OpenFlexure Microscope - epifluorescence acquisition

I just chatted briefly to one of my biological collaborators about sybr-safe, and she mentioned it was way brighter under UV light. Are you sure 460-470nm is the right wavelength? The filter set described in that thread was for GFP/fluorescein, but perhaps you actually need a UV LED (and corresponding filters). You might get away with a UV LED and the existing filters, but perhaps you should remove the excitation filter, that might be attenuating the light. Have you checked the spectra of the filters and the dye? Sorry if that’s an obvious question, but sometimes obvious questions are worth asking! Posting/linking to the relevant spectra here would be helpful for anyone else reproducing what you are doing.

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You could also try lowering the framerate further to allow even longer integration times. If your microscope is properly vibrationally isolated, it should be more or less equivalent. I appreciate this is only a pit-stop solution, but given our current roadmap, averaging may well not be included for a while yet.

Let us know how it goes!

Indeed the sybr-safe is especially used for gel band staining as an alternative to EtBr, and would be brighter with UV light, as officially its excitation max peaks are at 280 and 502nm when bound to DNA. However, most lab transilluminators are at about 365nm to see bands in gels, and that works fine for sybr-safe stained gels too. For our cheek cell comet assays, however, we were always very successful imaging (on a lab scope) with the GFP filter set, so that’s what I thought would be best to try in this OpenFlexure context. I also had some concern in our open public lab that the UV might pose more hasards for users… I tried the 3W blue LED yesterday without the excitation filter, and that again was not satisfactory for the stained cells, though brighter… Btw we have modified the LED holder so I can hopefully fit various options better, and may also try an LED with a UV peak today… (Clearly, from the slides I imaged with our DIY transilluminator, using blue LEDs, the sybr cells look more yellow than the fluorescein, confirming that going for green isn’t ideal. Probably I could also look into getting another filter set for a more yellow emission.) Thanks for your help, again!

I understand, Joel… Our OpenFlexure build is not on an air-table, however - I doubt it would be considered ‘properly vibrationally isolated’ at all. So, already the 500ms integration time is probably quite a lot for these conditions. Also, if I try to change the frame rate right down to 1 (one frame per second), won’t all my ‘bright field’ images with the white LED on top be quite overexposed?? Thanks for your feedback, and help.

just to show yesterday’s slides under the transilluminator: fluorescein on tissue is above, water on tissue in middle, and cells with a lot of Sybr-safe (more yellow than fluorescein) below…

and here is the official word on excitation emission from thermo fisher for sybr-safe bound to DNA
thermofisher

If you want good vibration isolation on the cheap:
Get an inner-tube for a bike tyre and pump it about 3/4 full. Then get a concrete paving slab and put it on top. Put the microscope on this.

In general you want something squishy that reacts slowly, with something that cannot store vibrations on top. To test if something stores vibrations just clunk it with something metal. If it goes “diiiiiiiiiiiiiiinnnnnnnng” like a bell or a gong it is bad, if it goes “thudk” it is good.

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If you are trying to use the same exposure time for bright-field and fluorescence imaging, you will always either overexpose the bright field images, or get really dim fluorescence images. You will definitely need very different camera settings for the two imaging modes.

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yes, I have been doubling and tripling exposure times for the fluorescence vs the ‘bright-field’ acquisitions. (i.e. 5k for bf and 15k for blue led) As it is still surprising how weak the fluorescein image comes out, my hoping for the cells to show up may be too over-ambitious.
when I tried merging 5 identical images of the cells yesterday, the nuclei certainly did not pop out for me… :frowning:

to go back to the choice of gfp filter cube, I think it is pretty clear from the manufacturer’s data (in addition to our previous experience on lab epi-fluor rigs) that the sybr-safe should be excited (when bound to DNA) well by 460-470nm blue led (that can also have a hidden uv component) for emission of green light… sigh

two more points have come out today. 1) I put a 150 ohm resistor on the 3W blue LED, and am just running it from the 5V pins on the raspberry pi. Maybe I need a separate power supply (and much smaller resistor) to get any extra excitation with this led? and 2) averaging 5 images isn’t likely to be enough to make a big difference (noise will reduce by the square root of the number of replicates)…

just something more to mention (3oct controls):
Now I know all my samples were just fine, for imaging on a standard lab scope (Zeiss Axioscope at UNIL DMF).
I will attach a sample image from one of yesterday’s wet mount slides… (all worked well in today’s controls…)


And here is one of the dry cells stained with sybr-safe:

  • the white lines in both these images is the 100micrometer scale bar.

