Quinine was the first known fluorophoreQuinine was the first known fluorophorediscovered by discovered by Sir John Frederick

William Herschel.

Sir John Frederick William Herschel

Principles of SpectrometryPrinciples of SpectrometryPrinciples ofPrinciples ofSpectrometrySpectrometry

Absorbance Absorbance !! absorption of a photon by a absorption of a photon by asubstance, electronically excites.substance, electronically excites.

Luminescence Luminescence !! emission of a photon from a emission of a photon from asubstance, substance, occurs from electronically excited states.occurs from electronically excited states.

Two categories Two categories !! fluorescence & phosphorescence; fluorescence & phosphorescence;Depends on the nature of the excited state.Depends on the nature of the excited state.Focus on fluorescence.Focus on fluorescence.

"" A molecule absorbs light and an electron is excited andA molecule absorbs light and an electron is excited andpromoted to an unoccupied orbital.promoted to an unoccupied orbital.

–– The energy difference between the ground (SThe energy difference between the ground (S00) and excited singlet) and excited singletstates (Sstates (S11, S, S22, or higher) determines the wavelength at which light is, or higher) determines the wavelength at which light isabsorbed.absorbed. Stedmon et al., 2003

Absorption: general principlesAbsorption: general principles Absorption: general principlesAbsorption: general principles

The absorption strength of a molecule is determined by:The absorption strength of a molecule is determined by:

Beer-Lambert Law Beer-Lambert Law

"" IIoo is reference intensity is reference intensity"" I is intensity leaving the sampleI is intensity leaving the sample"" !! is molar absorption coefficient or molar is molar absorption coefficient or molar absorptivityabsorptivity"" C is concentrationC is concentration"" l is pathlength of the samplel is pathlength of the sample

Absorption maxima:Absorption maxima:The importance of conjugationThe importance of conjugation

"" The wavelength value of the absorption maximum and the molarThe wavelength value of the absorption maximum and the molarabsorbtivity are determined by the degree of conjugatation ofabsorbtivity are determined by the degree of conjugatation ofp-bonds.p-bonds.

"" Increasing the number of double bonds shifts the absorption toIncreasing the number of double bonds shifts the absorption tolower energy with a higher intensity.lower energy with a higher intensity.

Absorption maxima:Absorption maxima:Increasing the number of aromatic ringsIncreasing the number of aromatic rings

increases the absorption maximumincreases the absorption maximum

"" As the degree of conjugation increases (i.e. the number of electronsAs the degree of conjugation increases (i.e. the number of electronsinvolved in the delocalized p-orbitals) the absorption energy decreasesinvolved in the delocalized p-orbitals) the absorption energy decreases(> l, the energy between the ground and excited state decreases) and the(> l, the energy between the ground and excited state decreases) and theabsorption becomes more intense (>e, increased probability of absorption).absorption becomes more intense (>e, increased probability of absorption).

"" Excitation is followed by Excitation is followed by nonradiativenonradiative relaxation to the lowest relaxation to the lowestsublevel of the Ssublevel of the S11 state, vial vibrational relaxation and internal state, vial vibrational relaxation and internalconversions.conversions.

"" Emission occurs during relaxation to the ground state, whereEmission occurs during relaxation to the ground state, wherethe wavelength of the fluorescence emission is determined bythe wavelength of the fluorescence emission is determined bythe difference in energy between Sthe difference in energy between S11 and S and S00 states. states. Stedmon et al., 2003

Emission: general principlesEmission: general principles

–– Emission has lower energyEmission has lower energycompared to absorption.compared to absorption.## The fluorescence spectrumThe fluorescence spectrum

lies at longer wavelengthslies at longer wavelengthsthan the absorption.than the absorption.

–– Emission and absorptionEmission and absorptionspectra exhibit a stokesspectra exhibit a stokesshift and areshift and areapproximately mirrorapproximately mirrorimages.images.## The spacings of the energyThe spacings of the energy

levels in the vibrationallevels in the vibrationalmanifolds of the groundmanifolds of the groundstate and first excitedstate and first excitedelectronic states are usuallyelectronic states are usuallysimilar.similar.

General rules of emission spectra:General rules of emission spectra:

The fluorescence emission spectrumThe fluorescence emission spectrum

"" Emission mainly occurs from aromatic compounds.Emission mainly occurs from aromatic compounds.

