Methods of Photosynthesis Spectrometry Christophe Six Dfinitions - - PowerPoint PPT Presentation

Master 2ème année : Sciences et Technologies Mention : Sciences de l’Univers, Environnement, Ecologie Spécialité : Océanographie et Environnement Marin Spécialité : Océanographie et Environnement Marin Unité d’Enseignement : Evolution du Phytoplancton Marin et Biogéochimie

Methods of Photosynthesis Spectrometry

Christophe Six

Définitions

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

• f systems transferring energy

Qualitative and quantitative studies of spectra derived from the interaction 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. (or of a whole unicell organism) in solution, function of the light wavelength. Spectrofluorimetry is an analytic, quantitative method that consists in measuring p y y , q g 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) /λ

E : Photon energy

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 .Very useful for assays

Using colorimetric assays Using colorimetric assays

Concept of absorbance and measurement

Sample Photomultiplicator Light Source

I0 I

>

Absorbance Transmittance

A = log (I0 / I) T = I / I0 A = -log T

Spectrophotometers

Components : .One or several light source(s)

Extended Visible (350-900 nm) : Tungsten, Halogen

Components :

( ) g g 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

• r monochromator

D.O.

• r monochromator

λ (

)

400 500

λ (nm)

400 500

Single beam spectrophotometers

. 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, solvant, etc) except the absorbing substance that you want to measure. It is actually rare to be able to use a perfect true blank but one should approach it as much as possible. approach it as much as possible.

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

V i l i t i ( t i l i id i t t ) Various colorimetric assays (proteins, nucleic acids, pigments, etc.)

. Main problems

The decrease of lamp intensity is not compensed In single wavelength mode, one cannot check for artefacts

I0 I

(fix) (measured) The making of these instruments is usually less careful

Double beam spectrophotometers

M h t

Reference Cuvette

Monochromator

I0

Chopper Sample Cuvette Chopper 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

(Eli i ti f l t b ti ) (Elimination of solvent absorption)

. Correction of the variations of the light sources

Artefacts

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

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

=> Aλ

. Other optical phenomenons linked to diffusion, reflexion and diffraction

• f light may also distort the measurement.

. Other optical phenomenons linked to diffusion, reflexion and diffraction

• f light may also distort the measurement.

Artefacts : Light diffusion

Turbid solutions cell suspensions . Turbid solutions, cell suspensions

=> Aλ

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

σS =

F( d, n)

λ4

=

Diffusion of Rayleigh

λ

d : Diameter of particules n : Refraction index

λ : Wavelength Diffusion also depends on :

• Particule concentration
• Particule shape

Impact of diffusion on absorption spectra

4,0E-11 5,0E-11

1

1 0E-11 2,0E-11 3,0E-11

y = 1 x4 Diffusion is λ-dependent

0,0E+00 1,0E-11

350 450 550 650 750 850 Longueur d'onde (nm) g ( )

=> Aλ ok => Aλ

Impact of diffusion on absorption spectra

Example : absorption spectrum of a phycoerythrin I

Spectrum with diffusion Fitting a correction curve Final spectrum

ce sorbanc Abs Wavelength (nm)

Measuring absorbance in a diffusing sample

=> Aλ

Bringing the detector Bringing the detector nearer to the cuvette Increasing the surface g

• f the detector

Measuring absorbance in a diffusing sample

Echantillon homogène Détecteur du photomultiplicateur

Homogeneous sample Light detector

DO (nm) DO (nm)

A

(nm) (nm) DO DO

B

Suspension de cellules

(nm) (nm)

Cell suspension

Source Rayon lumineux

DO (nm)

C

Light beam Light source and

lumineuse et monochromateur Sphère d’intégration

Integration sphere Light 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 Condensation on the cuvette Gaz formation (diffusion)

Sample drops on the outside of the cuvette . 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 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

Aλ = ελ . l . C

A : Absorbance (no unit) λ : Wavelength (nm) l : Optical path (cm) C : C

t ti ( l L 1)

λ λ

C : Concentration (mol L-1)

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

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

A = ε l C + ε l C + ε l C + + ε l C Aλ = ελ,1 . l . C1 + ελ,2 . l . C2 + ελ3 . l . C3 + … + ελ,n . l . Cn

. For l = 1 cm :

Aλ = ελ . C => C = A / ελ A = ε C + ε C + ε C + + ε C Aλ = ελ,1 . C1 + ελ,2 . C2 + ελ3 . C3 + … + ελ,n . Cn

Fluorescence: what is it ?

Stokes shift

Intensity of fluorescence emission

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

The measurement depends strongly on :

• The nature of the fluorescent system that is studied

y

• The device used to quantify fluorescence (light source intensity, optics configuration, etc.)

Need to use standard curves to quantify molecules by fluorescence Need to use standard curves to quantify molecules by fluorescence

. 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 (λ, 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 On average, there is 10 times less photons that hit the detector of a spectrofluorimeter than in a spectrophotometer

• A light source : Mercury or xenon lamp

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 Photomultiplicator Monochromator shutter Monochromator Monochromator Slit Mirror Lens Lens Sample

Emission and Excitation spectra of fluorescence

Monochromator scanning

Emission spectrum

Fix monochromator : Monochromator scanning all wavelengths Quantification of the fluorescence emitted ∼ 15 nm Fix monochromator : One given λ Emission fluorescence emitted by the excitation of a given λ Excitation Sample At which λ is the maximum of fluorescence emission of the compound ? 600 500 700 400 p Fix monochromator : One given λ

Excitation spectrum

Emission Monochromateur scanning all wavelengths Quantification of the fluorescence emitted many wavelengths λ Excitation Sample 600 500 700 400 600 500 700 400 Which λ gives rise to the fluorescence emission at a given λ ? (Excitation spectra are often similar to absorption spectra)

Fluorescence of marine picocyanobacteria : Synechococcus spp.

Marine phycoerythrins & spectrofluorimetry

. There are several types of phycoerythrins (PE) Excitation In the Emission Variable Excitation Emission In the blue-green region, at 500nm between 560-580 nm depending on the between 400 and 550 nm Emission at 580 nm

(for instance)

Excitation spectrum Emission spectrum

(for instance)

type of PE

One or two major maxima

495 545 600 500 700 400 600 500 700 400

Phycoerythrin structure and excitation spectra

Phycobiliprotein = Apoprotein + pigment Phycobiliprotein = Apoprotein + pigment Pigment = chromophore phycobilin One or two types of phycobilin are bound to marine phycoerythrins

Excitation spectrum

545

One or two major maxima

495 600 500 700 400