A DIY LED ring for darkfield

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Hobbyst46
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A DIY LED ring for darkfield

#1 Post by Hobbyst46 » Sun Mar 18, 2018 1:40 pm

So it has been discussed, explained and demonstrated in the "Dark field illumination: Questions and answers" post
(thanks a lot Micro-Bob, Apochronaut , MicroBob, charlie g, wporter, MichaelG and others!)

that quality darkfield (DF), especially with high NA objectives, requires intense illumination, dedicated condenser (reflective, cardiodide etc) and an iris diaphragm or funnel stop inside the objective. And that phase contrast objectives are unsuitable.

On the other hand, the Zeiss brightfield-darkfield-phase contrast Achromat-Aplanat 1.4 condenser (Cat No 465277) is expected, according to the Catalogue, to provide good darkfield - up to NA=1.0, like the dedicated DF Ultracondenser. However, with my pre-owned, used, worn-out condenser and microscope, darkfield is poor even for dry objectives of NA=0.30. My experiments with alternatives, i. e. circular stops in the filter tray and even in the spare holes of the condenser itself were unpromising.

So here is a temporary remedy: LED ring that rides on the condenser top lens, under the slide. It was hand-fabricated from plastic screw-caps of bottles or small jars, and a consumer self-adhesive LED flexible strip. The latter is attached to the inner surface of the top compartment of the ring, such that the LEDs shine radially inwards, towards the center. The strip specification is 10-12Watts/meter. In the ring there are only about 11cm (six LEDs), so they consume less than 1.5W. Wires are soldered to the strip and pass through small holes drilled in the wall. I power it directly with a 12V, 1A small wall-plug power-supply. There is no brightness control as yet.

The beam width from each LED is supposed to be 120 degrees (indirect info). The bottom compartment of the ring fits closely over the condenser top lens. Hence, the ring is horizontally fixed and fairly centered, and can be raised towards the slide by raising the condenser. The ring is small and short enough to pass between the condenser and the stage, in order to be placed on top of the condenser. Another option is to pass it through the stage opening, below the nose-piece.

The bottom of the LED compartment, below the LED strip, blocks the light from reaching the condenser top lens (and reflection). In the center of the LED compartment, a black opaque cylinder is glued to the disc. It should confine the light beam and make it hollow. The diameter of the cylinder is 22-23mm (sorry, omitted from the scheme).

On my initial testing, there was no serious heating of the gadget or the slide, even after an hour. The expenditure, including power supply, was about $30.
The DF images obtained with the LED ring and 40x objectives are far better than those with the condenser itself, although they are no match to DF dedicated optics. The contrast with the iris 40x/1.0 Planapo oil immersion objective, after closing the iris down to NA=0.6, is better than those with 40x/0.65 Plan (not shown) and the 40x/0.75 Neofluar Ph2 (as predicted by MicroBob, immersion performs better). The same LED ring is used for the 6.3x-10x/0.30-16x/0.40-25x/0.45-40x/1.0_Oil (albeit not with the same level of success), raising or lowering the ring as needed. Chromatic aberrations are obvious especially from the combination of Olympus objectives and the photo Zeiss KPL eyepieces (and camera lens).

(Citation: "...One Ring to rule them all...and in the darkness bind them" - J.R. Tolkien).

The DF images are of a 70 micrometers long diatom in a (too) dense strew slide. All were recorded afocally at iso 1600, -3 correction, aperture priority. They are single frames and were zoomed to reach the same diatom size, cropped and resized, no other processing.

