Help Needed: Cree XT-E Build for Biofluoroscopy

Flashlight Modding and DIY Parts
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OK, this is something which I can potentially provide some helpful info: I am a biochemist whose work deals a lot with fluorescent materials (including a number of fluorescent proteins).

A bit of background for those trying to help, it is not uncommon for highly efficient fluorescent proteins to only have 5-20nm of difference between peak absorbance and peak emission. The image below is the absorbance and emission spectrum for a common fluorescent protein Phycoerythrin: Ab(max)=565nm and Em(max)=575nm. Along with this close relationship between the absorbance and emission, you can see there is also a rather steep roll-off when it comes to absorbance to the red side of the peak. As such, light which is 10nm from Ab(max) is almost entirely unabsorbed.

Here is my recommendation:

Understand your absorbance curve for the biofluorescent proteins and use it to your advantage. While the above image has multiple peaks, even if your protein of interest only has one peak, it will have its sharpest roll off in absorbance to the “red” side of the absorbance curve. This means the “redder” the light is, the less it absorbs. Your focus on the FWHM of the LED is giving you only a part of the picture, as there is more than one way to target this mismatch. For example, targeting bluer wavelengths with wider emission FWHM might provide you with a better match (say 440±20nm).

Additionally, if you want optimal performance then you are going to HAVE to use filters to ensure the light you are capturing comes only from the fluorescence you are targeting. Typically, we use a combination of a Band Pass filter on the excitation source combined with a Long Pass (aka High Pass) and a Band Pass filter on the emission detection side to ensure that all excitation light is excluded and we are ONLY capturing the fluorescent emission.

An example filter set which would result in your excitation source illuminating only within 400-450nm and the image sensor only collecting light between 460nm and 480nm. Note that these are research grade and aren’t exactly cheap:

Excitation Filter (425/50): AT425/50x | Chroma Technology Corp
Emission Long-Pass (460nm): ET460lp | Chroma Technology Corp
Emission Band-Pass (455/50): ET455/50m | Chroma Technology Corp

In your case, it appears you have multiple emission wavelengths in which you are interested (side note: that yellow-orange glowing newt likely has another absorbance peak around 500-540nm) so you might need more than one filter set or just drop the Band Pass filter on the emission side. That being said, you typically want at least 5nm separation between your excitation filter and your emission filter.

Feel free to ask me any questions!

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Great info. I hope you will keep us up on the evolution of your project Biofluofunfun.

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Pedro: First off, let me say thank you for your thoughtful response.

You're right that here isn't just one Green Fluorescent Protein (GFP) of particular interest here that I can cater to. I'd like to use this light (likely along with other lights) to examine many organisms both terrestrial and marine.

I should also note this work is both of research interest (publishing observations of undocumented biofluorescence in species), and for leisure/education (getting people interested in field biology/conservation by showing them fantastical fluorescent videos and photos). In the future I may also base grad degrees on this work.

In regards to amphibians in general, Lamb and Davis did some work in 2020 regarding the emission spectra of these species in response to blue excitation light (440-460nm)

Image details from paper: "Amphibian biofluorescent emission spectra. The peak emission wavelengths of the biofluorescent light emitted by salamanders, frogs (Ceratophrys cranwelli), and caecelians (Typhlonectes natans) are green to greenish-yellow. Biofluorescent emissions were measured with a FLAME spectrometer through a yellow long pass filter (500 nm) from either the dorsal (Ceratophrys, Ambystoma, Dicamptodon) or ventral surfaces (Typhlonectes, Amphiuma, Cynops, Icthyosaura), but we did not focus on any specific part of the anatomy (see Methods for further detail). Relative intensities of these biofluorescent emissions varied substantially across taxa. Pictured are white light images of T. natans and C. cranwelli above images of these species biofluorescing. Biofluorescence was imaged by exposing individuals to blue light (440–460 nm) and viewing them through a yellow long pass filter (500 nm)."

Research into sea anemones (another area of interest for me), has found a number of active fluorescent proteins that vary from species to species (this is likely also true for amphibians). Here's an exert from a 2016 paper examining the active fluorescent protein in a deep-sea anemone species:

"Despite the close relationship between cjFP510 and the shallow-water actiniarian fluorescent proteins, the long excitation spectrum of cjFP510 (this is the protein of interest in this study) makes the protein distinct from others. The excitation peak of cjFP510 (500 nm) was longer than that of asFP499, cgFP496 and hcFP500, whose excitation peaks were 403/480, 399/482 and 405/481 nm. Johnsen et al.3 reported that the wavelengths of deep-sea bioluminescence tended to shift towards green, so the long excitation peak of cjFP510 may be an adaptation to deep-sea greenish bioluminescence, which could be the excitation light source."

Here's a graph of the cjFP510 excitation/emission behavior:

Graph details from the paper: "(a) Recombinant cjFP510 purified from the bacterial cell. (b) SDS-PAGE of purified recombinant cjFP510. (c) Excitation (---) and emission (—) spectra. (d) Tolerance of cjFP510 (○) and EmGFP (•) to high temperatures. (e) Tolerance to freeze–thaw cycles. In both stability experiments, 0.1 μg/μL of cjFP510 or EmGFP was prepared in 10 mM phosphate buffer (pH 7.4). RFI: Relative fluorescence intensity. Vertical bars represent the standard errors."

