A DIY LED ring for darkfield

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wporter · #31 Post by **wporter** » Fri Mar 30, 2018 5:32 pm

Jumping on the "edge-lit slide" bandwagon, here is a cheap way that I came up with, to experiment with the technique, which won't cost an arm and a leg. It involves modification of a pocket LED flashlight. As the test exposures show, it gives acceptable results. The fabrication involves edge-gluing a slide to the window of the pocket light to be able to funnel the light to another slide that has the specimen on it.

First, procure the pocket light (image 1)

The light has a strip LED which is quite bright (130 lumens) (image 2)

Next, get a slide, and superglue one long edge to the window of the light (image 3)

After the glue has set (I gave it a few hours; breathe on the joint periodically to accelerate the cure), add a fillet of black silicone RTV over the glue joint for additional support and to block the light from the LED from coming out anywhere other than through the slide (image 4 & 5)

More in my next post, see below:

wporter · #32 Post by **wporter** » Fri Mar 30, 2018 5:56 pm

Page 2:

After overnight, test the light about 1/8" (3mm) off a piece of paper, and note that the light emitted is restricted to a very narrow band, the thickness of the slide (image 5)

Mount an interesting specimen on the microscope, and lay the edge of the light-slide against the specimen slide and prop it in position. One could oil the slides where they touch, for better light transmission, but for the following images, I did not do this (image 6). Note the fingerprints visible on the specimen slide, very well lit-up.

Here is an image taken with the edge lite (image 7)

Here is one of the same specimen (an abused slide micrometer scale) taken using an AO-Reichert 1096 immersion darkfield condenser (image 8)

Taken on a Reichert Diastar with a 10x plan fl apo. Not really a fair 'comparison', as the light is coming from different directions, have different intensities, & there are different effects expected, etc., but interesting nonetheless.

MichaelG. · #33 Post by **MichaelG.** » Fri Mar 30, 2018 6:13 pm

Nice demonstration ... Thanks !!

MichaelG.

Hobbyst46 · #34 Post by **Hobbyst46** » Sat Mar 31, 2018 11:56 am

wporter wrote:Page 2:
Taken on a Reichert Diastar with a 10x plan fl apo. Not really a fair 'comparison', as the light is coming from different directions, have different intensities, & there are different effects expected, etc., but interesting nonetheless.

This is a simple and effective demonstration, much easier to do than my fiber optic experiment!
The challenge as I understand it is to provide a lot of light but tailor the angle of incidence to prevent direct rays into the objective. You have demonstrated that for the plan apo 10x (NA round 0.30 I guess?) the DF condenser performs somewhat better.

Could it be that for a high NA objective, the flahlight+slide illuminator performs better than that particular condenser?
It might also be instructive to repeat you setup but with a thinner slide, say 0.5mm thickness, and see subtler differences (with different objectives). Or alternatively, instead of such non-standard slides, with coverslips no. 4 (are they being sold??), thickness ca 0.4mm, as illuminator plates.

I mimicked your setup with a stage micrometer as specimen and a single sawn-off 18cd 5mm LED, instead of flashlight. Thereby obtained co-planarity of the two slides. Illumination seemed to be fine. However, DF was very weak. When the light slide was inclined, similar to your experiment, DF with a 16x0.40 objective was better, but with a 40x0.75 it was again very weak. I suspect that fine tuning to achieve really good DF relies on narrower beams than the specimen slide thickness.

Using oil interface between the two slides will probably help, but I think it is not very convenient as a routine.

Hobbyst46 · #35 Post by **Hobbyst46** » Mon Apr 02, 2018 10:09 pm

Here are some more darkfield images. Obtained with the LED ring, that rides on top of the condenser, without any other illumination. The ring height was optimized for each objective, by using the condenser focusing knob.

The sample is fresh onion epidermis, after a 5min fixation in 70->50 degC water (following W. Dioni), rinsing in 40% fructose and mounting in 85% fructose.

Thus, cells have not been cleared. Images are single frames, resized and cropped to decrease the image by 13% to remove corners. for comparison, one image was AutoCorrected in Microsoft Picture Manager. The LED ring appears to provide somewhat better DF with the 40x1.0 (actually, 40x0.6) oil objective than with the lower NAs. For the 40x0.6 oil, a and b represent two different fields.

Notes: 1. When using the LED ring, I place a 10x14cm dark red filter (transparency) in front of the nosepiece,
to protect against the super-bright LEDs light.
2. This ring design could possibly be improved somewhat, but the big advantage is anticipated from the
"light guide slide" approach. Fiber optic based gadgets work but are too cumbersome with my scope.

MichaelG. · #36 Post by **MichaelG.** » Mon Apr 02, 2018 10:42 pm

Thanks for posting those
... The images from the 40x are particularly pleasing.

MichaelG.

Hobbyst46 · #37 Post by **Hobbyst46** » Tue Apr 03, 2018 3:17 pm

A DF of milk: still image below and a clip that demonstrates Brownian motion - how molecules randomly push the oil droplets to and fro - in the link.
https://youtu.be/Ta1d2t3-r6M

40x1.0 Planapo Oil immersion (iris closed to NA 0.6)
LED ring illumination
A drop of 1% fat milk
raw image, and auto corrected image in Microsoft Picture Manager

MichaelG. · #38 Post by **MichaelG.** » Tue Apr 03, 2018 4:51 pm

Hobbyst46 wrote:... and a clip that demonstrates Brownian motion - how molecules randomly push the oil droplets to and fro - in the link.
https://youtu.be/Ta1d2t3-r6M

Sorry ... It may because I am using iPad, but that video link displays as a black screen, with the words 'This video is unavailable.' in white text.
MichaelG.

Hobbyst46 · #39 Post by **Hobbyst46** » Tue Apr 03, 2018 5:41 pm

@MichaelG

Yes, this is really weird. The clip is opened and displayed correctly on a Windows PC, but not on an Android phone/tablet, and the same error note.

MichaelG. · #40 Post by **MichaelG.** » Tue Apr 03, 2018 9:23 pm

Hobbyst46 wrote:@MichaelG

Yes, this is really weird. The clip is opened and displayed correctly on a Windows PC, but not on an Android phone/tablet, and the same error note.

A quick google search suggests that this has something to do with copyright, and/or advertisements ... The devious mechanics of it are beyond me, I'm afraid.

I will try your link on the Mac.

MichaelG.
.
Edit: Nope! that doesn't work either.
... It begins to look like the youtu.be hyperlink is Windows-specific.

Edit: Just seen your follow-up
... Link now works fine : Thank You.

Hobbyst46 · #41 Post by **Hobbyst46** » Tue Apr 03, 2018 9:36 pm

@MichaelG
The problem with the link to video clip was solved. It was my mistake apparently.

Hobbyst46 · #42 Post by **Hobbyst46** » Wed Apr 18, 2018 11:57 am

The dense LED strip arrived (top one in the photo). Can be cut into three LED, 15mm sections, hopefully will make better rings for darkfield.
Below is the previous strip, used to construct the first ring. And - who knows - shine directly into the slide...

MichaelG. · #43 Post by **MichaelG.** » Wed Apr 18, 2018 1:05 pm

That's a useful-looking strip

One quick question: Is the first LED hidden under the heatshrink sleeve ?

MichaelG.

Hobbyst46 · #44 Post by **Hobbyst46** » Wed Apr 18, 2018 1:21 pm

MichaelG. wrote:That's a useful-looking strip

One quick question: Is the first LED hidden under the heatshrink sleeve ?

MichaelG.

Yes, so it is.

MichaelG. · #45 Post by **MichaelG.** » Wed Apr 18, 2018 1:49 pm

Hobbyst46 · #46 Post by **Hobbyst46** » Tue Apr 24, 2018 8:01 pm

(Continued - hopefully not to bore too much

)

The above described LED ring is useful in that it rides on the top (front) lens of the Achromat-Aplanat condenser, thus its proximity to the slide can be adjusted, which in turn produces acceptable (to me at least) darkfields with 10x0.30, 16x0.32-0.40, 40x0.75 or 40x0.6 iris_oil objectives. So it relies mechanically on the condenser.

Yet, direct illumination into the slide edge has the potential to perform better. Using a 0.6mm (silica) fiber optic attached to a 5mm LED, I had managed to shine into the slide edge in a way that produced even better DF, and some DF even from a 63x1.25 (irisless) objective. But that illumination device is difficult to make (DIY) and cumbersome in use. Tiny ~0.5mm LEDs adjacent to the slide edge would be better, I thought.