Suggestions for other test objects, besides diatoms and spider webs, and criticism are welcome...

results are shown below
Attachments
LED ring for DF.jpg
LED ring for DF.jpg (47.69 KiB) Viewed 10489 times
Micro_20180317_213157.jpg
Micro_20180317_213157.jpg (165.2 KiB) Viewed 10489 times
Micro_20180317_213401.jpg
Micro_20180317_213401.jpg (166.1 KiB) Viewed 10489 times
Micro_20180317_195306.jpg
Micro_20180317_195306.jpg (120.73 KiB) Viewed 10489 times
Last edited by Hobbyst46 on Sun Mar 18, 2018 1:54 pm, edited 2 times in total.
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Re: A DIY LED ring for darkfield

#2 Post by Hobbyst46 » Sun Mar 18, 2018 1:43 pm

recorded images
Attachments
OlympusSPlan10x0.30.jpg
OlympusSPlan10x0.30.jpg (32.17 KiB) Viewed 10487 times
ZeissNeofluar16x0.40_Ph2.jpg
ZeissNeofluar16x0.40_Ph2.jpg (34.16 KiB) Viewed 10487 times
ZeissNeofluar40x0.75_Ph2.jpg
ZeissNeofluar40x0.75_Ph2.jpg (29.74 KiB) Viewed 10487 times
ZeissPlanapo40x0.6-1.0_oil.jpg
ZeissPlanapo40x0.6-1.0_oil.jpg (38.92 KiB) Viewed 10487 times
ZeissPlanapo40x0.6-1.0_oil uncropped.jpg
ZeissPlanapo40x0.6-1.0_oil uncropped.jpg (59.8 KiB) Viewed 10487 times
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Re: A DIY LED ring for darkfield

#3 Post by Hobbyst46 » Sun Mar 18, 2018 1:45 pm

And another one, this one with an Olympus 40x1.0 Planapo Oil with iris

I should have stressed that this gadget is empirical, not based on any calculaions ("it is experimetal" - says Q).
Attachments
OlympusDP40x1.0PlanapoI_iris.JPG
OlympusDP40x1.0PlanapoI_iris.JPG (40.31 KiB) Viewed 10486 times
Last edited by Hobbyst46 on Sun Mar 18, 2018 4:14 pm, edited 1 time in total.
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Re: A DIY LED ring for darkfield

#4 Post by MichaelG. » Sun Mar 18, 2018 2:06 pm

Looking good !!

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Re: A DIY LED ring for darkfield

#5 Post by Hobbyst46 » Sun Mar 18, 2018 2:41 pm

Thanks michael
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Re: A DIY LED ring for darkfield

#6 Post by MicroBob » Sun Mar 18, 2018 7:11 pm

Hi Doron,

as always a very interesting and innovative post from you! I'm happy to see that your experiment was a success. There is always a point when a practical test is more useful than trying to get the problem evaluated in a theoretical way.
I like your idea with the central cylinder. I can imagine that experiments with reflecting surface of the cylinder or other shapes than a cylinder could further improve the performance.

May be you continue with an immersion dark field version.

Bob

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Re: A DIY LED ring for darkfield

#7 Post by Hobbyst46 » Sun Mar 18, 2018 7:37 pm

MicroBob wrote:Hi Doron,
I can imagine that experiments with reflecting surface of the cylinder or other shapes than a cylinder could further improve the performance.
Thanks Bob!
Yes, I already tried a reflective aluminum foil wrap the cylinder's outer surface but it did not change anything. Apparently a curved sophisticated reflector (i.e. cardiodide.. :lol: )
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Re: A DIY LED ring for darkfield

#8 Post by Hobbyst46 » Mon Mar 26, 2018 8:28 pm

The latest discussed item in this post is the Whole Slide Imaging darkfield version, where side-ways illumination into the slide and the resulting total internal reflection of the light beam inside the slide illuminate the specimen indirectly and create DF without condenser, as described ("DIRI technique") in a 2013 article by Kawano et al. It is said to provide very good DF.
If a commercial version of the apparatus is expensive, then why not DIY...
I tried the following: drilled a hole in the dome of a superbright 5mm diameter white LED (18,000 mcd, 30 degrees beam, about 16mA in the circuit). Stuck one end of a 600micrometer silica (glass) fiber optic inside, placed a diatom slide on the stage, and juxtaposed the free end of the fiber against the edge at a right angle.
Some weak DF was observed, but the light intensity was too low. Apparently the LEDs must touch the slide.
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Re: A DIY LED ring for darkfield

#9 Post by MichaelG. » Mon Mar 26, 2018 8:36 pm

Hobbyst46 wrote:... Some weak DF was observed, but the light intensity was too low. Apparently the LEDs must touch the slide.
Thanks for trying the experiment
... a 'failure' is still informative !!