So in order to really examine as many organisms as I would like I would indeed need to purchase many different filters and lights. But I know that the 455nm lights (with yellow eyewear and a yellow camera filter) work well for many amphibians and the species of anemones I have observed here where I live in the Pacific Northwest. So I think it would be great to try other wavelengths, but currently I am operating on my own dollar, if I had a research grant I would gladly spend thousands on filters.

But I am working with a friend of mine, a physicist with knowledge of robotics, to examine if we can make an array of different wavelength LEDs that can be programmed to combine different levels of brightness to produce different wavelengths, much like RGB chips. This way in the field I could switch through different wavelengths on the fly to see what has the highest excitation value. Maybe you are aware of a simpler way to do this?

Anyway in short I think starting with a light of 455nm will work relatively well for the footage I am trying to capture, but I am also open to trying other wavelengths provided I can build relatively cheap lights. I appreciate your recommendations.

I will definitely keep everyone appraised of my progress!

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Also if anyone is aware of bandpass filters (ideally ≈ 470nm) that don't cost $200-300 I would much appreciate suggestions, I've been looking at filters all day.

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In the meantime I found this Willcrew DF60 dive light that has 6 XP-L2s:

I'm going to order it and see if I can pull the PCB and reflow XTEs in.

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I’ll save you some work.

Seriously… try this first.

Before buying XTEs and swapping them, just remove the domes and phosphor layer fro the XPL2s. You can literally do this with your bare fingers. I’d personally start by grabbing the dome with tweezers and twisting it off. I’d then scrape the rest with my finger nail. This will avoid damage to the underlying chip. Maybe a stiff bristled toothbrush and IPA to finish.

I’ll bet you the output is way higher too and the spectrum nearly identical. (Assuming they are genuine CREE emitters)

Thank me later :innocent: :+1:

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Ummm, the XP-Ls (L2 chips) still have bond-wires, no? They ain’t flip-chips like the evil G3.

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Okay I think I understand now. I’m still wrapping my head around all of the LED components terminology. I will definitely give that a shot, thank you.

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Is it difficult to tell if they have bond wires? Will scraping the phosphor damage these wires?

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XP-L gen1 have bond wires, but XP-L2s certainly are G3 style flip chips…

Koef3 actually demonstrates the phosphor/dome removal in that thread

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Right, but you said they were XP-Ls, not XP-L2s.

More confusing because XP-L uses the L2 chips, and XP-L2 uses L3 chips.

(Predictably, though, XM-L uses Ls, XM-L2 uses L2s.)

Ugh, my head hurts.

52

If you see a discrete yellow chip (square) in the clear covering, it’s the older style with bond-wires.

If the entire thing is yellow that it looks like the whole mess was spraypainted with phosphor, it’s the flip-chip without bond wires.

Those teeny tiny pure Au wires are what delivers current to the chip. If gone, it’s like snipping off the power-cord to your toaster.

Flip-chips have the contact area at the bottom of the chip, and light comes out the opposite side, on top. Or the current comes in the top, and light comes out the bottom. Depends how you look at it.

Here ya go…

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I would do some Amperage draw test before mounting XTEs —- XPLs can handle a lot more current —— you might end up with Blue Smoke you don’t want

More than likely the MCPCB is aluminum

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Is it just the XM-L2 this phosphor trick works on?

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No. This works on most ‘flip-chip’ emitters. XML2 would not be included in this classification.

Examples that are.
CREE:

XTE
XPG3
XPL2
‘HE’ models
MTG2

Most if not all Nichia’s relevant to blf

None from Luminus, though the SFT40 can be safely scraped clean.

Luxeon HL2X, V, V2 etc

Samsung LH351x

56

Where did I say that?

Also, the XML3 is not a flip chip design. There are still bond wires, but they did cover the entire package with phosphor.

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Right, I meant to write XPL2. I’ll give it a go. Might have another XPL2 around somewhere I can try it on too.

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Where do you guys order your 365nm UV chips? I’m gonna get going on building a UV dive light, as well.

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You can get the Seoul Viosys from Simon at Convoy with choice of board size https://www.aliexpress.com/item/32951600559.html?spm=a2g0o.store_pc_groupList.8148356.38.2e9024bfHcSUY5. You may be able to get individual diodes at Mouser or Digikey if you want multiple emitters. The best 365nm leds are the Nichias but I don’t know where to get them these days. Once you pick a host search on Aliexpress for a ZWB2 filter in the proper size. Hmm on second thought I don’t know how that would go with a dive light where strength is important. May need to stick with the chosen front glass.

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Scientist, thanks for the recommendations, I have been looking at ZWB2 filters. I was just checking out your UV triple build post the other day. Great info.

So I received the first dive light in the mail. For the life of me I cannot take this damn think apart to get at the emitters, I have tried clamping where there look to be seams and trying to twist it open but it just won't give. I even tried heating some areas with a small heat gun, to no avail. You guys have any tips?

Some images of the head: