Methods of Photosynthesis SpectrometryFor Phytoplankton

Christophe Six

Spectrometry = spectroscopy :

Methods of spectral analysis allowing to understand the composition, the structure of matter and/or the study

of systems transferring energy

Qualitative and quantitative studies of spectra derived from the interaction between the matter and the wavy radiations of different frequences .

Definitions

Spectrophotometry is an analytic, quantitative method that consists in measuring the absorbance (= absorption ~ optical density) of a given chemical substance (or of a whole unicell organism) in solution, function of the light wavelength.

Spectrofluorimetry is an analytic, quantitative method that consists in measuring the emission and excitation levels of fluorescence of a given chemical substance (or of a whole unicell organism) in solution, function of the light wavelength.

Energy and wavelength

E = (h . c) /

E : Photon energyh : Plank constant factorc : Light celerityl : Photon wavelength

X-rays U.V. Visible Infrared Radio wavelengths

400 nm

800 nm

Wavelength in nanometers

Absorbance of molecules and molecular complexes

.Understanding photophysics and photobiology

.Very useful for assays

Using colorimetric assays

Concept of the absorbance measurement

A = log (I0 / I)

I0 I>

Sample PhotomultiplicatorLight Source

Absorbance

T = I / I0

Transmittance

A = -log T

Spectrophotometers

.One or several light source(s)

Extended Visible (350-900 nm) : Tungsten, HalogenUV (<400 nm) : Deuterium

. One monochromator : Selection of wavelengths

. One sample compartment

. One detector : photomultiplicator or photodiode detector

. A result display system

Components :

Single beam spectrophotometers

or monochromator

(nm)

D.O.

400 500

. A simple compartment for a single sample cuvette

. The simplest system

. The reference = blank is measured before the samples for zeroing the device

Blank : all chemical components (buffer, solvent, etc) except the absorbing substance that you want to measure.It is actually rare to be able to use a perfectly true blank but one should approach it as much as possible.

. Instrument useful for simple routine applications (single or few wavelengths)

Various colorimetric assays (proteins, nucleic acids, pigments, etc.)

. Main problems

The decrease of lamp intensity is not compensed

I0 I

In single wavelength mode, one cannot check for artefacts

(fixed) (measured)

Single beam spectrophotometers

The making of these instruments is usually less careful

Double beam spectrophotometers

ReferenceCuvette

SampleCuvette

I0

I

Chopper

Chopper

Monochromator

. Correction of the variations of the light sources

. For each wavelength, one mesures the absorbance of the sample AND the absorbance of the reference (blank)

. Good reliability of the measurements, ideal for absorption spectra(Elimination of solvent absorption)

Double beam spectrophotometers

. Devices generally better than single beam ones

Artefacts

. Other optical phenomenons linked to diffusion, reflexion and diffraction of light may also distort the measurement.

Refraction : deviation of a wave when its speed changes (interface between 2 media)

=> A

Diopter (surface of the cuvette and surface of the sample)

. Other optical phenomenons linked to diffusion, reflexion and diffraction of light may also distort the measurement.

=> A

Artefacts : Light diffusion

. Turbid solutions, cell suspensions

Diffusion occurs when some light is deflected by particules and therefore does not reach the detector

S = F( d, n)

4=Diffusion of Rayleigh

d : Diameter of particulesn : Refraction index : Wavelength

Diffusion also depends on :

- Particule concentration - Particule shape

Impact of diffusion on absorption spectra

0,0E+00

1,0E-11

2,0E-11

3,0E-11

4,0E-11

5,0E-11

350 450 550 650 750 850

Longueur d'onde (nm)

y = 1x4

=> A ok

=> A

Diffusion is -dependent

Ab

sorb

ance

Wavelength (nm)

Spectrum with diffusion Fitting a correction curve Final spectrum

Impact of diffusion on absorption spectra

Example : absorption spectrum of a phycoerythrin I

Measuring absorbance in a diffusing sample

=> A

Bringing the detector nearer to the cuvette

Increasing the surface of the detector

Measuring absorbance in a diffusing sample

Source lumineuse et

monochromateur

Rayon lumineux

Suspension de cellules

Sphèred’intégration

Echantillonhomogène

Détecteur du photomultiplicateur

DO

(nm)

