Laser Remote Sensing

Laser Remote Sensing http://www.nat.vu.nl/envphysexp

Introduction

Laser remote sensing (LRS) is the general term to describe the procedure to gain physical information on systems from a large distance with the aid of lasers. The technique is also referred to as LIDAR: light detection and ranging. The principle is simple: light from a laser strikes the system of interest and the returning light is detected by a telescope and analysed. The technology is developing from the 1970's but especially the rapid advances in laser technology and computers over the past ten years opened up a wide variety of applications. Most commonly LRS is used in atmospheric and environmental studies applied from an airplane, minivan or satellite, measuring concentrations of pollutants, mapping cloud formation and monitoring agricultural crops for stress. The latter example is being dealt with in the experiment chosen here. This is a special case of LRS since instead of investigating scatter properties one monitors the laser-induced fluorescence (LIF) of photosynthetic systems.

Photosynthesis is the process by which plants, algae, cyanobacteria and some other bacteria chemically fix the energy from solar light. This process involves a high number of different protein complexes and reaction steps, all tuned for maximal energy conversion (meaning minimal heat loss) and directionality. Photosynthesis is optimised for low-light conditions and several mechanisms exist to dispose of excess energy. Here, we will investigate the fluorescence pathway.

Learning objectives:

Apply light spectroscopy on a living biological system. Investigate the possibilities and limitations of a new, important technology. Learn practical applications of laser spectroscopy, lock-in modulation technique and photomultipliers.

1 Contents:

Theory Photosynthesis Interaction of light with biological matter Fluorescence Fluorescence of plants

Experimental design and measurement procedure System design Illumination Detection

Signal processing and Data analysis Signal processing Optimisation Data processing and analyses

Example Introduction Measurements Analyses

Complications

Suggestions for other experiments

Teachers section Guidance and student activities Maintenance requirements Software suggestions Costs

Literature

2 Theory

Photosynthesis Interaction of light with biological matter Fluorescence Fluorescence of plants

Photosynthesis

Photosynthesis efficiently converts solar energy into a chemical form that can be used by living organisms. The primary processes of photosynthesis are the absorption of light by chromophores or pigments (= light absorbing molecules), subsequent energy transfer among many pigments and electron transfer, which finally results in proton translocation over a membrane. The photosynthetic membrane then acts like a biological battery, enabling to use the energy stored as a potential difference to create energy rich chemical bonds, which are used to drive life processes. In figure 2.1 the reaction steps of this process are schematically drawn. The photosynthetic apparatus consists of a membrane-associated network of interconnected proteins that serve to hold the pigments in an ordered state. The different reaction steps are compartmentalised for regulation purposes and to ensure uni-directionality of the reactions. Light absorbing and energy transferring pigments are embedded in Light Harvesting Complexes (LHC's). When such a pigment absorbs a photon the excited state energy is transferred to a nearby pigment. Eventually such excitation reaches pigments of another type of photosynthetic complex: a Reaction Center (RC). Instead of further energy transfer, electrons are transferred within this complex from one side of the complex to the other side. These electrons are collected by carrier pigments, which also take up, and subsequently transport, protons. These can flow backwards mechanically whereby the energy is stored chemically. light

LHC RC protonpump protonpump/ chemical fixation Figure 2.1. simplified diagram of the photosynthetic process. The different functional complexes are represented as blocks within a membrane. These complexes consist of many proteins and pigments. Energy transfer routes are shown as blue arrows, electron transfer as red arrows, proton transfer as green arrows and chemical fixation as a black arrow. The chromophores of the plant RC’s and LHC’s are chlorophylls or chlorophyll-like pigments and

3 carotenoids. By using different types of chlorophylls and carotenoids a wider window of absorption is created and energy and electrons can be transferred in an organised fashion. The most prominent pigments in plant photosynthesis are the chlorophyll's a and b and -carotene. In figure 2.2 the structure and absorption spectrum of the most abundant LHC in plant photosynthesis is shown: the LHC2 complex. Roughly, the absorption spectrum is a sum of those of the constituting pigments. Chlorophyll a has absorption maxima around 670 and 430 nm and chlorophyll b at 650 and 470 nm. The absorption spectrum of the carotenoids overlaps with that of the chlorophyll's in the blue/UV part starting at 500 nm.