Not sure if I will be able to get this ‘diy’ setup to work for fluorescence of the stained cells… :frowning:
The filter cube should be good as it sees the fluorescein signal, but I guess it is a problem of the camera sensitivity to see the stained nuclei… Am still trying more LED tests (funnily, for smaller resistance, it is a much bigger beast! =)

For (1) I think it would be well worth consulting the LED’s datasheet. If you are using a 150 ohm resistor, with a 5v supply, and a typical blue LED (which has a junction voltage of somewhere in the region of 3.5V, that means you’ve got 1.5v (supply voltage - LED junction voltage) dropped over 150 ohms, so your current is about 10 mA. That means you’re really running it at something like 35 mW (power=volts x current=3.5V x 10mA), not 3W, i.e. 1% of it’s rated power.

Do not try to run a high-power LED off the Pi GPIO pins - at best it won’t work and you’ll blow a polyfuse. At worst you will completely and permanently destroy your Pi.

With a separate power supply, you can calculate the resistor you’ll need, based on the input voltage, LED voltage, and LED current requirements. That sets the voltage and current through the resistor, and you can use Ohm’s law to figure out the right resistance. That said, a constant-current driver is usually a much better way to drive big LEDs like this. We have not designed and documented one, we might do in a few months’ time and rest assured we will post about it when we do.

There are plenty people who have made constant-current circuits for LEDs though, e.g.:

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I have been doing a bunch of tests with an external power supply, the ‘diffused’ LED hooked to the same ‘power resistor’ as used before (4.7 ohms) and could get the blue LED as high as 9.65V (1Amp). bright! (5V gives about 300mA current for a more reasonable level to not fully saturate the camera. 7V gives about 600mA.)
Thanks for your further info above, Richard! (confirming also what we now have seen empirically too)
However, epifluor from the sybr-stained cells is still not convincing, although I did two exposure and voltage series today, that with a bit of adjustment (fingers crossed) might get the nuclei to ‘pop out’ if I am at all lucky.
I had also worried that the RPi camera simply isn’t able to pick out the signal due to resolution issues, but I got images of a hemocytometer grid and did the calculations- we get 2 pixels per micrometer with the RaspberryPi camera at full resolution, which is not so far off from the lab system with 10x objective… The nuclei of these 50um dia cheek cells are probably only about 10um in diameter (max) of course, so maybe the this is still the problem. sigh. The other worry is that the ccd camera in the lab is simply way more sensitive, and that that is what is needed to see what we need to see… More soon, I hope!

Note added 13oct: (I need some response to this thread to say anything more in a new post! is anyone seeing this? :slight_smile:)
it is clear the filter set is the main problem.

Those Comar ones I ordered are not correct.

1 550nm dichroic mirror, 25x16mm (GFP) Mfr: Comar; Part No. 550 1Y 116
1 490nm excitation filter, 25x16mm (GFP) Mfr: Comar; Part No. 495 1K 116
1 500nm emission filter, 25x16mm (GFP) Mfr: Comar; Part No. 515 1B 116

I should not have followed the recommendation in the issue thread, I guess…

When these ‘excitation and emission’ ones are put together, and I look at a normal fluor bulb’s light, turquoise blue comes through.

I have to find the correct band-pass filters (that hopefully won’t be too expensive).
Anyone have a good idea about this?
ciao for now

Joel just checked the ones we are currently using (which may or may not match the ones I used in the past), and the excitation and dichroic are the same as yours, but the emission filter we have is a 510 IY. He also reckons it’s not impossible that there is a little leakage, and that we’re relying on the dichroic to cut that down.