DO

(nm)

DO

(nm)

DO

(nm)

DO

(nm)

A

B

C

Homogeneous sample

Light detector

Cell suspension

Integration sphere

Light beamLight source and

monochromator

If the absorbance of a sample is not stable…

. Sample much colder than the atmosphere of the compartment

Condensation on the cuvette

Gaz formation (diffusion)

. Sample drops on the outside of the cuvette

. There’s not enough sample in the cuvette and the beam passes through the meniscus

. Cuvettes not adapted (micro-cuvettes)

. The sample contains absorbing particules that sink in the cuvette

The Beer-Lambert law

At a given wavelength, the absorbance of a solution is proportional to the concentration of the absorbing chemical species that are present in this solution, and to the optical path

A = . l . C

A : Absorbance (no unit)l : Wavelength (nm)l : Optical path (cm)C : Concentration (mol L-1)

: Extinction coefficient (L mol-1 cm-1)

. The Beer-Lambert law is additive. Pour n chemical species :

A = ,1 . l . C1 + ,2 . l . C2 + 3 . l . C3 + … + ,n . l . Cn

. For l = 1 cm : A = . C => C = A /

A = ,1 . C1 + ,2 . C2 + 3 . C3 + … + ,n . Cn

Fluorescence: what is it ?

Stokes shift

. With fluorescence, there’s no such general relation as the absorbance Beer-lambert law

The measurement depends strongly on : - The nature of the fluorescent system that is studied - The device used to quantify fluorescence (light source intensity, optics configuration, etc.)

Intensity of fluorescence emission

. It is possible to quantify the fluorescence energy when a fluorescence quantum yield Qf :

Energy of fluorescence emitted (If) = Absorbed energy (Ia) x Qf

Qf = f (, T°C, pH, ions, etc.)

Need to use standard curves to quantify molecules by fluorescence (in absolute units)

Spectrofluorimeters

. None photon from the excitation light must be detected by the detector excitation at 90°

On average, there is 106 times less photons that hit the detector of a spectrofluorimeter than in a spectrophotometer

- A light source : Mercury or xenon lamp

- Two monochromators selecting either the emission or excitation precise wavelengths

- A dark compartment with the cuvette in a 90° excitation/emission cuvette holder

Main components :

- A photomultiplicator

Diagrammic representation of a spectrofluorimeter

Xenon lamp

Lens

LensLens

Slit

Entrance Slit Exit slit

Photomultiplicator

Sample

Mirror

shutter Monochromator

Monochromator

Emission and Excitation spectra of fluorescence

Fix monochromator : One given

Excitation

Emission

Sample

Monochromator scanning all wavelengths

Emission spectrum

Quantification of the fluorescence emitted by the excitation of

a given

At which is the maximum of fluorescence emission of the compound ?

600500 700400

15 nm

Fix monochromator : One given

Excitation

Emission

Sample

Monochromateur scanning all wavelengths

600500 700400

Quantification of the fluorescence emitted many wavelengths

Which (s) give(s) rise to the Fluorescence emission at a given ?(Excitation spectra are often similar to absorption spectra)

Excitation spectrum

Fluorescence of marine picocyanobacteria : Synechococcus spp.