n o i t p

r o s b A

300 400 500 600 700 800 Wavelength (nm)

Figure 2.2. left: a structural model of the LHC2. The protein is depicted in dark grey and the 2 carotenoids (crossed in the center of the complex) as light grey. Chlorophyll’s a are blue and chlorophyll’s b yellow. Right: Absorption spectrum of the LHC2 complex at room temperature. (figure used with permission from dr. J. Salverda, thesis Vrije Universiteit).

In plants there are two clusters of RC's and LHC's, who work in series in order to maximise the energy conversion of incoming photons into energy rich chemical bonds. These clusters are called photosystems and are named photosystem 1 (PS1) and photosystem 2 (PS2). PS1 and PS2 consist of many antennae or light harvesting complexes (LHC’s) which surround a few reaction centers (RC’s). The above-depicted LHC2 complex for instance is mainly associated with the PS2 complex. The two photosystems contain about 400 chlorophyll molecules; the vast majority of these belong to a LHC transferring their excitation to the RC’s. For all plants, the yield of photosynthesis can approach the theoretical maximum value, even under field conditions. However, because of fluctuations in light intensity in environmental conditions, photosynthesis frequently occurs at rates much below the maximum capacity provided by the available

4 light, and under such conditions excess light has the potential to severely and irreversibly damage the pigment-protein complexes, leading to cell death. Therefore plants have evolved various photoprotective mechanisms, one of which actually converts excess absorbed radiation into heat, which can be harmlessly dispersed.

Interaction of light with biological matter

Matter can interact with an incoming lightbeam in various manners: light can be defracted, scattered and absorbed. Here we will deal with the absorption of light and the reactions, which follow absorption. Biological molecules have a high tendency to absorb UV radiation. The amino- and nucleic acids of respectively proteins and DNA show distinct transitions in the near UV (200-300 nm). Many proteins include groups other than amino acids: chromophores. These are often, but not always covalently linked to a polypeptide chain and absorb in the visible region: 400-750 nm. Examples are flavins, iron porphyrins, rhodopsin and chlorins. The latter group contains the chlorophyll molecules, which are the dominant chromophores, or pigments in photosynthesis. The absorption spectra of biological chromophores are considerably broader than the theoretically cal- culated homogeneously- or lifetime broadened linewidth, which is only dependent on the excited state lifetime of the molecule. The reason for this is first of all the occurrence of vibronic transitions accompanying an electronic absorption. Depending on temperature this leads to a number of variations of transitions for every single chromophore. The other reason is inhomogeneous broadening which depend on the differences in local environment of every chromophore, which will influence both the electronic and vibronic transitions. When measuring the absorption of a bulk sample every chromophore will thus have a slightly different transition- and vibrational energy, leading to broadened spectra. Photosynthesis starts off with the absorption of a photon by a chlorophyll or carotenoid molecule, which will be in the singlet-excited state (total spin S=0). An excited state is always unstable and will eventually return to the ground state. The ways by which excitation energy can be lost are: spontaneous and stimulated emission, internal conversion, decay to a triplet state (total spin S=1), energy transfer and photochemistry. It is clear that for efficient photosynthesis the latter two processes should be highly favourable over the others. From all the processes leading to loss of excited states, the emission of light is the most easily detectable parameter.