"" The concentration and chemical composition of a substanceThe concentration and chemical composition of a substanceinfluences the intensity and shape of the fluorescenceinfluences the intensity and shape of the fluorescencespectra and is dependant on environmental parameters (i.e.spectra and is dependant on environmental parameters (i.e.ph, temperature, ph, temperature, redoxredox conditions, etc). conditions, etc).

An application of fluorescenceAn application of fluorescencespectroscopy inspectroscopy in

oceanographic researchoceanographic research

Chromophoric dissolvedChromophoric dissolvedorganic matter (CDOM)organic matter (CDOM)

Dissolved organic matter (DOM)Dissolved organic matter (DOM)

- Heterogeneous mixture of organic carbon (OC)- Heterogeneous mixture of organic carbon (OC)that remains largely uncharacterizedthat remains largely uncharacterized

- Plays active role in the microbial loop- Plays active role in the microbial loop

-- Dissolved organic carbon (DOC) in the ocean isDissolved organic carbon (DOC) in the ocean isof the same magnitude as atmospheric COof the same magnitude as atmospheric CO22..

Small changes in ocean DOC concentrations canSmall changes in ocean DOC concentrations canhave a significant effect on the biogeochemistryhave a significant effect on the biogeochemistryof the global carbon cycle.of the global carbon cycle.

Environmental significance of DOMEnvironmental significance of DOM

"" The concentration and composition of DOM inThe concentration and composition of DOM inaquatic environments can directly and indirectlyaquatic environments can directly and indirectlyinfluence:influence:

–– BiologyBiology !! microbial and plankton ecologymicrobial and plankton ecology

–– ChemistryChemistry !! trace metal speciation and transporttrace metal speciation and transport

–– PhysicsPhysics !! optical propertiesoptical properties

Stedmon et al., 2003

Chromophoric dissolved organicChromophoric dissolved organicmatter (CDOM)matter (CDOM)

-CDOM -CDOM !! the light absorbing constituent of the DOM pool the light absorbing constituent of the DOM poolin natural waters.in natural waters.

-Constitutes a small fraction on the total DOM pool.-Constitutes a small fraction on the total DOM pool.-Absorbs light in the ultraviolet and visible wavelength-Absorbs light in the ultraviolet and visible wavelength

range.range.

Two distinct sources of CDOM to the oceanTwo distinct sources of CDOM to the ocean

1.)1.) Terrestrially derivedTerrestrially derived !! river runoff river runoff !! more refractory. more refractory.

2.)2.) Marine derivedMarine derived !! primary/secondary production and primary/secondary production and remineralizationremineralization !! more labile (biologically more labile (biologically reactive).reactive).

Proxies using fluorescence to determineProxies using fluorescence to determineCDOM sourcesCDOM sources

"" Microbially derived DOM has fluorophores with a moreMicrobially derived DOM has fluorophores with a moresharply defined emission peaks occurring at lowersharply defined emission peaks occurring at lowerwavelengths than terrestrially derived fluorophores.wavelengths than terrestrially derived fluorophores.

–– Protein or amino acid signal:Protein or amino acid signal:emem 300-305 nm (tyrosine-like);340-350 nm 300-305 nm (tyrosine-like);340-350 nm((tryptophantryptophan-like) / ex 220-275 nm.-like) / ex 220-275 nm.

–– Humic-like signal:Humic-like signal:emem 420-450 nm / ex 230-260; 320-350 nm 420-450 nm / ex 230-260; 320-350 nm

Proxies using fluorescence toProxies using fluorescence todetermine the presence of CDOMdetermine the presence of CDOM

and its sourceand its source

"" The absorption coefficient at 350 nm (a350) is used toThe absorption coefficient at 350 nm (a350) is used totrace DOC in natural environments.trace DOC in natural environments.

a350 = (2.303*aa350 = (2.303*a"") / l, where a) / l, where a"" is the absorbance reading at any is the absorbance reading at anygiven wavelength and l is the pathlength in meters.given wavelength and l is the pathlength in meters.

"" The fluorescence index (FI) is the ratio of theThe fluorescence index (FI) is the ratio of theemission intensity at 450 nm to that at 500 nm,emission intensity at 450 nm to that at 500 nm,obtained at excitation of 370 nm, serves as aobtained at excitation of 370 nm, serves as asimple index to distinguish DOM sources.simple index to distinguish DOM sources.