So here is a feasibility test.
The 204LED/m flexibel strip is shown in the thread above. I cut a 3.5cm piece and soldered wires to the + and - ends. Fed from a small wall 12V, 1A power supply, it is the illuminator. Made a carrier for it, by cutting and bending a flat 1mm thick aluminum plate. It becomes a 60x60mm plate, with a vertical 10mm vertical edge. The right side of the plate is cut to allow space for the mobile arm of the slide holder of the stage. The illuminator strip, being a self-adhesive tape, is affixed to the vertical edge. When the plate is flat on the stage, the illuminator is easily positioned to illuminate directly into the edge of the slide. The wires pass through the opening in the stage, between the stage and the condenser and away to the power supply. These should be soft and flexible wires - I think gauge 18-20 would be fine. The aluminum carrier is also the heat sink (not much heat anyway).
The LEDs dies are 3mm high. I tried to narrow the light beams by masking with an opaque black PVC electrical isolation tape.
Below are photos of the gadget. In a later post there are images of my (usual) over-crowded diatoms.
The plate height was optimized for each objective. It was either placed directly on the stage, or slightly higher by using a 6mm Lexan oplate as spacer. With 40x0.75 Neofluar, 40x0.6 iris_oil the DF is again acceptable. With 40x0.95 Planapo (dry) contrast is worse, each bright item is surrounded by a wide bright halo. Not recommended (and not shown). With 63x1.25 (irisless) Neofluar the background is gray, not dark enough.
In the last photo of the device, the shadow on the left is just a small metal weight to keep the plate temporarily in place.
One can see the reflected light from the 5 LEDs on the objective.

pros: (1) The LED strip can produce DF at NA of 0.6-0.75, without any condenser. It can cope with lower NAs as well, by making the vertical edge of the carrier somewhat longer so the LEDs will be below the slide edge. (2) It is cheap and is easy to make, provided that you can solder wires onto the tiny end points of the strip. The height of the LED strip can be tailored so as to shine light directly into the slide edge.
cons: (1) no DF with higher NA objectives, (2) not symmetrical so somewhat oblique - but I trust that a similar strip can be placed against the other edge of the slide.

NOTE: EACH OF THESE TINY LEDS IS AN INTENSE LIGHT SOURCE. DO NOT LOOK DIRECTLY INTO IT. Prevent reflections.

Hobbyst46 · #47 Post by **Hobbyst46** » Tue Apr 24, 2018 8:07 pm

(continued - results)

MichaelG. · #48 Post by **MichaelG.** » Tue Apr 24, 2018 9:45 pm

Great demonstration of the concept ... Thanks

MichaelG.

billbillt · #49 Post by **billbillt** » Tue Apr 24, 2018 10:17 pm

Thanks for sharing.. This looks real useful to me... a small prisim may work also...

BillT

coominya · #50 Post by **coominya** » Fri Apr 27, 2018 12:24 am

Nice bit of R&D Hobbyst46, following you progress with interest.

Hobbyst46 · #51 Post by **Hobbyst46** » Fri Apr 27, 2018 7:48 am

@coominya
Thanks, trying to translate other's ideas into DIY gadgets. So far, three of them appear to be reliable: (1) A ~36mm diameter ring, consisting of six LEDs, from 5050-type 60 LEDs/m strip, riding on the top lens of condenser; (2) A ~35mm straight strip, consisting of 4-5 LEDs, from 3014-type 204 LEDs/m strip, affixed to a carrier&heat sink on top of the stage and shining into the edge of the slide; (3) A ~55mm ring, from the 204 LEDs/m strip, on a carrier&heat sink metal ring below the slide - this one is still experimental (quote "Q"), unfinished and undocumented.

coominya · #52 Post by **coominya** » Fri Apr 27, 2018 3:38 pm

Hobbyst46 wrote:@coominya
Thanks, trying to translate other's ideas into DIY gadgets.
- this one is still experimental (quote "Q"), unfinished and undocumented.

Yes... I have a few of those. My lamp (the one I overhauled and cleaned) developed a loose lens-flip mechanism and rather than looking into it I overhauled another one I had on the shelf. I seem to spend a fifth of my time repairing and tinkering, a fifth collecting samples and housing them, a fifth actually observing and 2/5 reading and posting here. When I was using a microscope as a hobby a couple of decades ago I spent ALL my time observing and drawing what I saw. Funny how the internet and money to buy extra stuff actually draws you away from what you got into the hobby in the first place for.

manu de hanoi · #53 Post by **manu de hanoi** » Thu May 03, 2018 8:49 am

In order to get darkfield on x40 and x 100, I tried blasting light (just 3w led) on the edge of a slide a couple months ago, without much success. Even though I got great improvements by oiling the edge.
My understanding is that you get internal reflection (like an optics fiber) in the glass slide if the light has a low angle... then most of the light exits on the other end (as seen in the fixture above), on the other hand light having a big angle will exit the slide too early.

