Introduction: A DIY Imaging Fluorometer
The idea of having an imaging fluorometer at home takes to a complete new level those of us with a keen eye for plants. Imagine, for instance, that you want to develop a more efficient LED lamp for your specialty crop, or that you are looking for ways to understand the underlying causes of stress under different external conditions such as lack of, or too much, humidity, extreme temperatures or lack of nutrients. But what does it make fluorescence to be above other well stablished techniques such as multispectral or hyper spectral imaging and/or ground sensors?
In order to answer the above question, we would have to lay some of the fundamentals about fluorescence first. When photons reach the surface of a plant, they make the molecules of chlorophyll to get 'excited'. Literally, excitation means that the molecules absorb electromagnetic radiation from the UV-visible range of light, making the electrons to jump from a ground energy state to a higher energy state (See image below). Now, classical biology tells us that those ‘excited’ electrons will travel through other nearby Chl molecules by virtue of FRET (Föster Resonance Energy Transfer) until they reach the Wholly Grail of the Light Dependent Reactions, that is, the Reaction Centre (CR), where they will finally get knocked off from the Chl molecules and transferred to the acceptor Plastoquinone. Let us bear in mind that Chlorophyll has two major absorption bands and therefore, it has two different energy levels other than ground: 1st excited singlet state (occasioned by red light absorption), and 2nd excited singlet state (a higher energy level attained from absorbing blue light, which is more energy intensive).
During this process, some of the energy produced by the excited molecules will necessarily be lost; as is the case when the amount of energy absorbed by Chl molecules exceeds the light utilisation of photosynthesis; such excess of energy is dissipated as heat as part of the Chl molecules returning to their ground state. Heat dissipation is always the result of vibrational relaxations that arise from the second excited singlet state. This photo-protective process is called Non Photochemical Quenching (NPQ).
But heat dissipation is not the only way in which a Chl molecule can return to the ground state. Another mechanism involves the emission of a photon while the Chl molecule decays to ground state. Such emission is the result of decay from the first singlet state, and it has a longer wavelength (it will always be in the red portion of the visible spectrum) than the absorbed light. This final process is called fluorescence. Now, the three processes, electron transfer, heat dissipation and fluorescence, occur against one another, which means that out of 100% of energy being captured by the Ch molecules, more or less 80% will be transferred to the RCs, around 18% will be dissipated as heat, and around 2% will be release as fluorescence. Any increment in any single one of them will mean a reduction in the other two. The above has huge implications when studying plants, for one thing is to measure external factors such as light intensity, humidity levels or nutrient content, and another one completely different is to be able to know exactly how many electrons are being used by a plant to sustain glucose production. At the heart of measuring Chlorophyll fluorescence is the examination of photosynthesis performance, the single most important process occurring inside a plant.
Kautsky & Hirsch Effect
In the early 1930s Professor Hans Kautsky and his collaborator A. Hirsch observed an increase of fluorescence intensity when dark adapted photosynthetically active samplea were illuminated. They published their discovery in Naturwissenschaften with the title Neue Versuche zur Kohlensäureassimilation (New Experiments on Carbon Dioxide Assimilation). The immediate conclusion of the authors was that Chl fluorescence rises rapidly to a maximum, then declines to finally reach a steady level within minutes (I would say seconds in fact, see video below). Furthermore, such decline was found to be inversely correlated with the increase in the rate of CO2 assimilation. After that experiment and the consequent publication, everyone went down the rabbit hole.
