Methods of Photosynthesis Spectrometry For Phytoplankton Christophe Six.
-
Upload
melvin-garey-franklin -
Category
Documents
-
view
218 -
download
0
Transcript of Methods of Photosynthesis Spectrometry For Phytoplankton Christophe Six.
![Page 1: Methods of Photosynthesis Spectrometry For Phytoplankton Christophe Six.](https://reader033.fdocuments.in/reader033/viewer/2022052509/56649d0b5503460f949df780/html5/thumbnails/1.jpg)
Methods of Photosynthesis SpectrometryFor Phytoplankton
Christophe Six
![Page 2: Methods of Photosynthesis Spectrometry For Phytoplankton Christophe Six.](https://reader033.fdocuments.in/reader033/viewer/2022052509/56649d0b5503460f949df780/html5/thumbnails/2.jpg)
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.
![Page 3: Methods of Photosynthesis Spectrometry For Phytoplankton Christophe Six.](https://reader033.fdocuments.in/reader033/viewer/2022052509/56649d0b5503460f949df780/html5/thumbnails/3.jpg)
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
![Page 4: Methods of Photosynthesis Spectrometry For Phytoplankton Christophe Six.](https://reader033.fdocuments.in/reader033/viewer/2022052509/56649d0b5503460f949df780/html5/thumbnails/4.jpg)
Absorbance of molecules and molecular complexes
.Understanding photophysics and photobiology
.Very useful for assays
Using colorimetric assays
![Page 5: Methods of Photosynthesis Spectrometry For Phytoplankton Christophe Six.](https://reader033.fdocuments.in/reader033/viewer/2022052509/56649d0b5503460f949df780/html5/thumbnails/5.jpg)
Concept of the absorbance measurement
A = log (I0 / I)
I0 I>
Sample PhotomultiplicatorLight Source
Absorbance
T = I / I0
Transmittance
A = -log T
![Page 6: Methods of Photosynthesis Spectrometry For Phytoplankton Christophe Six.](https://reader033.fdocuments.in/reader033/viewer/2022052509/56649d0b5503460f949df780/html5/thumbnails/6.jpg)
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 :
![Page 7: Methods of Photosynthesis Spectrometry For Phytoplankton Christophe Six.](https://reader033.fdocuments.in/reader033/viewer/2022052509/56649d0b5503460f949df780/html5/thumbnails/7.jpg)
Single beam spectrophotometers
or monochromator
(nm)
D.O.
400 500
![Page 8: Methods of Photosynthesis Spectrometry For Phytoplankton Christophe Six.](https://reader033.fdocuments.in/reader033/viewer/2022052509/56649d0b5503460f949df780/html5/thumbnails/8.jpg)
. 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
![Page 9: Methods of Photosynthesis Spectrometry For Phytoplankton Christophe Six.](https://reader033.fdocuments.in/reader033/viewer/2022052509/56649d0b5503460f949df780/html5/thumbnails/9.jpg)
Double beam spectrophotometers
ReferenceCuvette
SampleCuvette
I0
I
Chopper
Chopper
Monochromator
![Page 10: Methods of Photosynthesis Spectrometry For Phytoplankton Christophe Six.](https://reader033.fdocuments.in/reader033/viewer/2022052509/56649d0b5503460f949df780/html5/thumbnails/10.jpg)
. 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
![Page 11: Methods of Photosynthesis Spectrometry For Phytoplankton Christophe Six.](https://reader033.fdocuments.in/reader033/viewer/2022052509/56649d0b5503460f949df780/html5/thumbnails/11.jpg)
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.
![Page 12: Methods of Photosynthesis Spectrometry For Phytoplankton Christophe Six.](https://reader033.fdocuments.in/reader033/viewer/2022052509/56649d0b5503460f949df780/html5/thumbnails/12.jpg)
=> 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
![Page 13: Methods of Photosynthesis Spectrometry For Phytoplankton Christophe Six.](https://reader033.fdocuments.in/reader033/viewer/2022052509/56649d0b5503460f949df780/html5/thumbnails/13.jpg)
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
![Page 14: Methods of Photosynthesis Spectrometry For Phytoplankton Christophe Six.](https://reader033.fdocuments.in/reader033/viewer/2022052509/56649d0b5503460f949df780/html5/thumbnails/14.jpg)
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
![Page 15: Methods of Photosynthesis Spectrometry For Phytoplankton Christophe Six.](https://reader033.fdocuments.in/reader033/viewer/2022052509/56649d0b5503460f949df780/html5/thumbnails/15.jpg)
Measuring absorbance in a diffusing sample
=> A
Bringing the detector nearer to the cuvette
Increasing the surface of the detector
![Page 16: Methods of Photosynthesis Spectrometry For Phytoplankton Christophe Six.](https://reader033.fdocuments.in/reader033/viewer/2022052509/56649d0b5503460f949df780/html5/thumbnails/16.jpg)
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
![Page 17: Methods of Photosynthesis Spectrometry For Phytoplankton Christophe Six.](https://reader033.fdocuments.in/reader033/viewer/2022052509/56649d0b5503460f949df780/html5/thumbnails/17.jpg)
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
![Page 18: Methods of Photosynthesis Spectrometry For Phytoplankton Christophe Six.](https://reader033.fdocuments.in/reader033/viewer/2022052509/56649d0b5503460f949df780/html5/thumbnails/18.jpg)
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
![Page 19: Methods of Photosynthesis Spectrometry For Phytoplankton Christophe Six.](https://reader033.fdocuments.in/reader033/viewer/2022052509/56649d0b5503460f949df780/html5/thumbnails/19.jpg)
Fluorescence: what is it ?