5 Fluorescence

The emission of light from an excited electronic state is generally referred to as fluorescence. When the fluorescent molecule is the same as the absorbing molecule the lineshape of the fluorescence spectrum is expected to be a mirror image of the absorption spectrum because the same electronic and vibrational transitions are being probed. However, after excitation the molecule looses some energy by internal vibrations. Therefore the fluorescence spectrum is shifted to lower energy than the absorption spectrum. The fluorescence characteristics are also strongly dependent on the solvent. Since the excited state interacts often differently with the solvent than the ground state, more spectral shifts can be induced. This can also cause the fluorescence linewidth to be broader than the absorption linewidth because of an increased heterogeneity in the excited state. Because fluorescence occurs at lower frequency than that of the incident light it can easily be detected since there is no background signal

(scatter) from the excitation source. Therefore it is often possible to record fluorescence at concentrations two orders of magnitude lower than required for recording absorption spectra. If a sample is illuminated to produce fluorescent excited states and the light is switched off the fluorescence decays. This decay is generally first order and the intensity of fluorescence [IF] would be characterised by the following rate law: d  I   – ( E x c ) = k  E x c  F d t F equation 2.1 with [Exc] the concentration of fluorescent states

The decay would be exponential with time with a rate constant kF. The inverse of this rate constant is called the radiative lifetime F:

F = 1 / kF equation 2.2

Thus, the radiative lifetime would be the lifetime of the emission if there would be no other decay possibilities of the excited state. Note that the lifetime refers to a bulk property that measures how long an average molecule exists in a particular state. In practice, however, fluorescence is just one of the ways by which the excited state returns to the ground state. The overall rate constant (k) for the depopulation of the excited state is obtained by summing the individual rate constants for all the competing processes (which are assumed to be simple processes like in eq. 2.1): equationk = k F +2.3 k i

6 in which ki represents the various competing radiationless decay processes of the excited state.

The overall lifetime of the excited state ( ) is again the reciprocal of the overall rate constant:

 = 1 / k equation 2.4

The quantum yield (FF) of fluorescence is the fraction of absorbed photons that lead to fluorescence. This is the number of photons fluoresced divided by the number absorbed. This again equals the ratio of the rate of fluorescence to the rate of absorbance. Since in a steady state the rate of absorbance equals the rate of decay of the excited state, the quantum yield can be described as: k   = ------F------= ---- F  k F + k i F equation 2.5

Absolute values of F are difficult to measure experimentally because instrument correction factors have to be known. In practice, they are obtained by comparison with a standard with known quantum yield.

Fluorescence of green plants

Fluorescence spectroscopy provides an excellent means to investigate photosynthetic processes in green plants. Spectrally the photosystems PS1 and PS2 can be distinguished since the PS1 antenna contains a number of chlorophylls that absorb at longer wavelength (less energy) than most of its other chlorophylls and of the chlorophylls embedded in the PS2 complex. Excitation energy in the PS1 antenna tends to focus on these special chlorophylls, which give rise to fluorescence at considerably longer wavelengths than those of PS2. The normal circumstances for a plant is a low-light environment because most of its leaves will be in the shade of the few highest ones. In a so called dark-adapted plant the fluorescence quantum yield is very low. The main reason is that the usual decay channels of the singlet-excited state of chlorophyll (fluorescence, internal conversion into heat, intersystem crossing into a triplet state with total spin S=1) are circumvented by the energy and charge separation processes of photosynthesis. When the amount of available light increases, however, the photosynthetic pathway becomes saturated and the plant reaches the maximal rate at which CO2 and H2O can be converted into O2 and organic

7 matter. Under these conditions, the fluorescence yield increases, because the excited state of the chlorophylls can not decay anymore photosynthetically and the decay routes via the other processes (fluorescence, internal conversion, intersystem crossing) will increase. With even more light, however, the fluorescence yield decreases again. The reason is that all decay channels become saturated including the occurrence of a singlet-to-triplet conversion of the excited state. These amounts of light are quite harmful for the plant, because the triplet state of chlorophyll can react with oxygen (which is a triplet in its ground state) to form singlet oxygen, an extremely reactive and hazardous species that will react with many components of the plant cell. The deleterious effects of chlorophyll triplets are circumvented because of the presence of carotenoids, which not only serve to increase the absorption cross section but which also effectively can take over the triplet state of the chlorophylls. The energetics are such that a carotenoid triplet can not react with singlet oxygen. Since there are more chlorophyll's then carotenoids this quenching is limited to a certain amount of excited chlorophylls and thus to the light intensity.

