Methods of Photosynthesis Spectrometry
For Phytoplankton
Christophe Six
Definitions
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 .
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) /
800 nm
E : Photon energy
h : Plank constant factor
c : Light celerity
l : Photon wavelength
Wavelength
in nanometers
400 nm
X-rays
U.V.
Visible
Infrared
Radio wavelengths
Absorbance of molecules and molecular complexes
.Understanding photophysics and photobiology
.Very useful for assays
 Using colorimetric assays
Concept of the absorbance measurement
Light Source
Photomultiplicator
Sample
I0
>
I
Transmittance
Absorbance
T = I / I0
A = log (I0 / I)
A = -log T
Spectrophotometers
Components :
.One or several light source(s)
Extended Visible (350-900 nm) : Tungsten, Halogen
UV (<400 nm) : Deuterium
. One monochromator : Selection of wavelengths
. One sample compartment
. One detector : photomultiplicator or photodiode detector
. A result display system
Single beam spectrophotometers
D.O.
or monochromator
400
500
 (nm)
Single beam spectrophotometers
. The simplest system
. A simple compartment for a single sample cuvette
. 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
In single wavelength mode, one cannot check for artefacts
The decrease of lamp intensity is not compensed
The making of these instruments is usually less careful
I0
(fixed)
I
(measured)
Double beam spectrophotometers
Monochromator
Reference
Cuvette
I0
Chopper
Chopper
Sample
Cuvette
I
Double beam spectrophotometers
. 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)
. Correction of the variations of the light sources
. Devices generally better than single beam ones
Artefacts
Refraction : deviation of a wave when its speed changes (interface between 2 media)
Diopter (surface of the cuvette and surface of the sample)
=> A
. Other optical phenomenons linked to diffusion, reflexion and diffraction
of light may also distort the measurement.
Artefacts : Light diffusion
. Turbid solutions, cell suspensions
=> A
Diffusion occurs when some light is deflected by particules and therefore does
not reach the detector
Diffusion of Rayleigh
S =
Diffusion also depends on :
- Particule concentration
- Particule shape
=
F( d, n)
4
d : Diameter of particules
n : Refraction index
 : Wavelength
Impact of diffusion on absorption spectra
5,0E-11
4,0E-11
3,0E-11
y=
2,0E-11
1
x4
Diffusion is -dependent
1,0E-11
0,0E+00
350
450
550
650
750
850
Longueur d'onde (nm)
=> A ok
=> A
Impact of diffusion on absorption spectra
Example : absorption spectrum of a phycoerythrin I
Fitting a correction curve
Absorbance
Spectrum with diffusion
Wavelength (nm)
Final spectrum
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
Homogeneous
Echantillon
sample
homogène
Détecteur
Light du
photomultiplicateur
detector
A
DO
(nm)
B
DO
Suspension
Cell
de cellules
suspension
(nm)
C
Light
Rayon
beam
Light Source
source and lumineux
lumineuse et
monochromator
monochromateur
DO
(nm)
Sphère
Integration
d’intégration
sphere
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
. The sample contains absorbing particules that sink in the cuvette
. There’s not enough sample in the cuvette and the beam passes through the meniscus
. Cuvettes not adapted (micro-cuvettes)
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)
 : 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 =
. For l = 1 cm :
,1 . l . C1 + ,2 . l . C2 + 3 . l . C3 + … + ,n . l . Cn
A =
 . C
=> C = A / 
A =
,1 . C1 + ,2 . C2 + 3 . C3 + … + ,n . Cn
Fluorescence: what is it ?
Stokes shift
Intensity of fluorescence emission
. 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.)
 Need to use standard curves to quantify molecules by fluorescence (in absolute units)
. 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.)
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
Main components :
- 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
- A photomultiplicator
Diagrammic representation of a spectrofluorimeter
Photomultiplicator
Xenon lamp
Lens
Entrance Slit
Exit slit
Monochromator
shutter
Monochromator
Slit
Mirror
Lens
Lens
Sample
Emission and Excitation spectra of fluorescence
Emission spectrum
Monochromator scanning
all wavelengths
Fix monochromator :
One given 
Excitation
 15 nm
Emission
Quantification of the
fluorescence emitted
by the excitation of
a given 
Sample
400
500
600
700
At which  is the maximum of fluorescence
emission of the compound ?