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Re: A DIY LED ring for darkfield

#10 Post by Hobbyst46 » Mon Mar 26, 2018 9:29 pm

Maybe I will try to contact Everlight. But in the meantime:

From their Technical Data Sheet, I could not figure out whether the LEDs are separate units, so each one must be handled individually (have to find a Stereo microscope for that) or are they in a LED strip. The latter possibility is unclear since in the scheme of the reel, page 11, the LEDs themselves are parallel to each other (??) so they cannot be arranged along the slide edge? On the other hand, if the number of LEDs per reel varies and can be specified by the customer, does that mean that the basic unit is a strip of several LEDs? I am confused. I will give them a chance to explain...
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Re: A DIY LED ring for darkfield

#11 Post by MichaelG. » Tue Mar 27, 2018 4:14 am

The reels [as supplied by Everlight and similar] are for use in assembly machines [*]
... The tape is just a carrier for the components: Think of a machine-gun belt !!

MichaelG.
.

[*] Skim through this video https://m.youtube.com/watch?v=dXlhHkuh-6Q
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Re: A DIY LED ring for darkfield

#12 Post by MichaelG. » Tue Mar 27, 2018 5:30 am

Hobbyst46 wrote:I tried the following: drilled a hole in the dome of a superbright 5mm diameter white LED (18,000 mcd, 30 degrees beam, about 16mA in the circuit). Stuck one end of a 600micrometer silica (glass) fiber optic inside, placed a diatom slide on the stage, and juxtaposed the free end of the fiber against the edge at a right angle.
As an experiment: It may be worth coupling the fibre to the slide with immersion oil.

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Re: A DIY LED ring for darkfield

#13 Post by Hobbyst46 » Tue Mar 27, 2018 6:45 am

MichaelG:
Thanks for the explanation about the reel.
: It may be worth coupling the fibre to the slide with immersion oil
Yes. I thought of polishing the ends of the fiber but oil is far easier.
So I tried oil and, indeed, the light intensity was enhanced somewhat (can't tell quantitatively).

And another trick - cutting off the epoxy ball lens of the LED, so that the LED die itself is very near the front surface of the transparent epoxy package. I polish the surface, then temporarily attach the fiber tip to the surface. This way, the slide is much more illuminated (visually) and I see a fairly nice DF with the 40x0.75 dry objective. And the 40x1.0 oil, with iris reduced to 0.6. Not with the 63x1.4 oil without iris (as expected). So, a 600micron is usable - but the fiber beam is narrow, unlike the side-view LEDs.

Moreover, I see some DF with the 63x1.25 Oil Neofluar Ph3 THAT HAS NO IRIS. So DF at NA=1.25 oil immersion without condenser. Although it is a non-uniform DF, since only one fiber illuminates the slide, still... So maybe remove the lens from 6-8 LEDs as above, attach 6-8 respective fibers, align them all in an array on a flat thin metal platform, like a communication flat ribbon, and position perpendicular and touching the long side of the slide. However, it will remain sort of oblique, the beams coming just from one side. On the other hand, tiny 0.4mm LEDs can perhaps be positioned against the front as well as the back side of the slide.
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Re: A DIY LED ring for darkfield

#14 Post by Hobbyst46 » Tue Mar 27, 2018 9:12 pm

Having contacted both Everlight and Citizen, now waiting for quotations...
The advantage of the tiny LEDs appears to be that they can be placed on both opposing sides of the slide; the 0.6mm core fiber optic gave DF even with NA of 1.25 oil, but can only reach the slide from the front...
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Re: A DIY LED ring for darkfield

#15 Post by MichaelG. » Tue Mar 27, 2018 9:44 pm

Thanks for the updates ... That's great progress.
.
MichaelG.

sorry; I'm not in a position to offer practical support at the moment
... but I'm with you in spirit !!
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Re: A DIY LED ring for darkfield

#16 Post by MichaelG. » Tue Mar 27, 2018 11:50 pm

Update:

I have just found an interesting follow-up paper:
http://journals.plos.org/plosone/articl ... =printable

This covers 'Color DIRI' and uses larger LED assemblies.
... NeoPixel sticks, from AdaFruit

MichaelG.
.