The Basics of Fluorescence Measurements
As explained above, the excited electrons need to reach the Reaction Centres in order to be transferred to the Calvin cycle via the acceptor Plastoquinone. But, as the plant has been dark-adapted, the enzymes responsible for driving the whole cycle are not 'activated', which means that the first electrons reaching the plastoquinone acceptor will effectively close the reaction centres. That is, no other electrons will be accepted until the enzymes are progressively activated by light and the electron transfer can occur. We can safely say that the plant is in 'sleep' mode. That in turn means that all the electrons that are not transferred will have to be dissipated or 'burnt' in order to prevent tissue damage. Therefore, Chl fluorescence rises rapidly, along with a massive increase in heat dissipation. Once the enzymes start progressively to activate, more and more reaction centres will then open, allowing the electron transfer to occur and both heat dissipation and Chl fluorescence decline until reaching a steady level. All the above sounds fine and exciting but, can we gain any practical knowledge from it? The answer is yes, but in order to gain such useful knowledge, we need to separate both photochemical and non-photochemical quenching. Remember that any increase or decrease in one of the factors (electron transfer, heat or fluorescence) will directly affect the other two. So the trick of any fluorescence measurement is to be able to close, or reduce to almost zero, two of the processes, so the one left can be estimated. The most common way of doing this is by preventing the occurrence of photochemical and non-photochemical quenching, by using a short pulse of light. The pulse needs to be very short (no more than half a second) and very strong, preferably in the order of +4,000 μmol·m-2·s-1. The light pulse will be too short to enable any electron transport or heat dissipation, and in the absence of both, the maximum level of fluorescence can be determined. But if we are to measure the maximum level of fluorescence, we also need to measure a baseline, or minimum level. Ideally, the minimum level of Fluorescence is measure by switching a light that is way too weak for any photosynthesis-related process to occur. That is achieved by making sure that the Measuring Light (ML) does not exceeds 1 μmol·m-2·s-1. Once we have measured the minimum and the maximum levels of Chl fluorescence, we also need to measure the yield of Chl fluorescence during light conditions; that is, in the presence of both photochemical and non-photochemical quenching. Below you can see a typical fluorescence measuring technique:
Three types of illumination are used in the experiment above:
- Measuring Light (ML) or MB: 1 μmol·m-2·s-1
- Saturating Pulse (SP): Over 4,000 μmol·m-2·s-1
- Actinic Light (AL): Usually at steady 600 μmol·m-2·s-1
The terminology used for the measurements as follows:
- Fo: Minimum level of Fluorescence attained by switching on ML.
- Fom: Maximum level of Fluorescence attained by applying SP.
- Fp: Maximum level of Fluorescence attained in the presence of PQ and NPQ immediately after switching AL on.
- F’m: Maximum level of Fluorescence attained while applying SP in the presence of PQ and NPQ.
- Ft: The level of Fluorescence immediately before applying the SP while the AL is on.
- F’o: Minimum level of Fluorescence after switching off AL (preferably applying far-red light simultaneously).
The most common used parameters used in Fluorescence measurements are detailed as follows:
Photochemical Quenching Equation
- 𝚽PSII: The quantum yield of Photosystem II. (F’m - Ft) / F’m
- qP: Proportion of open PSII. (F’m - Ft) / (F’m - F’o)
- Fv/Fm: Maximum quantum yield of PSII. (Fm - Fo) /Fm
Non Photochemical Quenching Equation
NPQ: Non-Photochemical Quenching. (Fom - F’m) / F’m
From 𝚽PSII we can extract even more useful information about the Photochemical processes; for example:
Linear Electron Transport Rate (J) or ETR: J = 𝚽PSII X PFDa X (0.5)
Where PFDa is absorbed light in μmol·m-2·s-1, and 0.5 the factor that accounts for the energy partition between PSI and PSII.
Another very important parameter for us will be Fv/Fm since this parameter reflects the potential quantum efficiency of PSII and it's used as an indicator of photosynthesis performance, with an optimal value of 0.83 across species.
This project will seek to reproduce the fluorescence trace of the image above and calculate Electron Transport Rate (ETR) values, as well as Fv/Fm using the DIY equipment listed in the following sections.
