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A DIY Imaging Fluorometer

Is it possible to build a precise Fluorescence Imaging Device at home?

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Apart from being extremely fascinating, measuring plant fluorescence is also difficult. Most DIY fluorescence projects are built for educational purposes, and few of them are built with precision in mind. In the majority of cases, these projects are built around photodiodes arrays that collect unidimensional data. But what about imaging fluorometers? Well, these systems are complex and difficult to build, and depend on expensive LED arrays and even more expensive optics that can filter spurious light.
The device I am proposing here is far from cheap in the DIY sense, but it is substantially cheaper than a professional fluorometer imaging system from Waltz or any other reputed scientific instruments manufacturer. That is not to say that those systems are overpriced; surely they are much more advanced. But analysing fluorescence can also be done by using python, opencv and scikit-learn.

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).

Chlorophyll energy states. Image taken from Buchanan et al. (2015) Photochemistry & Molecular Biology of Plants. Wiley (Oxford). pp 115

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...

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LED_Panel_Fluorometer.zip

Eagle schematic and CAM files for the 4 custom LED panels

Zip Archive - 136.98 kB - 09/23/2020 at 12:24

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  • 1 × Heatsink 300 X 300 mm RS-Online Stock No 264-670
  • 2 × Bosch Rexroth Strut 20 X 20 mm, 3,000 Length RS-Online Stock No 466-7219
  • 8 × Bosch Rexroth Strut Profile Corner Cube, 20 mm Groove 6mm RS-Online Stock No 466-7433
  • 1 × Bosch Rexroth Strut Profile T-Slot Nut, M4 Thread, pack of 10 RS-Online Stock No 466-7281
  • 1 × Bosch Rexroth Mounting Rim 6mm Slot RS-Online Stock No 227-674

View all 26 components

  • Improving the Light homogeneity within the box

    Mayke09/30/2020 at 20:33 0 comments

    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. 

    Looking good! I can't wait to obliterate some alien plant with a shot of 4000 μmoles as soon as this gets done.

  • Filters and their challenges

    Mayke09/26/2020 at 12:05 0 comments

    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, below 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.

  • First Experiment

    Mayke09/23/2020 at 14:48 0 comments

    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!

    Below you can see my setup on the Jetson TX2:

View all 3 project logs

  • 1
    Building the Box

    You will need the following cuts:

    • 12 x 300mm
    • 4 x 520mm

    Upper 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.

  • 2
    LED PCB Assembling Boards

    Part 1:

    • 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:

    If you do not have the oven at home, you will have to send the parts to be assembled elsewhere. Another option is to request assembling when requesting the PCB boards from your favourite PCB manufacturer. 

  • 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. 

                      Image taken from Photon Systems Instruments Instructions Manual p 17.

    Another question you may have is what are the small PCBs for? Well, the lamps are too powerful to fulfil the requirement of the Measuring Light (ML), which is illuminating the sample with a beam of less than 1 umol. I will deal with that in one of the Project Logs.

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