Edit: The NeoPixel ID given in the paper [14268] appears to be wrong,
but I am almost certain that this is the one
https://www.adafruit.com/product/1426

Edit: Another variant of the NeoPixel array is now available
where each unit contains Red Green Blue and White LEDs
https://www.adafruit.com/product/2868

This all looks VERY promising
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Re: A DIY LED ring for darkfield

#17 Post by Hobbyst46 » Wed Mar 28, 2018 12:33 pm

MichaelG
This all looks VERY promising
There are the altogether three Kawano et al. papers that involve the WSI. In the first one (K1) they utilized the 0.4mm LEDS for a slide. Either unidirectional or multi-directional Illumination - I am not sure. In the second paper (K2) they switched to a dense rigid LED strip (the Adafruit) for microfluidics. The microfluidic slab is a PDMS (kind of silicon rubber) slab, of thickness 2mm. The LEDs, however, appear to be large - 5x5mm if I am correct. In the third paper (K3) they remained with the strip and apparently back to the slide. It was unidirectional illumination - just from one edge of the slide. They admit, and I can easily see it in their results (K3), that they obtained non-symmetrical oblique illumination, because of that.

For our present purpose, ignore the RGB vs white vs RGB+white. IMO, a high-contrast DF requires an accurate beam sideways, into the slide, and as little as possible above-the-slide and below-the-slide beams. From my crude experiments by holding either a 5mm LED or a 0.6mm fiber optic against the slide edge, it is a challenge. Larger LEDs are not necessarily better. The Adafruit is similar to my flexible LED strip (except that it is 2.5x as dense). But placing the flexible strip against the edge of the slide did not provide good DF. Unless the LED die itself is much smaller than 5x5mm in the Adafruit...And how warm would the slide be when in touch with the LEDS?

When I look at the picture of the Neopixel 8-LED stick in the Adafruit site, it appears that when the stick is positioned so that the LEDs shine horizontally into the slide, the height of the LED die above the stage is 4-5mm. Can the light enter the slide without being reflected from above as well, directly into the objective?
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Re: A DIY LED ring for darkfield

#18 Post by MichaelG. » Wed Mar 28, 2018 3:55 pm

Hobbyst46 wrote:The LEDs, however, appear to be large - 5x5mm if I am correct. [ ... ] Unless the LED die itself is much smaller than 5x5mm in the Adafruit...And how warm would the slide be when in touch with the LEDS?
1. Quoting directly from the paper: The distance between the blue and green LEDs is about 1.2 mm. The red LED is placed in between the blue and green LEDs. [*]

2. From personal experience: The heat output from the illuminating side of most LEDs is negligible; any stray heat is output in the other direction and can be easily dissipated.

3. As mentioned yesterday, I am not currently in a position to do any practical experiments; but I hope to try these devices in a couple of months time. ... Meanwhile I follow your investigations with great interest.

MichaelG.
.

[*] I note however, that there are design variations appearing ... so we may need to be selective !
This appears to be the useful layout, that is referenced in the paper:
https://www.sertronics-shop.de/Baueleme ... LED-weiss/
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Re: A DIY LED ring for darkfield

#19 Post by Hobbyst46 » Wed Mar 28, 2018 6:45 pm

MichaelG
1. Quoting directly from the paper: The distance between the blue and green LEDs is about 1.2 mm. The red LED is placed in between the blue and green LEDs. [*]
I think that there is no contradiction, the 3 small R G and B LEDs reside inside the 5x5mm square, but they are still high (3mm?) above the stage. I re-checked the layouts in the Adafruit datasheets. And of course I may be wrong.
3. As mentioned yesterday...
Your contribution so far is great and very valuable, thanks! mind storming yields understanding of the challenge. Experiments to be continued when the tiny LEDs become available.
I agree that the designs in K1-K3 are tailored and should be scrutinized.

