# Multicolour Led Light For Fluorescence Microscope



## mattthemuppet2 (Jan 8, 2016)

Next project on the list, that's been waiting in the wings since the summer.

I work on a nematode called C. elegans, which is very cool in many ways, but one of the best (and Nobel prize winning) tricks it has is that you can make transgenic worms which glow green. Not just for fun, but also to see where genes are expressed or how proteins are being regulated. The thing that makes them glow green is a protein called Green Fluorescent Protein, GFP for short, which was discovered in a jelly fish A. victoria (https://en.wikipedia.org/wiki/Green_fluorescent_protein). Since then a bunch of other fluorophores were discovered, Red, Yellow, Cyan etc from jelly fish and coral (I think), so you can have multiple different colours in the same animal. All of these fluorophores share the same attributes - they're all excited by a specific wavelength of light and re-emit that light at a different, higher wavelength.

GFP needs between 395 (UV) and 475nm (blue) for excitation and emits at 509nm (lower end of green spectrum). So to work with GFP, you need a UV or blue light source and a filter that blocks out all light below ~500nm. Light sources used to be white mercury lamps or metal halide bulbs (what I used in my last job, what a PITA), with filters to let only blue light through. With LEDs, which can emit at very narrow frequencies, alot of manufacturers are now using different colour LEDs as the light source (typically individual LEDs in a carousel) which is more efficient because a) LEDs are more efficient than filament or HID bulbs and b) you only generate the wavelength you want instead of stripping it out of a whole bunch you don't want.

Anyway, long blurb short, where I work now (temp professor at Whitman College) has an LED blue light source on their inverted scope, which is pretty cool, but you can only look at objects on glass slides with it. I do a lot of work (genetic crosses etc) on agar plates, which I can't easily do without a blue light source on a dissecting scope. So I used some of my research funds from last year and the summer to buy an adapter for the dissecting scopes we have at work and a green band pass filter, plus a multicolour Cree LED (white, blue, green and red on one LED!), some switches and with a whole bunch of bits'n'pieces in my garage, I'll be making a multicolour LED light source!

It's going to be really basic, with 4 individually switched channels, each with 2 x 350mA AMC7135 current regulators for 700mA total. I hope that is both enough and not too much. If it's not enough, I'll add another chip, if it's too much I'll see if I can add an extra channel for each colour with only 1 chip. We'll see.

First task, make a plate with the chips JB welded to it. They're really fiddly to handle, so this will hold them in place, plus the heatsinking won't hurt. Used a random piece of scrap and tapped the existing holes 6-32.


Each chip has a ground leg (to negative leg of power source, be it battery or 5V DC) which is the large center one, a negative drain leg (to negative side of LED) and a Vdd leg which turns the chip on and allows current to flow. I can never remember which is which so I'll dig out a diagram before wiring them up.


made a little adapter collar for the fiber optic arm things that will carry the light to the sample. Should have been more careful with my cuts, but it polished up well enough with a series of finer grit sandpapers wrapped around a pencil and then a polish with Mothers.



lightly JB Welded in place. The idea here is that it will help capture stray light that comes out of the sides of the LED and hopefully direct it down the fiber optic.



those have to set overnight plus it's Friday Family Film night, so that's probably it until tomorrow. Next is to mount the LED on the heatsink (out of focus on the right in the above picture) and the optic on some kind of base, so I can start working on the case.

here's some pics of worms I have made 

some head muscle, neurons and the intestine (top right)



a whole bunch of neurons in the head of a worm



dye filling some neurons


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## rpmMan (Jan 8, 2016)

pretty neat stuff...not sure if you are aware but the large pad (common) connection on the regulator is designed to accommodate the heat sink thermal connection..i.e. ideally you would solder it directly to a pad  of copper which acts as the heat sink..


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## mattthemuppet2 (Jan 8, 2016)

thanks Rich! You're right, the chips are normally heatsinked through the board they're mounted too, but that wasn't an option on this build. Heat shouldn't be much of an issue, the power levels are reasonable and the voltage drop isn't too bad. The red LED will have a Schottky diode or two in series to reduce the voltage drop in the regulator as its forward voltage is a lot lower, around 2V vs. 3V or so for the others. One day if I learn Eagle and start designing my own boards this would be fairly trivial. Lots of other things to learn before that though!


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## brino (Jan 8, 2016)

Hey Matt,

What is the actual size of those little guys?
Or another way, what magnification ranges do you work with?

Thanks,
-brino


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## mattthemuppet2 (Jan 8, 2016)

hi brino, they're about 1mm long and 50um wide fully grown. picking them up takes a bit of practice


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## mattthemuppet2 (Jan 13, 2016)

alright, have to give this a major push to get it done before the start of the semester next Tuesday.

wormie helping out


fibre optic screwed and pinned in place. The heatsink with the LED on will be tweaked to center it in the hole.