In theory the change from air surface to water should help the light get out of the glass slide into the specimen, but.... that doesnt guarantee you'll get the optimal angle, and most of the light having the proper angle will enter at the outer edges of the sample and not the center etc.... So good luck

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Hobbyst46 · #54 Post by **Hobbyst46** » Thu May 03, 2018 9:17 am

manu de hanoi wrote:In order to get darkfield on x40 and x 100, I tried blasting light (just 3w led) on the edge of a slide a couple months ago, without much success. Even though I got great improvements by oiling the edge...
In theory the change from air surface to water should help the light get out of the glass slide into the specimen, but.... that doesnt guarantee you'll get the optimal angle, and most of the light having the proper angle will enter at the outer edges of the sample and not the center etc.... So good luck

How wide is the light beam from your 3W LED?
To get good darkfield, one needs a narrow beam that shines ONLY into the edge on the slide, not above or below the edge, otherwise direct light rays enter the objective, create a brightly lit background, so no darkfield! Kawano et al have demonstrated it (in articles and commercial devices or prototypes) with 0.4mm LEDs, and there are demonstrations in this thread (above) using an auxiliary slide as light source, and a 0.6mm fiber optic (a decade or so ago others achieved the same or better). It is a fairly challenging job.

Regarding the drawing, please notice that light rays like the rightmost light ray in your drawing, that are not internally reflected but exit the slide, such that they neither hit the specimen nor enter the objective, are irrelevant. The total illumination from the source is so intense that "lost" parts, such as the rightmost light ray in your drawing, are not important. They are not seen, similar to ambient light in the room that (mostly) is not seen through the microscope. The problem is rays that directly, or by reflection from the slide, enter the objective and create a bright background.

manu de hanoi · #55 Post by **manu de hanoi** » Thu May 03, 2018 9:27 am

Hobbyst46 wrote:
manu de hanoi wrote:In order to get darkfield on x40 and x 100, I tried blasting light (just 3w led) on the edge of a slide a couple months ago, without much success. Even though I got great improvements by oiling the edge...
In theory the change from air surface to water should help the light get out of the glass slide into the specimen, but.... that doesnt guarantee you'll get the optimal angle, and most of the light having the proper angle will enter at the outer edges of the sample and not the center etc.... So good luck
How wide is the light beam from your 3W LED?
To get good darkfield, one needs a narrow beam that shines ONLY into the edge on the slide, not above or below the edge, otherwise direct light rays enter the objective, create a brightly lit background, so no darkfield! Kawano et al have demonstrated it (in articles and commercial devices or prototypes) with 0.4mm LEDs, and there are demonstrations in this thread (above) using an auxiliary slide as light source, and a 0.6mm fiber optic (a decade or so ago others achieved the same or better). It is a fairly challenging job.

it was quick and dirty and surely light was going top and below the slide (led was about 3.5mm diameter), I dont recall much of the details but I believe I was testing 40x and 100x (because my regular darkfield setup works fine for 4 and 10x), for these, such top light dont enter the objective (the objective is nearly touching the coverslip)

Hobbyst46 · #56 Post by **Hobbyst46** » Thu May 03, 2018 9:36 am

manu de hanoi wrote:
Hobbyst46 wrote:
manu de hanoi wrote:In order to get darkfield on x40 and x 100, I tried blasting light (just 3w led) on the edge of a slide a couple months ago, without much success. Even though I got great improvements by oiling the edge...
In theory the change from air surface to water should help the light get out of the glass slide into the specimen, but.... that doesnt guarantee you'll get the optimal angle, and most of the light having the proper angle will enter at the outer edges of the sample and not the center etc.... So good luck
How wide is the light beam from your 3W LED?
To get good darkfield, one needs a narrow beam that shines ONLY into the edge on the slide, not above or below the edge, otherwise direct light rays enter the objective, create a brightly lit background, so no darkfield! Kawano et al have demonstrated it (in articles and commercial devices or prototypes) with 0.4mm LEDs, and there are demonstrations in this thread (above) using an auxiliary slide as light source, and a 0.6mm fiber optic (a decade or so ago others achieved the same or better). It is a fairly challenging job.
it was quick and dirty and surely light was going top and below the slide (led was about 3.5mm diameter), I dont recall much of the details but I believe I was testing 40x and 100x (because my regular darkfield setup works fine for 4 and 10x), for these, such top light dont enter the objective (the objective is nearly touching the coverslip)