--->Again, in paper K1, the tiny LEDs, do you think that they are used to illuminate the slide from all four edges, or just the front and back edges, or only the front? I could not decide, and it probable affect the design. How many edges are required to obtain decently symmetrical DF.
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Re: A DIY LED ring for darkfield

#20 Post by MichaelG. » Wed Mar 28, 2018 9:17 pm

Hobbyst46 wrote: 1. ... I think that there is no contradiction, the 3 small R G and B LEDs reside inside the 5x5mm square, but they are still high (3mm?) above the stage. I re-checked the layouts in the Adafruit datasheets. And of course I may be wrong.
I'm sure you are correct, but I see that only as a simple engineering issue [put the slide on raising blocks]
Hobbyst46 wrote: 3. ... mind storming yields understanding of the challenge. Experiments to be continued when the tiny LEDs become available.
I agree that the designs in K1-K3 are tailored and should be scrutinized.

--->Again, in paper K1, the tiny LEDs, do you think that they are used to illuminate the slide from all four edges, or just the front and back edges, or only the front? I could not decide, and it probable affect the design. How many edges are required to obtain decently symmetrical DF.
If I recall correctly; they first tried illuminating at both ends of the slide [effectively replicating the MVI DarkLite's configuration].
... I have a couple of 'bus journeys tomorrow, so will re-read the set of three papers in sequence.

Keep up the good work
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Re: A DIY LED ring for darkfield

#21 Post by Hobbyst46 » Wed Mar 28, 2018 9:39 pm

With the fibre optic I can illuminate from two perpendicular edges, the front and the left side.
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Re: A DIY LED ring for darkfield

#22 Post by zzffnn » Wed Mar 28, 2018 11:13 pm

Very nice work, Hobbyst46!

I thought about buying or making something similar to that MVI Darklite too. But I did not proceed, as it seemed difficult to make it universal in terms of mechanical fitting. What I did instead was modifying 3rd party darkfield condensers to fit on different scopes.
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Re: A DIY LED ring for darkfield

#23 Post by Hobbyst46 » Thu Mar 29, 2018 6:31 am

Thanks a lot zzffnn!
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Re: A DIY LED ring for darkfield

#24 Post by MichaelG. » Thu Mar 29, 2018 10:03 am

MichaelG. wrote:
Hobbyst46 wrote:If I recall correctly; they first tried illuminating at both ends of the slide [effectively replicating the MVI DarkLite's configuration].
... I have a couple of 'bus journeys tomorrow, so will re-read the set of three papers in sequence.
Apologies: My recollection was wrong
... In all three papers; a single row of LEDs is shown illuminating just one long edge of the slide.

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Re: A DIY LED ring for darkfield

#25 Post by Hobbyst46 » Thu Mar 29, 2018 10:38 am

MichaelG. wrote:... In all three papers; a single row of LEDs is shown illuminating just one long edge of the slide.
Then it was apparently oblique illumination, just sufficient to create DF like effects. To imitate it, unidirectional beam is adequate in principle.
Although internal reflections add some divergence.
The CL-435 diodes are not for sale in small quantities, but besides that, soldering individual 1-2mm LEDs is no piece of cake either.
So I am back to a "normal" LED strip. But not the Adafruit Neopixel, I am not enthusiastic about raising the slide on blocks.