Therefore all plants take extra precautions under high-light conditions by changing the antenna structure somewhat, with as final result a strongly increased decay by internal conversion. This prevents accumulation of triplet states, and has as a second effect that also the fluorescence yield decreases rather strongly. The ability of a plant to show these types of fluorescence quenching depends on an intact photosynthetic apparatus; single chlorophylls will not show this kind of behaviour. Thus, the fluorescence yield of green plants shows a number of dynamic variations that largely depend on the specific light conditions and on the capability of the plant to react on changes therein. By means of LRS one increases the light intensity locally and in a controlled condition. In that way one can obtain information on the quality of the vegetation investigated.

Experimental design and measurement procedure

System design Illumination Detection

System Design

8 An LRS system in principle consists of a laser as excitation source and a telescope for detection. Excitation and detection are commonly from a very similar angle in order to discriminate between excitation and other effects and to use the same alignment when the set-up moves. By means of a set of mirrors the excitation and detection light are guided such as to come from the same direction. Because detection is done over large distances the measured signals will be very small there fore great care has to be taken to obtain a good signal-to-noise ratio (S/N). The set up for studying Laser induced fluorescence of green plants (see figure 3.1) will be discussed in the following sections .

Illumination

Intense and monochromatic light is needed to excite over great distances and to discriminate between the several processes induced by light in plants. Ideally this is provided by a wide range tuneable laser but one with a few lines like an Argon laser will work too. Depending on the type of laser and the interest of research a colour is chosen from the laser. While aligning the set-up the laser is set to a minimal output power. The outcoming laser beam is guided through a pinhole to a positive lens (L1 in figure 3.1) and into the detectionarm by a mirror (M1). There a fixed mirror (M2) projects the beam in a forward direction. A larger or smaller surface of a leave of a plant can be illuminated by adjusting the distance between the two lenses.

Detection

Fluorescence is detected by means of a large parabolic mirror which focuses incoming light onto a fixed mirror (M3) which guides the light outside the excitation/detection arm onto a monochromator / photomultiplier(PM) system. So the fluorescence can be measured as a function of the wavelength. For a certain setting of the PM and monochromator a maximal signal can be obtained by tuning mirror M1.

Because the scattered excitation light is very intense great care has to be taken not to set the monochromator on the excitation wavelength, to avoid saturation of the PM. Since the scattered excitation light is always at higher energy than the fluorescence light they do not interfere in the fluorescence spectrum.

9 Signal processing and Data analysis Signal processing Optimisation Procedure Data processing and analysis

Signal Processing

In order to discrimate between fluorescence induced by the excitation laser and other light sources and to be able to detect very small signals the laser light is chopped by a chopper with a certain frequency; the output of the photomultiplier (PM) is fed into a lockin amplifier (LIA) set at the same frequency for ultimate detection. The PM signal is lead simultaneously into an oscilloscope for optimisation purposes. When optimising the signal one first only checks the signal on the oscilloscope. The signal- to-noise (S/N) ratio is visible as the variations of the signal compared to the depth of modulation. The output signal of the LIA can be collected by computer via a data acquisition card (DAC) for instance spectrally or timeresolved.

Optimisation

Besides optimising the signal by aligning the excitation and detection parts by means of the two lenses and mirrors, the signal can also be optimized by choosing a good combination of the chopper frequency with the RC-time of the LIA in relation to the timescale of the physical process of interest.

Data processing and analysis

At the physics lab of the VUA a LABVIEW programme exists which can plot the fluorescence intensity or time dependency versus wavelength.

10 Spectra can be analysed by means of a data analysis programme. Spectra can be fitted with gaussian functions to discriminate between the two different photosystems and one can derive the peak positions, maxima values and the full-width-at-half-maximum (FWHM) of these gaussians. These parameters may or may not change according to illuminating time, dark period or laserpower. Variations can be investigated for different types of plants.

Example Introduction

Measurements

Analyses

Introduction

Here we present a start up experiment to help students to get accustomed with the set-up and plant photosynthesis. We will measure the fluorescence dependency on the wavelength of excitation. This experiment can very easily be extended by measuring the power dependency of the fluorescence intensity and possible photodamage effects. Note: working with lasers can be very hazardous. Never look directly into the outcoming laserbeam! When aligning the set-up make sure the laserbeam can not harm other people entering or exiting the lab!