–– Where a value of ~1.9 indicates microbially derived DOM andWhere a value of ~1.9 indicates microbially derived DOM and~1.4 indicates terrestrially derived DOM~1.4 indicates terrestrially derived DOM Diane Mcknight et al., 2001

Kowalczuk et al., 2010

Excitation Emission Matrix (EEM)Excitation Emission Matrix (EEM)SpectroscopySpectroscopy

Qualitative analysisQualitative analysis useful for source identification and transport of useful for source identification and transport ofCDOMCDOM

Collects excitation and emission over a broad wavelength rage, withCollects excitation and emission over a broad wavelength rage, withintensity on the z-axis, to build a fluorescence landscape.intensity on the z-axis, to build a fluorescence landscape.

EEM - Data Acquisition:EEM - Data Acquisition:–– Photon Technologies International Fluorometer (Quanta Master-4 SE)Photon Technologies International Fluorometer (Quanta Master-4 SE)–– Excitation wavelengths: Excitation wavelengths: 220 to 450 nm220 to 450 nm (5nm increments) (5nm increments)–– Emission wavelengths: Emission wavelengths: 230 to 600 nm230 to 600 nm (2nm increments) (2nm increments)–– 1 cm quartz cuvette1 cm quartz cuvette–– Excitation and emission slit widths set to 5nmExcitation and emission slit widths set to 5nm–– Excitation and emission correction files applied to correct for instrumentExcitation and emission correction files applied to correct for instrument

specific biases; the ex/specific biases; the ex/emem signal was normalized to a reference detector to signal was normalized to a reference detector toaccount for fluctuations in light source characteristicsaccount for fluctuations in light source characteristics

Stedmon et al., 2003; Murphy et al., 2007; Stedmon and Bro, 2008; Lakawetz and Stedmon, 2009

EEMEEM’’s s –– Post Data Processing in Matlab Post Data Processing in Matlab

"" Remove Excitation < 240nm and emission < 300nmRemove Excitation < 240nm and emission < 300nm–– Due to deteriorating signal-to noise ratiosDue to deteriorating signal-to noise ratios

[Stedmon et al., 2003][Stedmon et al., 2003]

"" Apply Inner filter factor (I)Apply Inner filter factor (I)–– (I) = 10[(a_ex) (0.005)) + (((I) = 10[(a_ex) (0.005)) + ((a_ema_em) (0.005))]) (0.005))]

## where a is the absorption coefficientwhere a is the absorption coefficient–– Divide (I) into each ex/Divide (I) into each ex/emem pair within each EEM pair within each EEM

[[LakowiczLakowicz, 2006], 2006]

"" Raman calibrateRaman calibrate–– Normalize data to the area under the Raman scatter peak at excitationNormalize data to the area under the Raman scatter peak at excitation

wavelength 350nm of a Milli-Q water sample ran dailywavelength 350nm of a Milli-Q water sample ran daily–– Results in Raman units (R.U., nm-1)Results in Raman units (R.U., nm-1)

[[LawaetzLawaetz and Stedmon, 2009] and Stedmon, 2009]

"" Remove Raman scatterRemove Raman scatter–– Subtract the Raman normalized Milli-Q EEM from each sample EEMSubtract the Raman normalized Milli-Q EEM from each sample EEM–– [Stedmon et al., 2003][Stedmon et al., 2003]

"" Remove Rayleigh scatterRemove Rayleigh scatter–– Delete emission less than or equal to excitation + 20 nm.Delete emission less than or equal to excitation + 20 nm.–– [Stedmon et al., 2003][Stedmon et al., 2003]

Stedmon et al., 2003; Murphy et al., 2007; Stedmon and Bro, 2008; Lakawetz and Stedmon, 2009

EEMs EEMs –– Post Data Processing in Matlab Post Data Processing in Matlab

Organize EEM data for PARAFAC modeling processOrganize EEM data for PARAFAC modeling process

"" Remove regions of no fluorescence or scatterRemove regions of no fluorescence or scatter–– EEMs now range from 240 to 400nm along theEEMs now range from 240 to 400nm along the

excitation axis and 320 to 580nm along theexcitation axis and 320 to 580nm along theemission axis.emission axis.