1 × Heatsink 300 X 300 mm
2 x Bosch Rexroth Strut 20 X 20 mm, 3,000 Length
8 x Bosch Rexroth Strut Profile Corner Cube, 20 mm Groove 6mm
1 x Bosch Rexroth Strut Profile T-Slot Nut, M4 Thread, pack of 10
1 x Bosch Rexroth Mounting Rim 6mm Slot
1 x Bosch Rexroth Strut Profile Gusset, 20mm, groove 6mm
4 x Acrylic Mirror Sheet 310 x 410 mm
1 x Plywood board or MDF board 300 x 300 mm, 20mm thick
1 x Black 3.0
25 x Osram high Power LED Single Colour Blue 445nm
25 x Osram high Power LED Single Colour Red 660nm
25 x Osram high Power LED Single Colour White 70 CRI 6500K 320 - 350 Lm
25 x Osram high Power LED Single Colour Green
4 x LED PCB Board - Custom
4 x Ledil C12609_Virpi-W 25-Way LED Lens
1 x Mid-Opt LP695 Near-IR Longpass Filter or similarLongpass filter cutoff at 700nm 14 mm diameter for M12 lens
4 x Longpass filter cutoff at 700nm or similar 76 x 76 mm (mounted over the Virpi lens to filter spurious light)
1 x See3CAM_CU55M - 5MP Monochrome USB Camera
1 x NVIDIA Jetson TX2 Developer Kit or Raspberry Pi
1 x Rex Wire PTFE Black 24 AWG 25m
1 x Rex Wire PTFE Red 24 AWG 25m
1 x Arduino Uno
4 x Optocoupler through hole
4 x Potentiometer
4 x Mean Well LED Driver 40W LCM-40
10 x Resistors Assorted
Step 1: Build the Box
You will need the following cuts:
12 x 300mm4 x 520mmUpper part: Take 4 x 300mm, 4 corner cubes and the heatsink. With the heatsink flat surface pointing down, put the 4 profiles around and assemble them using the screws and the cubes. No need for drilling as the profiles will compress around the heatsink keeping it in place. Bottom part: Take 4 x 300mm profiles, 4 cubes and the 4 x 520mm profiles. Tight the screws in the three different directions. Place the other 4 300mm profiles at 100mm from the bottom up and use the Gussets to keep them in place. The Gussets should be placed below the profiles. Present the upper part at the top of the 4 520mm profiles. Do not tight the screws linking the whole box just yet.
Step 2: LED PCB Assembling Boards
Blue/Red Panel x 2
Part 2: White/Green Panel x 2
Part 1 assembling: Take 13 Blue LEDs and 12 Red LEDs for the first panel. Take 12 Blue LEDS and 13 Red LEDs for the second panel.
Part 2 assembling: Take 13 White LEDs and 12 Green LEDs for the third panel. Take 12 White LEDs and 13 Green LEDs for the fourth panel. Follow the order below:
Step 3: Camera and LED Panels Mounting
Here is where the fun starts.
Find the centre of the heatsink and drill a 16mm hole. This is to accommodate the USB 3 cable to connect the camera and the LEDs wiring. Put the USB cable through the hole and connect it to the camera. Please the camera onto position and draw the position for the 4 holes. For M2 screws, use an 1.8mm drill bit. Tap the threads with an M2 tap. Use metal spacers to place the camera.
From the centre hole, draw a square of 80 x 80 which will guide you to place the four boards aligned. Mark with a pencil the corner holes and drill them onto the heatsink using a 2.8mm drill.
Tap the thread using an M3 tap into each hole. Place the boards and secure them with M3 screws. The same colour panels are connected in parallel. That means that you need to connect the positive connector to the negative of the other board. To close the circuit, you need to connect the positive wire from the LED driver to the positive of the first board, then the negative of the first board to the positive of the second board and, finally, the negative of the second board to the negative of the LED driver. Your wiring does not need to be as ugly as mine. As a matter of fact, I originally created a whole PCB board of the size of the heatsink to prevent all these wires running amok. But in later stages I decided to go for individual PCBs for each lamp due to the lack of available lenses. I settled for the VIRPI lens of Ledil which, in turn has created problems of its own. Mainly the fact that I need to create an angle to illuminate the samples uniformly. See the Photon Systems Instruments Open FluorCam.