The LED itself. Isn't it cool? White, blue, green and red dies on a single substrate. No idea where the heatsink came from, whenever I find one I put it in a bag under my bench for situations like this 



all holes are drilled and cut. L>R on the top - 4 on-off switches for the individual dies, pre-existing holes that will be used to attach the heatsink, 4 holes for the current regulators, square hole for main on-off switch. On the side are the holes for the power plug and fuse, the fan and to secure the case to its base



once I've had my coffee it'll be time to draw up a wiring schematic and start putting it together!


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## WDG (Jan 13, 2016)

Did I ever tell anyone about the time I took a heart out of a pig and transplanted it into a tin can and once I was done they used it in a movie?  Just Kidding, my way of telling you your way over my ankles, but still verrrrry interesting.  Back when I was a young man I worked in a research laboratory, but that was then and this is now.  I just do little projects like putting up Christmas wreaths and taking them down, nothing too complicated like what you're doing.  I'm going to keep an eye on your project to see how you come out.  I've heard Red is the hardest color, if you get that then you can get the rest.  Is that Cyan?


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## mattthemuppet2 (Jan 15, 2016)

thanks WDG, much appreciated. At the end of the day, it's all pretty simple. Fixing electrical problems in a car, now that's the work of geniuses 

I'd like to say that this is finished (I thought it was) but there's some kind of fault that developed after taking it into work today, almost like the switches are arcing and letting enough current through to turn the chips on. Have to get the schedule done for Tuesday so I won't get to it today or possibly tomorrow. Also really want to get on with my next project too.

switches (grr) and heatsink in place


power supply - 5V 2.5A scrounged from somewhere


wiring diagram - very handy to refer to!



current limiters wired up. Each one has to be turned on (Vdd pin, controlled by a switch) which then allows current to flow from LED negative terminal to the negative leg of the DC power supply.


LED and switches wired up. The switches and LED share a common positive DC supply from the power supply, all the control happens downstream. The extra bit on the Red -ve line is a diode to drop another 0.7V so the current control chips don't have to burn off too much voltage as the Vf of the red LED is a fair bit lower than the others (~2V vs ~3.2V)


test run with my "test" li-ion battery





power supply, mains switch, plug+fuse and 12V fan wired in (red and blue wires). The fan will run pretty slowly at 5V, but it should provide enough of a breeze to keep everything cool. It's almost silent at that speed too!


all together!


I didn't feel like the blue was bright enough, so I added a couple more current regulator chips (air wired at the bottom of the pic), for a maximum of 1.4A. The spec sheets say a max of 1A, so it probably won't draw the full 1.4A anyway. Still, every little bit counts with fluorescent proteins.



white


blue


green



red


yellowy orangish (red and green together)


sort of cyan (blue and green together)



I have some troubleshooting to do over the weekend, but on the plus side this is a super simple circuit, so hopefully I'll get to the bottom of it quickly. May end up just ordering some new switches from Digikey.


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## WDG (Jan 16, 2016)

When you're done, I'm refurbishing some antique Coke machines you can help on..  Watching what you're doing makes me wish I had stayed at the research company.


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## mattthemuppet2 (Jan 16, 2016)

what do you need help with? When it comes to electricity I'm limited to pretty simple stuff


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## mattthemuppet2 (Jun 6, 2016)

alright, had to do some rejigging to get this to work reliably and then I found out that it wasn't bright enough 

rework plan includes a new fiber optic light guide, as the current one is ancient and probably not very efficient, and a 4up LED board, with 1 white LED, 1 Royal Blue LED (460nm) and 2 blue LEDs (485nm). First job was to make an adapter to use the new light guide.





this is the LED end, with a 60deg included angle to collect as much of the light as possible



this is what it looks like with the light guide screwed in




finished product (almost, have to drill and tap for a set screw). 4 holes on face are for bolts to secure it to the LED heatsink. 2 holes on top are for screws to secure it to the base of the light




next up is reflowing the LEDs onto their base, then drilling/tapping the bolt holes in the heatsink


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## mattthemuppet2 (Jun 11, 2016)

got the light all finished up..









..and unfortunately it doesn't excite the GFP protein. Nuts 

Spent 1/2h talking to the inventor of the Nightsea adapters, who we bought the filter from, and his opinion is that the fiber optic design is the issue. He reckons that most of the light is entering the optic at too acute an angle and not getting internally refracted, so the intensity isn't high enough to excite the protein.

Got lots of ideas though, so as soon as we get the Nightsea light, I'll tear this one down and start having a play.


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