Instead, I plan to lower the strip somewhat below the surface of the stage, so the center of each LED will be at exactly the same height as the slide's edge.
For that to happen, the LED strip should be attached to a vertical carrier or "wall" that is lower than the stage surface. I devised an appropriate simple carrier. And found a dense LED strip that can be cut to 2.5cm sections, 3 LEDs per section. The 2.5mm width is small enough to fit inside the hollow channel of the stage below the objective. It will not illuminate the whole slide like in the K1-K3 papers, just the area below the objective. I hope it all makes sense... The LEDs shall be masked with black plastic if necessary.
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Re: A DIY LED ring for darkfield

#26 Post by MichaelG. » Thu Mar 29, 2018 6:18 pm

Hobbyst46 wrote:
MichaelG. wrote:... In all three papers; a single row of LEDs is shown illuminating just one long edge of the slide.
Then it was apparently oblique illumination, just sufficient to create DF like effects. To imitate it, unidirectional beam is adequate in principle.
Although internal reflections add some divergence.
I had an interesting [but probably difficult and/or expensive] idea ...
With one long edge illuminated; the other three edges could be polished and silvered.
... This would bounce the light around inside the slide and should result in illumination from every point of the compass.

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Re: A DIY LED ring for darkfield

#27 Post by Hobbyst46 » Thu Mar 29, 2018 7:23 pm

I had an interesting [but probably difficult and/or expensive] idea ...
With one long edge illuminated; the other three edges could be polished and silvered.
... This would bounce the light around inside the slide and should result in illumination from every point of the compass.
Funny!! I thought about it yesterday, but declined it. Neither silvering nor coating to create total reflection are practical in this case, because of the chemicals involved, the need to treat the slide edge only, the need to preserve the coating for a while afterwards and the mere thought of dependence on specially treated slides...
Perhaps one can place a LED strip against the opposite edge as well. I hope that the strip will provide 3-4 LEDs per edge. The Adafruit Neopixel is wonderfully dense, 8 LEDs per 5cm, but 5cm is too long.
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Re: A DIY LED ring for darkfield

#28 Post by MichaelG. » Thu Mar 29, 2018 11:53 pm

Another 'side-emitting' LED of reasonably suitable size; with a good datasheet:
https://dammedia.osram.info/media/resou ... 20Y87C.pdf

I have just ordered Qty.20 for £4.99 including postage, from:
https://www.ebay.co.uk/itm/Neutral-Whit ... tem4679932

... I now need to learn to solder small things :shock:

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Re: A DIY LED ring for darkfield

#29 Post by Hobbyst46 » Fri Mar 30, 2018 7:01 am

WOW!!! my surfing did not find anything like that, maybe since I was only using the keyword "sideways".
These appear to be almost perfect, and the data sheet is superb. And you could get a small quantity! I am sure that luminosity of 450mcd is even an overkill.
They can be seated on a flat thin (0.2mm at the most) base plate on the stage, or (maybe better) - hung from a base plate. Soldered, then the plate is placed inverted on the stage. Then the base plate can be thicker, say 1-2mm or more.
And the really insane innovation: hung from a screw-driven plate, such that the height of the LED relative to the slide can be optimized per objective. But that can be left for the future.
I do not know why but all makers of tiny LEDs recommend to solder them ASAP after opening the package, and do that under dry atmosphere. Under a stream of nitrogen...they try to avoid both moisture and oxygen? I can try to think about alternatives, considering hand-soldering under a stereo scope...
if this tiny LED gadget is realized, I think that it will be much better than the LED strip track that I am following!
Apparently in the Kn papers they turned to LED strips in order gain programability, sequencing colors etc.
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Re: A DIY LED ring for darkfield

#30 Post by MichaelG. » Fri Mar 30, 2018 8:43 am

Hobbyst46 wrote:... I do not know why but all makers of tiny LEDs recommend to solder them ASAP after opening the package, and do that under dry atmosphere. Under a stream of nitrogen...they try to avoid both moisture and oxygen? I can try to think about alternatives, considering hand-soldering under a stereo scope...
Soldering, at these scales, has become a very sophisticated technical process and, for production purposes, they strive for 'Six Sigma' quality.
This 83 page document is way beyond my level, but it does give us a glimpse of how 'they' do things and helps me to understand why our modern mass-produced electronic devices are so reliable.
https://www.onsemi.com/pub/Collateral/SOLDERRM-D.PDF

Expediency, of course, dictates that I will be doing things differently ...

MichaelG.
.
.
Edit: Here is a useful note from Osram, about manual soldering of their LEDs :
https://dammedia.osram.info/media/resou ... uctors.pdf
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