"" Insert region of zeros into areas of no fluorescenceInsert region of zeros into areas of no fluorescence–– (where excitation >> emission)(where excitation >> emission)

"" Cut emission wavelengths > 550nmCut emission wavelengths > 550nm–– Helps to correct second order scatter and quickensHelps to correct second order scatter and quickens

PARAFAC modeling processPARAFAC modeling process

Corrected EEMs

Marine Signal Terrestrial Signal

Trans-polar driftShelf water

"" Several types of fluorescent signals observedSeveral types of fluorescent signals observed–– Humic-likeHumic-like–– Tyrosine-likeTyrosine-like–– TryptophanTryptophan-like-like

EEMsEEMs peak picking technique to peak picking technique todetermine CDOM sourcesdetermine CDOM sources

Coble 1996Coble 1996

Coble 1996Coble 1996 Coble 1996Coble 1996

Coble 1996Coble 1996

A multivariate technique ! qualitative and quantitative

Decomposes the combined CDOM fluorescence signal withinan EEM data array into fluorescent components thatcorrespond to a chemical analyte, or group of stronglycovarying analytes.

Used to- predict DOC concentrations- distinguish compositional characteristics- discriminate between terrestrial and marine DOM sources

Parallel Factor Analysis(PARAFAC)

Three main steps involved tovalidate a PARAFAC model

1.) Outlier identification1.) Outlier identification–– Compare sample weights in each modelCompare sample weights in each model–– Narrow down the range of how many components are adequate to describeNarrow down the range of how many components are adequate to describe

the overall fluorescence variation within in the EEM data arraythe overall fluorescence variation within in the EEM data array

2.) Split-half validation2.) Split-half validation–– Divides data into two random halves of equal size and runs the modelDivides data into two random halves of equal size and runs the model

independently on the two halves.independently on the two halves.–– The appropriate number of components is validated when there is a strongThe appropriate number of components is validated when there is a strong

overlapping of component loadings from the two halves.overlapping of component loadings from the two halves.

3.) Residual analysis3.) Residual analysis–– Assess how much fluorescence is not explained by the PARAFAC modelAssess how much fluorescence is not explained by the PARAFAC model–– Model validated when residuals reflect random noise with minimalModel validated when residuals reflect random noise with minimal

systematic variation.systematic variation.

Stedmon 2008

Outlier IdentificationOutlier Identification

Residual analysis

Subtraction of the modeled from the measured spectra should yield a residualfluorescence of at least an order of magnitude lower than the measured spectra.

Validated PARAFAC Model

Component abundanceAOS station 30 10m

Modeled sample EEMAOS station 30 10m

AOSC4 (ex:<240,275 / em:304)

0

0.1

0.2

0.3

0.4

240 340 440 540

Wavelength (nm)

nm

-1

AOSC5 (ex:<240,330 / em:400)

0

0.05

0.1

0.15

0.2

0.25

0.3

240 340 440 540

Wavelength (nm)

nm

-1

AOSC6 (ex:<240,295 / em:400)

0

0.1

0.2

0.3

0.4

240 340 440 540

Wavelength (nm)

nm

-1

AOSC1 (ex:<240,300 / em:336)

00.05

0.10.15

0.20.25

0.30.35

0.4

240 290 340 390 440 490 540

Wavelength (nm)

nm

-1

excitationemission

AOSC2 (ex:<240,285 / em400)

00.050.1

0.150.2

0.250.3

0.350.4

240 340 440 540

Wavelength (nm)

nm

-1

AOSC3 (ex:<240,360 / em:468)

0

0.05

0.1

0.15

0.2

0.25

0.3

240 340 440 540

Wavelength (nm)

nm

-1

C1 (ex:<240,300 / em:336) C2 (ex:<240,285 / em:400)

C6 (ex:<240,295/ em:400)C5 (ex:<240,330 / em:400)C4 (ex:<240,275 / em:304)

C3 (ex:<240,360 / em:468)

AOS PARAFAC ComponentsVanillic acid

0

0.1

0.2

0.3

0.4

0.5

0.6

240 290 340 390 440 490 540

Wavelength (nm)

Inte

nsity

(nm

-1)

Vanillic acid

0

0.1

0.2

0.3

0.4

0.5

0.6

240 290 340 390 440 490 540

Wavelength (nm)

Inte

nsity

(nm

-1)

Hydroquinone

0

0.1

0.2

0.3

0.4

0.5

0.6

240 290 340 390 440 490 540

Wavelength (nm)

Inte

nsity

(nm

-1)

1,4-Naphthoquinone

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

240 290 340 390 440 490 540

Wavength (nm)

Inte

nsi

ty (n

m-1

)

3-Hydroxybenzoic acid

00.050.1

0.150.2

0.250.3

0.350.4

0.450.5

240 290 340 390 440 490 540

Wavength (nm)