Starting up: Start with the laser at low power: 25 mW. There are two shutters: one connected to the PM, keep this first closed and open the other shutter between lens L1 (see figure 3.1) and mirror M1 . Align L1 and M1 such that (part of) a leave is illuminated. The shutter of the PM can be opened when no extra resistance is connected to the PM. For fast timeresolved measurements one might consider placing an extra resistance parallel to the internal (10 Mohm) one. Check specifications of the particular PM for details.

Check colour fluorescence

11 A bright green or blue spot can be seen on the leave. One expects more fluorescence with blue excitation then with green (why?, see below). Check whether the colour of fluorescence indeed being red by looking at the scattered light from the leave with coloured glass filters.

Optimise -check the oscilloscope for a signal from the chopper. - Scan the monochromator to 690 nm, open the PM shutter - The PM starts working at a voltage of ~ 600V. Turn to 800 V and a fluorescence signal should be visible on the second channel of the oscilloscope - The signal can be first optimised by rotating M1. Secondly by increasing the power of the laser. - Adjust L1 for an illuminating surface on a leave of 5x5 cm2. - Set the LIA as sensitive as possible and set the phase to maximize the signal.

Measurements.

514 nm excitation Tune the laser to the 514 nm line (green) and make a scan from 640 to 800 nm. Figure 5.1 shows a recorded fluorescence spectrum with 514 nm excitation, ranging from 640 to 800 nm. This spectrum is obtained with the following set up characteristics: laserpower: 25 mW, PM 700 V, chopper frequency: 187 Hz, RC-time LIA: 300 ms, sensitivity LIA: 100 mV

1.6 1.4 1.2 1.0 0.8 0.6 0.4 0.2

640 660 680 700 720 740 760 780 800

Figure 5.1 room temperature fluorescence spectrum of a plant leave obtained with 514 nm excitation light.

There are two distinguishable peaks visible, around 690 nm and 730 nm. These corresponds with the two photosystems of plants, respectively PS2 and PS1. Since plants are green none of these PS's should absorb the green light to a high extent (why?, see below). Still, some green light is absorbed by the pigments of both photosystems leading to far-red fluorescence.

12 488 nm excitation Tune the laser to the 488 nm line (blue) and scan the same wavelength region again. figure 5.2 shows the fluorescence spectrum with 488 nm excitation, same wavelength region and settings as before.

640 660 680 700 720 740 760 780 800

Figure 5.2 room temperature fluorescence spectrum of a plant leave obtained with 488 nm excitation light.

Again two distinct peaks are visible, similar as in figure 5.1. The yield of fluorescence increases for both photosystems but most for PS2. Blue light is absorbed by both photosystems as expected by the colour of the leaves (why?, see below).

Comparing the fluorescence spectra with the two excitation wavelengths. Figure 5.3 shows the spectra from fig. 5.1 and 5.2. It is important that both spectra are measured with similar settings of the set-up since these directly influence the actual value of the fluorescence measured.

3 ] . u . a [

y t i 2 s n e t n I

640 660 680 700 720 740 760 780 800 Wavelength [nm]

Figure 5.3 comparison of the fluorescence spectra of a plant leave obtained with 488 nm (blue) and 514 nm (green) excitation light.

13 Leaves are green because red and blue light are absorbed to a large extent while green light is not; (it's mainly scattered). When light is not absorbed it can also not lead to fluorescence; therefore 488 nm excitation leads to a higher fluorescence yield than the 514 nm excitation. But also the ratio of PS1 to PS2 fluorescence changes with colour. This is because the amount of chlorophyll a and b is different for both photosystems. PS2 has a higher chlorophyll b/chlorophyll a (Chl b/Chl a) ratio then PS1. And since chlorophyll b absorbs more 488 nm light than chlorophyll a, PS2 has a higher fluorescence yield at this excitation wavelength. Most likely, the difference in pigment content is responsible for the difference shown here.