600500 700400

Excitation spectrum

Marine phycoerythrins & spectrofluorimetry

600500 700400

Emission spectrum

ExcitationIn the

blue-greenregion,

at 500nm(for instance)

Emission between

560-580 nmdepending on the

type of PE

. There are several types of phycoerythrins (PE)

VariableExcitation between 400 and 550 nm

Emission at 580 nm(for instance)

One or two major maxima

495

545

Phycoerythrin structure and excitation spectra

Phycobiliprotein = Apoprotein + pigment

Pigment = chromophore phycobilin

One or two types of phycobilin are boundto marine phycoerythrins

600500 700400

Excitation spectrum

One or two major maxima

495

545

PAM Fluorimetry and photosynthetic organisms

Diving-PAM©

Monitoring-PAM© Junior-PAM©

Multicolor-PAM©

PAM Fluorimetry and photosynthetic organisms

Objectif : étudier la régulation de l’activité du photosystème II

PAM Fluorimetry and photosynthetic organisms

Objectif : étudier la régulation de l’activité du photosystème II

Absorbed light energy

=

Fluorescence energy+

Photochemistry energy+

Heat energyAntenna

Open/closed PSII centres

Chl a

Antenna

Cen

tre

réac

tio

nn

el

Photochemistry

Fluorescence

Chl a

Antenna

Cen

tre

réac

tio

nn

el

Photochemistry

Fluorescence

Chl a

Antenna

Cen

tre

réac

tio

nn

elPhotochemistry

Fluorescence

Chl a

Antenna

Cen

tre

réac

tio

nn

el

Photochemistry

Fluorescence

Chl a

Cen

tre

réac

tio

nn

el

Photochemistry

Fluorescence

Heat

Chl a

Cen

tre

réac

tio

nn

el

Photochemistry

Fluorescence

Heat

PAM Fluorimeters

. Two types of light : - Modulated light : intermittent, low irradiance non actinic - Actinic light : continuous

Actinic light

Modulated light

Photosystem IIFluorescence

(red light)

Sample

Fiber optics

Conceptual diagram of theJunior-PAM

Signal display

Photo-multiplicator

PAM Fluorimetry : light response curves

Flu

ore

scen

ce (

AU

)

Time

Modulated light ON

F0

Actinic light ON

(Irradiance 1)

(Irradiance 2)(Irradiance 3)

(Irradiance 4)(Irradiance 5)

(Irradiance 6)

Actinic light

FM’ FM’FM’

FM’ FM’ FM’

Flash saturant

FV’

Ft

PAM Fluorimetry : light response curves

When increasing irradiancePSII reaction centres get more and more closed

PSII relative Electron Transfer Rate (rETR) = Irradiance x (FM’-Ft)/FM’

= Irradiance x FV’/FM’

(Irradiance : µmol photons / m² / s)

Flu

ore

scen

ce (

AU

)

Time

Modulatedlight ON

F0

Actiniclight ON

(Irradiance 1)

(Irradiance 2)(Irradiance 3)

(Irradiance 4)(Irradiance 5)

(Irradiance 6)

Actinic light

FM’ FM’FM’

FM’ FM’ FM’

Flash saturant

FV’

Ft

Flu

ore

scen

ce (

AU

)

Time

Modulatedlight ON

F0

Actiniclight ON

(Irradiance 1)

(Irradiance 2)(Irradiance 3)

(Irradiance 4)(Irradiance 5)

(Irradiance 6)

Actinic light

FM’ FM’FM’

FM’ FM’ FM’

Flash saturant

FV’

Ft

PAM Fluorimetry : light response curves

PSII rETR = Irradiance x (FV’/FM’)

PS

II rE

TR

Irradiance (µmol photons/m²/s)

Courbe PSII rETR versus Irradiance

PAM Fluorimetry : light response curvesP

SII

rE

TR

Irradiance (µmol photons/m²/s)

Pas de saturation

PS

II r

ET

R

Irradiance (µmol photons/m²/s)

Saturation du rETR

PSII antenna size : α

PS

II r

ET

R

Irradiance (µmol photons/m²/s)

α > α

ISAT

ISAT < ISAT

PAM Fluorimetry : light response curves

PS

II r

ET

R

Irradiance (µmol photons/m²/s)

Saturation without photoinhibition

PS

II r

ET

R

Irradiance (µmol photons/m²/s)

Saturation and photoinhibition

PAM Fluorimetry : light response curves

Example of application : Prochlorococcus ecotypesP

SII

rE

TR

Irradiance (µmol photons/m²/s)