Inte

nsity

(nm

-1)

3-Hydroxybenzaldehyde

00.050.1

0.150.2

0.250.3

0.350.4

0.450.5

240 290 340 390 440 490 540

Wavength (nm)

Inte

nsity

(nm

-1)

Syringic acid

00.050.1

0.150.2

0.250.3

0.350.4

0.450.5

240 290 340 390 440 490 540

Wavelength (nm)

Inte

nsity

(nm

-1)

3,5-Dimethoxy-4-hydroxybenzaldehyde

0.00E+00

5.00E-02

1.00E-01

1.50E-01

2.00E-01

2.50E-01

3.00E-01

3.50E-01

4.00E-01

4.50E-01

240 290 340 390 440 490 540

Wavelength (nm)

Inte

nsi

ty (n

m-1

)

3-Hydroxyacetophenone

0

0.1

0.2

0.3

0.4

0.5

0.6

240 290 340 390 440 490 540

Wavelength (nm)

Inte

nsity

(nm

-1)

Ferulic acid

0

0.050.1

0.15

0.20.25

0.3

0.350.4

0.45

240 290 340 390 440 490 540

Wavelength (nm)

Inte

nsity

(nm

-1)

p-Coumaric acid

0

0.05

0.1

0.15

0.2

0.25

0.3

0.35

0.4

0.45

240 290 340 390 440 490 540

Wavelenth (nm)

Inte

nsi

ty (n

m-1

)

L-Tryptophan

0

0.05

0.1

0.15

0.2

0.25

0.3

0.35

0.4

0.45

240 290 340 390 440 490 540

Wavelength (nm)

Inte

nsi

ty (n

m-1

)

DL-Tyrosine

0

0.1

0.2

0.3

0.4

0.5

0.6

240 290 340 390 440 490 540

Wavelength (nm)

Inte

nsi

ty (n

m-1

)

Riboflavin

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

240 290 340 390 440 490 540

Wavelength (nm)

Inte

nsi

ty (n

m-1

)

Sodium salicylate

0

0.05

0.1

0.15

0.2

0.25

0.3

0.35

0.4

0.45

0.5

240 290 340 390 440 490 540

Wavelenth (nm)

Inte

nsi

ty (n

m-1

)

L-Phenylalanine

0

0.1

0.2

0.3

0.4

0.5

0.6

240 290 340 390 440 490 540Wavelength (nm)

Inte

nstiy

(nm

-1)

PARAFAC model of known fluorophores

Courtesy of Colin Stedmon

Lab QuestionsLab Questions"" 1. Why does the length of your cuvette matter when1. Why does the length of your cuvette matter when

collecting absorbance? Based on your answer, howcollecting absorbance? Based on your answer, howwould you adjust the length of the cuvette for a morewould you adjust the length of the cuvette for a moreconcentrated sample verses a lower concentratedconcentrated sample verses a lower concentratedsample and why?sample and why?

"" 2. Name two chemical characteristics that can2. Name two chemical characteristics that caninfluence the absorption maxima of a molecule.influence the absorption maxima of a molecule.

"" 3. Which has more energy involved, excitation or3. Which has more energy involved, excitation oremission and why?emission and why?

"" 4. Why is there a stokes shift between emission and4. Why is there a stokes shift between emission andexcitation maxima for any given molecule?excitation maxima for any given molecule?

"" 5. What types of molecules tend to fluoresce? List5. What types of molecules tend to fluoresce? Listsome parameters that can affect the intensity andsome parameters that can affect the intensity andshape of the emission spectra.shape of the emission spectra.

"" 6. Based on the absorption coefficient at 350nm6. Based on the absorption coefficient at 350nm(a350) and the Fluorescence Index (FI), where do(a350) and the Fluorescence Index (FI), where dothink unknown samples A, B, C, and D werethink unknown samples A, B, C, and D werecollected from and why?collected from and why?

"" 7. You are given results from a PARAFAC model7. You are given results from a PARAFAC modelshowing three components. Based on theirshowing three components. Based on theirspectra, do you think they are terrestrially orspectra, do you think they are terrestrially ormicrobially derived and why?microbially derived and why?

"" 8. What is the benefit of applying PARAFAC8. What is the benefit of applying PARAFACanalysis to a fluorescence data set verses justanalysis to a fluorescence data set verses justusing the peak picking technique from using the peak picking technique from EEMEEM’’ss??

Lab QuestionsLab Questions