Analyses

From the fluorescence spectra it can be observed that the two fluorescence bands of PS1 and PS2 overlap to a great extent. For a more quantitative analysis a fitting routine is needed.

As a model we both represent the fluorescence bands of PS1 and PS2 as single gaussians and fit both fluorescence spectra. The results are shown in figure 5.4 and 5.5.

Figure 5.4 result from fitting the fluorescence spectrum with 488 nm excitation with two independent gaussian functions. Data: red dots, fit result: blue line, gaussians: red line. The residuals (the measured spectrum minus fit result) are shown at the top.

14 Figure 5.5 result from fitting the fluorescence spectrum with 514 nm excitation with two independent gaussian functions. Data: red dots, fit result: blue line, gaussians: red line. The residuals are shown at the top.

fit results: table 5.1 results from fitting the fluorescence spectra with two gaussian functions.

Excitation wavelength Maximum wavelength Amplitude (a.u) 2

488 nm 684.0 (error) 3.11 (error) 0.60

725.9 (error) 2.07 (error)

514 nm 685.3 (error) 1.26 (error) 0.15

726.4 (error) 1.45 (error)

From the fit results one could conclude that not only the fluorescence yield is influenced by the wavelength of excitation but also the maxima of fluorescence of both photosystems. Especially PS2 experiences a notable, 1.3 nm, difference. From fluorescence measurements on isolated complexes it is known that too high concentrations can lead to self-absorption: the fluorescent light from one complex is absorbed again by another one. The concentration of complexes in leaves is so high that self- absorption occurs very frequently. Since the absorption is blueshifted compared to the fluorescence, the

15 blue shift of the 488 nm excitation fluorescence spectrum compared to the 514 nm excitation fluorescence spectrum could indicate that PS2 exhibits more self-absorption at 488 nm excitation then at 514 excitation. The reason for this difference could originate from the much higher number of excited complexes with 488 nm excitation compared to 514 nm excitation. Although smaller, a similar blue shift can be observed for PS1. This is in line with the smaller difference in fluorescence yield between the two excitation wavelengths for PS1. Also this self-absorption phenomenon can thus be deduced to originate from the difference in Chl b/Chl a ratio of the two photosystems. At 514 nm we excite at the red edge of the absorption bands of both complexes and probe mainly carotenoids and the lowest energy part of the distribution in energy of absorption of chlorophyll b. We expect for both complexes a small amount of self-absorption. At 488 nm excitation this is very different: now a large part of chlorophyll b is excited (and carotenoids, and low energy absorbing chlorophyll a pigments) and the self-absorption phenomenon becomes most prominent for the PS2 complex which contains large amounts of this pigment.

But whether or not the numbers presented here are within the error margin and can be used to confirm the above described mechanism cannot be concluded from a single measurement. In order to investigate the reproducibility more data should be obtained. Both on the same illuminated part of the plant as on different parts.

Complications

The trick of LRS is optimisation of the signal. The excitation/detection sources are generally not land based but located in moving objects. Besides, the object of observation moves too because of the wind. In the lab conditions are such that these considerations have not to be taken into account. Still, it is not recommended to illuminate the same surface constantly. One has to optimise the alignment such that a slight movement of the entire set-up does not affect the measuring capabilities. A simple way of increasing the S/N ratio is increasing the power output of the laser. For more sophisticated experiments one has to bear in mind possible photodamage effects though.

Suggestions for other experiments

16 It should be noted that the outcome of LRS experiments as described here is not fully investigated and/or understood. The student’s or researcher's aim is foremost to find a standard which depicts a 'healthy' plant. Then, the influence of parameters like laser intensity, light-dark periods (ranging from seconds to hours), background illumination (different colours) or plant-type variations (desert plants versus forest plants) for instance can be investigated. We suggest starting with the following experiments: 1. Simple dynamics: scan a new part of a leave. Measure fluorescence from 800 to 640 nm. Then repeat the measurement. Investigate if the illumination in the first experiment has any effect on the fluorescence yield and positions of maxima in the second experiment. Investigate if possible effects are equally observed for PS1 or PS2.

