Supplementary material
Computer processing of data
Following taking the spectral pictures you want, the room light can be switched on
again. Transfer the pictures to your computer. Open a picture with ImageJ (this
software is freely downloadable for Windows, Macintosh or other systems from
http://rsb.info.nih.gov/ij/.) This allows you to extract data for a spectrum that can be
transferred to and plotted in Excel. The detailed procedure for this is given here. With
the cursor select a suitable rectangular part of your spectral image and note the
coordinates, so you can select the corresponding part of other pictures to be processed
later. Then go to "Analyze" in the top toolbar, and from the pull-down menu select
"Plot Profile". In the lower left corner of the picture that appears is a push-button
marked "List". Move your cursor to this push-button and press the mouse button. A
table with two columns of figures appears. Go to "File" in the top toolbar and "Save
as ..." (give a suitable name and save the table as a text file). Repeat this with all the
photos your have taken, and then exit from ImageJ.
Start Excel and open a worksheet. Pull down the "Data" menu and select "Get
external data" and from there select "Import text file". The rest is ordinary Excel work.
I summed values over the wavelength ranges occupied by the reflected laser light and
fluorescence, respectively, and divided by the initial values to plot the time course of
the changes as shown in Figure 3. The wavelength scale for the recorded spectra was
calibrated by removal of the leaf, pointing the hole in the box where the leaf had been
attached toward a fluorescent lamp (another other source with known emission lines
can also be used) and photographing a spectrum. Mercury emission lines are visible in
ordinary old-fashioned fluorescent lamp light at 404.7, 435.8, 435.8, 547.0, and
577+579 nm, and also the second order imprint of the 404.7 line (corresponding to a
value on the scale of 809 nm) can be used. Once the size of the scale has been
determined in this way, the 532 nm laser line can be used as a routine check of the
alignment.
If you have time and wish to be careful, you can do a slightly more complicated
processing of the values. Instead of using the color pictures opened in ImageJ directly
for extraction of digital data, you can decompose the RGB (red-green-blue) pictures
into R, G, and B pictures by pulling down the "Image" menu to "Color" and select
"RGB Split". Then three grey-shade pictures will emerge, each one corresponding to
the red, green and blue channels of the camera. In case there would be any green
stray-light contamination of the fluorescence, you can get a cleaner version from the
red channel. More importantly, if you suspect that the reflected green light is out of
the linear range of the camera in the original RGB image, you can get a weaker signal
from the blue channel. Since no blue light is present in the experiment you can be sure
that the line you see there is green light from the laser.
Determination of the photon irradiance (PPFD) of the laser beam
Measuring the PAR (photosynthetically active radiation) that the laser produces at the
surface of the leaf requires some thought. The irradiance varies across the beam. To
determine the intensity profile across the beam, replace the leaf at the sample port
with a sheet of paper and take a photo of the spot, and also a photo of a ruler at the
same position in relation to the camera. Put a sheet of the red acetate foil used for the
viewing window between the paper and the exit port to decrease the light so the laser
spot and the mm ruling can be properly recorded in the same photo (Figure S1). Then
open the photo in ImageJ and select a thin rectangle going through the centre of the
image of the laser beam, and plot a profile just as you did for the spectrum. Figure S2
shows a beam profile obtained in this way. For comparison also a Gaussian function
is drawn, and you can see that this approximates the beam profile. The value 100 on
the vertical axis corresponds to the ambient light, necessary to record the mm scale on
the photo. Most of the beam is contained within ±0.5 mm from the center, i.e. within a
cross-section of *0.52 mm2 = 0.79 mm2.
Then you have to determine the total photon flux in the beam. You can use an
ordinary lux meter, or an instrument giving PAR directly. In both cases you must be
aware that the laser beam fills only a small fraction of the sensor aperture, so what
you read on the meter display is not the value in the beam. To get the (approximate)
average value for the beam, multiply the reading by the ratio of sensor area to beam
cross section. You must also be aware that the sensor may be more sensitive in the
center than near the perifery.