Stokes shift
![Page 20: Methods of Photosynthesis Spectrometry For Phytoplankton Christophe Six.](https://reader033.fdocuments.in/reader033/viewer/2022052509/56649d0b5503460f949df780/html5/thumbnails/20.jpg)
. 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)
![Page 21: Methods of Photosynthesis Spectrometry For Phytoplankton Christophe Six.](https://reader033.fdocuments.in/reader033/viewer/2022052509/56649d0b5503460f949df780/html5/thumbnails/21.jpg)
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
![Page 22: Methods of Photosynthesis Spectrometry For Phytoplankton Christophe Six.](https://reader033.fdocuments.in/reader033/viewer/2022052509/56649d0b5503460f949df780/html5/thumbnails/22.jpg)
Diagrammic representation of a spectrofluorimeter
Xenon lamp
Lens
LensLens
Slit
Entrance Slit Exit slit
Photomultiplicator
Sample
Mirror
shutter Monochromator
Monochromator
![Page 23: Methods of Photosynthesis Spectrometry For Phytoplankton Christophe Six.](https://reader033.fdocuments.in/reader033/viewer/2022052509/56649d0b5503460f949df780/html5/thumbnails/23.jpg)
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
![Page 24: Methods of Photosynthesis Spectrometry For Phytoplankton Christophe Six.](https://reader033.fdocuments.in/reader033/viewer/2022052509/56649d0b5503460f949df780/html5/thumbnails/24.jpg)
Fluorescence of marine picocyanobacteria : Synechococcus spp.
![Page 25: Methods of Photosynthesis Spectrometry For Phytoplankton Christophe Six.](https://reader033.fdocuments.in/reader033/viewer/2022052509/56649d0b5503460f949df780/html5/thumbnails/25.jpg)
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
![Page 26: Methods of Photosynthesis Spectrometry For Phytoplankton Christophe Six.](https://reader033.fdocuments.in/reader033/viewer/2022052509/56649d0b5503460f949df780/html5/thumbnails/26.jpg)
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
![Page 27: Methods of Photosynthesis Spectrometry For Phytoplankton Christophe Six.](https://reader033.fdocuments.in/reader033/viewer/2022052509/56649d0b5503460f949df780/html5/thumbnails/27.jpg)
PAM Fluorimetry and photosynthetic organisms
Diving-PAM©
Monitoring-PAM© Junior-PAM©
Multicolor-PAM©
![Page 28: Methods of Photosynthesis Spectrometry For Phytoplankton Christophe Six.](https://reader033.fdocuments.in/reader033/viewer/2022052509/56649d0b5503460f949df780/html5/thumbnails/28.jpg)
PAM Fluorimetry and photosynthetic organisms
Objectif : étudier la régulation de l’activité du photosystème II
![Page 29: Methods of Photosynthesis Spectrometry For Phytoplankton Christophe Six.](https://reader033.fdocuments.in/reader033/viewer/2022052509/56649d0b5503460f949df780/html5/thumbnails/29.jpg)
PAM Fluorimetry and photosynthetic organisms
Objectif : étudier la régulation de l’activité du photosystème II
Absorbed light energy
=
Fluorescence energy+
Photochemistry energy+
Heat energyAntenna
![Page 30: Methods of Photosynthesis Spectrometry For Phytoplankton Christophe Six.](https://reader033.fdocuments.in/reader033/viewer/2022052509/56649d0b5503460f949df780/html5/thumbnails/30.jpg)
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
![Page 31: Methods of Photosynthesis Spectrometry For Phytoplankton Christophe Six.](https://reader033.fdocuments.in/reader033/viewer/2022052509/56649d0b5503460f949df780/html5/thumbnails/31.jpg)
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
![Page 32: Methods of Photosynthesis Spectrometry For Phytoplankton Christophe Six.](https://reader033.fdocuments.in/reader033/viewer/2022052509/56649d0b5503460f949df780/html5/thumbnails/32.jpg)
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
![Page 33: Methods of Photosynthesis Spectrometry For Phytoplankton Christophe Six.](https://reader033.fdocuments.in/reader033/viewer/2022052509/56649d0b5503460f949df780/html5/thumbnails/33.jpg)
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
![Page 34: Methods of Photosynthesis Spectrometry For Phytoplankton Christophe Six.](https://reader033.fdocuments.in/reader033/viewer/2022052509/56649d0b5503460f949df780/html5/thumbnails/34.jpg)
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
![Page 35: Methods of Photosynthesis Spectrometry For Phytoplankton Christophe Six.](https://reader033.fdocuments.in/reader033/viewer/2022052509/56649d0b5503460f949df780/html5/thumbnails/35.jpg)
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
![Page 36: Methods of Photosynthesis Spectrometry For Phytoplankton Christophe Six.](https://reader033.fdocuments.in/reader033/viewer/2022052509/56649d0b5503460f949df780/html5/thumbnails/36.jpg)
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
![Page 37: Methods of Photosynthesis Spectrometry For Phytoplankton Christophe Six.](https://reader033.fdocuments.in/reader033/viewer/2022052509/56649d0b5503460f949df780/html5/thumbnails/37.jpg)
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
![Page 38: Methods of Photosynthesis Spectrometry For Phytoplankton Christophe Six.](https://reader033.fdocuments.in/reader033/viewer/2022052509/56649d0b5503460f949df780/html5/thumbnails/38.jpg)
PAM Fluorimetry : light response curves
Example of application : Prochlorococcus ecotypesP
SII
rE
TR
Irradiance (µmol photons/m²/s)