2. Measure real dynamics.

The time dependency of the fluorescence can be recorded in different time intervals. Compare long illuminated leaves with fresh ones. Investigate the effect of dark periods between measurements. Define the temporal behaviour of fluorescence of 'intact' leaves. (note: first seconds are thought to be important here).

Teachers section

Guidance and students activities.

This experiment is one of the experiments of course XYZ in the third year of the undergraduate student lab at the Vrije Universiteit Amsterdam (VUA). (see Overview of Students Laboratories). The students perform this experiment in 10 afternoons in the lab. They are supposed to spend another 5 afternoons for writing the report and performing possible additional experiments. In contrast to many other experimental studies performed by students during their education this experiment has no uniform outcome. The challenge is therefore to have students perform some pilot experiments as described above which enables them to define a subject of investigation and the possible limitations to ascribe certain effects to one free parameter.

17 As a start, students read parts of the relevant topics. The teacher can describe some aspects of the topics and of the set-up. The students have to align the experimental set-up themselves. It is assumed that the students have experience with the different components of the set-up (photomultiplier, lock-in amplifier, tuneable laser system) but should be given one or two hours to refresh their memory and get accustomed with the specific components used here. The teacher should warn them about the laser system and its hazards as well as the photomultiplier and its limitations. Some signal should be found within 2 hours, if not the tutor should advise about aligning the set-up some more. The experiment consists of the following steps: - Students will start with a pilot experiment as described above. The many free parameters which influences the fluorescence yield and/or temporal behaviour can be investigated. - Students choose the experimental problem they will investigate. Formulating hypothesis and expectations. Tutor helps the students to clarify the used concepts and to link theory to practice.

- Calculation and interpretation of preliminary results.

- Preparing work plan. - Work plan discussion between students and tutor. Determination of definite experimental problem and planning of activities. - Final measurements. - Interpretation of results. - Writing report. - Possibility for some more experiments when needed

Maintenance Requirements

Software Suggestions A LabVIEW ® program is used for the data acquisition. This program controls a digital spectrum analyser and collects the measured data. For data analyses the physics lab uses the IGOR programme.

Literature

Environmental Physics

18 Environmental Physics 2nd Edition (1999). Egbert Boeker and Rienk van Grondelle.John Wiley and Sons Ltd., Chichester, England. energy from photosynthesis: chapter 4.4.4 spectroscopy in environmental research: chapter 7

Molecular spectroscopy Physical Chemistry, Principles and Applications in Biological Sciences (2002). Tinoco. I., Sauer, K. Wang, J. C. and Puglisi J. D., Prentice-Hall, New Jersey, USA; absorption and fluorescence of biological molecules: chapter 10

Physical chemistry Physical Chemistry (1994) P. W. Atkins, Oxford University Press molecular quantum mechanics chapter 12 molecular orbital theory chapter 14 molecular symmetry chapter 15 electronic transitions chapter 17

Photosynthesis Molecular Biology of the Cell, fourth edition (2002). Alberts. B., Lewis J., Raff M., Roberts, K., Walter P., Garland Science Publishing introduction to the chloroplast: chapter 14 Photosynthetic excitons Photosynthetic excitons (2000) van Amerongen, H., Valkunas, L. and van Grondelle R., World Scientific Publishing Co. Pte. Ltd., Singapore photosynthetic pigments chapter 1 excited state energy transfer: chapter 1 excitonic coupled pigments in photosynthesis chapters 2 and 3

Web pages Laser safety: http://www.nat.vu.nl/~laser/safety.html

Photosynthesis research at the VU biophysics group: http://www.nat.vu.nl/bio/research-en.html

19 Photosynthesis on Internet: http://www.life.uiuc.edu/govindjee/photoweb/

Learning photosynthesis: http://photoscience.la.asu.edu/photosyn/

Introduction to LIDAR: http://pcl.physics.uwo.ca/pclhtml/introlidar/introlidarf.html

LIDAR links: http://www.lidar.com/links.htm