Fv/Fm) and PAR (μmol photons/s/m 2 ) vs. time. PAR Time Fv/Fm

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 photoreduction of QA to QA - and connectivity among Reaction Centers Slow rise (< minute) Fast rise (< second); #1 – low light; #2 – high light adapted; #3 DCMU photochemical, thermal and other quenching

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!