Laser Remote Sensing
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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!
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
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.
- 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.
The 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
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.
The 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.
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. Consequently the a.u. in figure 5.1 - 5.5 and table 5.1 are all the same
Figure 5.3: Comparison of the fluorescence spectra of a plant leave obtained with 488 nm (blue) and 514 nm (green) excitation light.
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.
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.
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.
Table 5.1: Results from fitting the fluorescence spectra with two gaussian functions.
Exitation wavelength Maximum wavelength Amplitude [a.u.] c2 488 nm 684.0 (error) 3.11 (error) 0.60 nm 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 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.
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