If you use a lux meter, it is useful to know that the definition of the lux (by CIE in
1931 as well as by the more recent definition by CIE in 1978) is such that at 532 nm
one W m-2 s-1 corresponds to 603.2 lux (CIE stands for the Comité International de
l'Eclairage, a recognized body for international unit definitions; the value we use was
obtained from interpolation in the column for "Photopic conversion lm/W" at
http://hyperphysics.phy-astr.gsu.edu/hbase/vision/efficacy.html or
http://webx.ubi.pt/~hgil/FotoMetria/HandBook/ch07.html). Lux meters can show
great deviations from this ideal value. Another difficulty is that modern lux-meters,
which use a small photodiode as light sensor under a large diffusing screen are more
sensitive to light hitting the center than the perifery of the screen (this is probably less
troublesome with old-fashioned lux-meters, which have a large-area barrier-layer cell
as sensor). To convert from W to mole of photons, remember that the energy of one
photon is Planck's constant multiplied by the velocity of light and divided by the
wavelength, and that there are 6.022*1023 photons in a mole. Planck's constant is
6.626*10-34 J s, and the speed of light 2.998*108 m s-1 in a vacuum (the wavelength
value for the laser light, 532 nm, refers to a vacuum, and of course you must convert
that to meter by multiplication by 10-9 to get the photon fluence rate or PPFD in mol
m-2 s-1).
The following is a detailed description for how the PPFD of the green laser beam of
0.79 mm2 cross section was estimated using a lux meter: First the light from an
ordinary lamp was measured using the whole sensor area. The result was 12200 lux.
Then a sheet of black cardboard with a hole in it on was put on top of the sensor. The
hole, of diameter 4.2 mm was at the center of the sensor surface, which had a
diameter of 38.5 mm. The same lamp was measured at the same distance as before.
The new reading was 1/13.8 of the first one, while the ratio between the whole sensor
area and the area of the hole was (38.5/4.2)2 = 84.0. Thus the sensitivity at the center
of the light probe was 84/13.8=6.09 times the average sensitivity over the whole
surface. The laser beam aimed at the center of the probe surface gave the reading
12620 lux. To get the true value for the 0.79 mm2 cross section of the beam we must
now divide the reading by 6.09 and multiply it by the ratio of the whole probe area for
which the probe was calibrated and 0.79 mm2, so the true illuminance value for the
beam was 12620*3.14*(38.5/2)2/0.79/6.09 lux = 3.05*106 lux. This in turn is
3.052*106/603.2 W m-2 = 5060 W m-2
= 5060*532*10-9/2.998*108/6.626*10-34/6.022*1023 mol m-2 s-1=22500 µmol m-2 s-1.
Then a "PAR meter" designed to give values of photosynthetically active radiation
was used. Using a metal washer with a diameter of 2.4 mm the ratio between the
sensitivity in the center of the probe and the average sensitivity was determined to be
1.60. The laser beam gave a reading of 885 µmol m-2 s-1. Since the beam cross section
(0.79 mm2) was smaller than the receiving area on the meter (47.8 mm2, calculated
from a measured diameter of 7.8 mm) an appropriate correction factor
(47.8/0.79=60.5) had to be applied. Thus the average PPFD over the cross section of
the beam was estimated to be 885*60.5/1.60 = 33500 µmol m-2 s-1.
An even more approximate way to estimate the PPFD of the laser beam is to use the
nominal power rating of the laser pointer, in our case 5 mW. By multiplying by the
wavelength in nm and dividing by the speed of light, the Planck constant, and
Avogadro's number we find that this corresponds to
5*10-3*532*10-9/(2.998*108*6.626*10-34*6.022*1023) mol s-1= 2.224*10-8 mol s-1 =
0.0222 µmol s-1. For a cross section of 0.79 mm2 this corresponds to 28100 µmol m-2
s-1.
In summary: The average PPFD across the beam was estimated by three methods.
The lux meter gave the lowest value, 22500 µmol m-2 s-1, the PAR meter 33500 µmol
m-2 s-1, and calculation from the nominal laser power 28100 µmol m-2 s-1. The PPFD
in the centre of the beam was about twice these values. A more reliable method for
measuring the beam would probably be to use a bolometer, but this is an instrument
that is not easily available to an amateur. If available, it can be calibrated
approximately in a very simple way (Björn LO [1971] Simple methods for the
calibration of light measuring equipment. Physiol Plantarum 25, 300–307), although
more accurately in a scientific laboratory.
Figure S1. Laser beam image in the margin of mm-ruled paper, used for producing
Figure S2.
Figure S2. The intensity of the laser beam compared to a Gaussian function. The
value 100 on the vertical axis corresponds to ambient light necessary to image the mm
ruler on the same photo as the beam.