I’ve never tried fluorescence imaging with a 10x objective; note that the difference between cheap and expensive 10x lenses in terms of Numerical Aperture is a lot bigger than at higher magnifications. That means a posh 10x will get a lot more fluorescence signal than a cheap one. I’m sure the Pi Camera is not as sensitive as it could be (the bayer filter is a pain) but your objective lens may not be helping. I’d not realised you were working at such low magnificiation…

Thank you for your further investigations!
When you hold the 510 IY next to the 495 IK and look at a light - what do you see? how much light, if any (and what color, if so?) comes through??
This may indeed help. (I just went to the first OpenFlexure epifluor issue thread - where I got my Comar order numbers - to try to note all this also… please see: https://gitlab.com/openflexure/openflexure-microscope/issues/43#note_237046645)

I also just looked again on the Comar site, and the transmission of the 515 1B filter looks much narrower than desired - green/green yellow is being cut out (so that also explains the fluorescein not being so bright) - also vs. the 511 1B possibility) but the 510 1Y definitely looks better - as it lets all light longer than 500 through!

So, I will just await your answer on the combination of the two (what is seen)! thx

In terms of our 10x objective - it’s a good one, I think - Fluotar … (I can take a picture again for you, if you like) - and with just 4x or 5x objectives we see our comets well, ordinarily (on the lab scope with ccd). Of course, as the max resolution of the Rpi Cam was calc’d to be about 2pixels per micrometer (using an image of a hemocytometer grid at full res), we are indeed talking about just a few pixels per nuclei in the intact cells. So, a more powerful objective does indeed seem likely to help… (should be ok -even if more images will be required to take the full ‘panorama’ of our samples…)

I’ll leave checking the filters in the lab to Joe or Joel, but to answer your point about 2 pixels per nucleus, remember it’s a colour camera with a Bayer filter. That means only every other pixel is sensitive to green light. I’d recommend increasing your magnification - if you’re picking up something that’s only a couple of pixels wide, and it’s centred on a red or blue pixel, you will really struggle to see it.

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Today’s update:
I am very glad to have already printed the reflection illuminator set, and started on the main body with the extra room for it to insert readily! Luckily, went ahead and ordered the long pass filter from Comar, even without your verdict on the combination test (exc and emission filters together should not let pass blue light), since apparently the co is not going to keep single items in stock any longer… whew, for me, for the moment, but - more sourcing should be done…

Also, to clarify the size/pixel issue - the inner cheek cells are about 50microns in diameter, so the nuclei are about 5-10micron in the fixed preps, a bit smaller in wet mounts… Not only are there more than 2 pixels per nucleus (maybe up to 20), but in fact with the 10x lab scope we only had 0.645microns/pixel, while the full res RpiCam gives 0.5microns/pixel, not so very much different.
Your point regarding the Bayer filter is well taken, however, esp in comparison to the lab rig CCD. Perhaps this is another reason to avoid red fluor in particular with the RpiCam, since at least there are 50% of pixels that are green ! :wink:
Thanks again for the feedback!

the new long pass filter indeed made a difference!
got my first images that show the nuclei of sybr-safe stained nuclei, still with the 10x objective! see also…

and here’s one more example from the center of the images…
croppedsybrnuclei2smaller|690x293

and one more from today!


Hoping it will be even better in version 6 of the OpenFlexure, with the different reflector insertion rig…

Great to see it is working!

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Still happy to have seen nuclei in cheek cells with the OpenFlexure, and looking forward to not only finishing the v6 build (with better accessibility for the filter cube and reflection illumination), but seeing some comets.
Just to clarify, for future reference, the quoted list of Comar filters above only was wrong for the emission filter. In fact a more ‘long-pass’ filter, the Comar 510 IY 116 is what was needed to make this work (sybr-safe stained cells). The images from a couple of days ago were still with our old 10x Fluotar objective, for instance.

one more point - the LED was a simple blue one, just powered by the 5V pins of the Raspberry Pi!