Step 4: Frontal Panels
The box was originally design to have mirror panels for better light distribution. This did not work well with the camera.
For that reason I had to invert the panels, and paint them with a black coating to avoid interference with the camera and also to be able to create the masks.
I also had bought a calibration panel to determine both the minimum and maximum brightness levels which, in the end, was not necessary.
So the final version is an all-black chamber. You can see the picture of the apricot seedling above.
Step 5: First Experiment
Curious whether the Fluorometer was any good after assembling, I decided to run one short experiment with some random weed plant from the garden.
After dark adapting the plant for 25 minutes, I applied the Actinic Light (White LEDs) at around 600 μmols per second per metre squared. Amazingly, the Kautsky effect is clearly captured by the script. I also applied a short pulse of blue light (SP) after few seconds, which is also clearly noticeable at the end of the graph. So far so good! You can see my setup on the Jetson TX2 on the pictures above.
Step 6: Stay Tuned: This Isn't Over Just Yet!
There are several steps I have not finished, but thinking that the community could potentially give some good ideas about the best approaches, I decided to publish it.
Below some challenges/things to do/correct:
- LED panels: They need to have a 20 degree angle towards the centre of the box in order to fully illuminate the subject.
- Filters: I need to add the long pass filters over the lights and block any spurious light leaking from the sides
- Electronics: They are all scattered out and need to be added in the bottom part of the box.
- Enclosure: I need to finish the outer panels and the door, so the box is sealed
- Thermal imaging: I need to install the Lepton Flir Camera to monitor heat
- Photodiode measurements: Also, in the making.
- Spectrometer: I would finally add a DIY spectrometer to have a full control of light and be able to calibrate
Please let me know if you have any comments, questions, or potential additions to the box.
Thanks for reading and keep an eye at this instructable.
Step 7: Filters and Their Challenges
As you can see above, the curves of the Longpass filters do not match, and that's what happens when you buy cheap filters :)
Ideally, the blue filter should have a steep curve down the 700nm, while the orange one should have a steep curve upwards the 700nm, meaning that the blue let's light below 700nm but blocks anything beyond it, while the orange blocks anything below 700nm while letting through anything above. The orange filter goes onto the camera lens, above a picture of a filter from MIdOpt, and the curve of the filter used in this project.
Usually, the steeper the curve the more expensive your filter is. I have added the red LED emission curve (in red) for you to see that the blue Longpass filter should cover it, which means that should allow most of it, which is not the case.
I have requested some quotations from Chinese manufacturers as a filter 76X76mm in size would cost a fortune in the UK or the USA. It is not that the Chinese will give it for free, but we can expect a 50% reduction in price, probably at the expense of some quality.
Step 8: Improving the Light Homogeneity Within the Box
I have decided to tackle 'head on' the issue with illuminating the samples in the box. As you can see from the picture above, the PCB LED panels lay flat on the heatsink; that means that quite a bit of light is wasted illuminating the lateral panels, instead of the samples. To solve the issue, I decided to mount the boards on aluminium blocks cut at a 20 degree angle.
I had the misfortune of cutting these pieces from a 4'X4' block using a mitre saw. Please do not even attempt it. Use instead a swivel bandsaw. The result will be much nicer and precise. I just couldn't justify myself to spend £500 quid on a swivel bandsaw to cut four aluminium pieces, but the motivation is there, and very likely, sooner or later I will succumb to the temptation :)
You can also see a Russian Yasen class submarine lurking in the background. As winter closes its claws around us, and being myself from a Tropical country, I need to keep my mind busy during the coming dark months.
You can see the preview above! I say preview as I have not finished drilling and tapping the holes for the PCB boards and I will need to rewire the whole thing. Although by the looks of it, it will be much easier than the spaghetti mess from the original. I can't wait to obliterate some alien plant with a shot of 4000 μmoles as soon as this gets done.