Excitation spectrum
Fix monochromator :
One given 
Monochromateur scanning
all wavelengths
Excitation
Emission
Quantification of the
fluorescence emitted
many wavelengths 
Sample
400
(Excitation spectra are often similar to absorption spectra)
500
600
700
Which (s) give(s) rise to the
Fluorescence emission at a given  ?
Fluorescence of marine picocyanobacteria : Synechococcus spp.
Marine phycoerythrins & spectrofluorimetry
. There are several types of
phycoerythrins (PE)
Variable
Emission
Excitation
between
between
560-580 nm
400 and
depending on the
550 nm
type of PE
Excitation
In the
blue-green
region,
at 500nm
(for instance)
Emission spectrum
Emission
at 580 nm
(for instance)
Excitation spectrum
545
495
400
500
600
700
400
500
 One or two
major maxima
600
700
Phycoerythrin structure and excitation spectra
Phycobiliprotein = Apoprotein + pigment
Pigment = chromophore  phycobilin
One or two types of phycobilin are bound
to marine phycoerythrins
Excitation spectrum
545
495
400
500
 One or two
major maxima
600
700
PAM Fluorimetry and photosynthetic organisms
Monitoring-PAM©
Diving-PAM©
Multicolor-PAM©
Junior-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
=
Antenna
Fluorescence energy
+
Photochemistry energy
+
Heat energy
Photochemistry
Fluorescence
Chl a
Photochemistry
Fluorescence
Centre réactionnel
Chl a
Centre réactionnel
Centre réactionnel
Open/closed PSII centres
Chl a
Fluorescence
Photochemistry
Fluorescence
Photochemistry
Chl a
Fluorescence
Photochemistry
Heat
Centre réactionnel
Chl a
Centre réactionnel
Centre réactionnel
Heat
Chl a
Fluorescence
Photochemistry
PAM Fluorimeters
. Two types of light :
- Modulated light : intermittent, low irradiance  non actinic
- Actinic light : continuous
Actinic
light
Modulated
light
Photosystem II
Fluorescence
(red light)
Photomultiplicator
Fiber optics
Conceptual diagram of the
Junior-PAM
Sample
PAM Fluorimetry : light response curves
Fluorescence (AU)
FM’
FM’
FM’
FM’
FM’
FM’
FV’
Ft
F0
Flash
saturant
Actinic light
Time
Modulated
light ON
Actinic (Irradiance 2)
(Irradiance 4)
(Irradiance 6)
light ON
(Irradiance 3)
(Irradiance 5)
(Irradiance 1)
PAM Fluorimetry : light response curves
Fluorescence (AU)
FM’
FM’
FM’
FM’
FM’
FM’
FV’
When increasing irradiance
PSII reaction centres get
more and more closed
Ft
F0
Flash
saturant
Actinic light
Time
Modulated
light ON
Actinic (Irradiance 2)
(Irradiance 4)
(Irradiance 6)
light ON
(Irradiance 3)
(Irradiance 5)
(Irradiance 1)
PSII relative Electron Transfer Rate (rETR) = Irradiance x (FM’-Ft)/FM’
= Irradiance x FV’/FM’
(Irradiance : µmol photons / m² / s)
PAM Fluorimetry : light response curves
Fluorescence (AU)
FM’
FM’
FM’
FM’
FM’
FM’
FV’
PSII rETR = Irradiance x (FV’/FM’)
Ft
F0
Flash
saturant
Actinic light
Time
Actinic (Irradiance 2)
(Irradiance 4)
(Irradiance 6)
light ON
(Irradiance 3)
(Irradiance 5)
(Irradiance 1)
PSII rETR
Modulated
light ON
 Courbe PSII rETR versus Irradiance
Irradiance (µmol photons/m²/s)
PAM Fluorimetry : light response curves
Saturation du rETR
PSII rETR
PSII rETR
Pas de saturation
Irradiance (µmol photons/m²/s)
ISAT
Irradiance (µmol photons/m²/s)
PSII antenna size : α
PSII rETR
α>α
ISAT < ISAT
Irradiance (µmol photons/m²/s)
PAM Fluorimetry : light response curves
PSII rETR
Saturation without photoinhibition
Irradiance (µmol photons/m²/s)
PSII rETR
Saturation and photoinhibition
Irradiance (µmol photons/m²/s)
PAM Fluorimetry : light response curves
PSII rETR
Example of application : Prochlorococcus ecotypes
Irradiance (µmol photons/m²/s)