From my experience, even with the smaller 3014 type LEDs (the chip is 3.0x1.4mm) send too much light into the objective and mask the darkfield effects. There are many reflective surfaces around the slide, and it may be difficult to prevent reflections of the source into the objective. Refracted light from the specimen is inherently weak, since a very small fraction of the field of view emits light. Hence, getting a good darkfield, especially with high NA, is quite tricky IMO.

manu de hanoi · #57 Post by **manu de hanoi** » Thu May 03, 2018 9:40 am

@hobbyist your results pictured above are consistent with mine. You had to push the iso (noise...) to see something, that's because most of the light is wasted. I suspect positive results were obtained using a much stronger illumination than mine in the connecting slide fixture, but i'm not even sure, because in the pics shown we see a ruler illuminated. The light enters the glass ruler because there is diffraction at ruler engravings, that's a different situation than a bio specimen on a flat slide.

My experiments, even with the smaller 3014 type LEDs (the chip is 3.0x1.4mm) indicate that too much light enters the objective and masks the darkfield effects

That would be easily dealt with, just put a cardboard tube around the objective

billbillt · #58 Post by **billbillt** » Thu May 03, 2018 8:50 pm

manu de hanoi wrote:@hobbyist your results pictured above are consistent with mine. You had to push the iso (noise...) to see something, that's because most of the light is wasted. I suspect positive results were obtained using a much stronger illumination than mine in the connecting slide fixture, but i'm not even sure, because in the pics shown we see a ruler illuminated. The light enters the glass ruler because there is diffraction at ruler engravings, that's a different situation than a bio specimen on a flat slide.
My experiments, even with the smaller 3014 type LEDs (the chip is 3.0x1.4mm) indicate that too much light enters the objective and masks the darkfield effects
That would be easily dealt with, just put a cardboard tube around the objective

Thanks for the tip!...

Regards,
BillT

Hobbyst46 · #59 Post by **Hobbyst46** » Thu May 31, 2018 5:51 pm

Hello all - back again to this old chewed up thread.

Epilogue of DIY illuminator for darkfield

After more slight improvements of the LED strip, which shines into the edge of the slide, I hope that a summary of its construction is in order, in spite of some repetition of previous text from this post.

A sideways oriented LED strip and an opposing sideways mirror, each on its respective carrier plate, are placed within the stage opening, flush with the two opposing long edges of the slide. There is no need to remove the microscope condenser, just to lower it to make ample space for the LED strip and mirror.

A 204LED/m flexible self-adhesive strip of warm-white 3014-type LEDs is used. A ~30mm piece is cut from the reel, and thin flexible wires are soldered to the + and - terminals. When the LED carrier is mounted on the stage, the wires pass through and below the stage opening, in the space between the stage and the condenser, and terminate at a simple dimmer fed from a small 12V, 1A wall power supply.
The LED strip is affixed, either by its own adhesive back or with a double-sided, 1mm thick, heat resistant mirror-mounting tape, to the 1.5mm-thick aluminum carrier plate. The carrier plate is trimmed and shaped to allow free movement of the spring-loaded slide holder arm, as well as free rotation of the objectives. The carrier plate is drilled and M5 screw nuts are glued to it, to accommodate three height-adjustment M5 screws. Tweaking with the screws helps, but is not essential. Good illumination into the specimen slide is basically obtained with flat, 1.5mm thick metal (preferably) or plastic spacers under the carrier. A pile of 2-4 such spacers raises the LED light beam such that it directly enters the edge of the slide. Darkfield with 10x0.30, 16x0.40, 40x0.75, 25x0.80 (all dry, neofluars) and 40x0.7, 63x1.25 planapo oil objectives (the 63x DF is not as good) was obtained.

The mirror on the mirror carrier should reflect the beam back into the slide, and hopefully provide more uniform illumination. A 1st-surface thin metal mirror (excess material left over from the mirror-replacement project reported in this Forum) is affixed to the carrier with a double sided tape, and is placed against the opposing edge of the slide. The 7mm recess of the Zeiss slide holder made it easy.

An essential feature of the device is the ~0.5mm "slit". The LEDs on the strip are 3.0mm tall - too tall for a 1mm thick slide. Also, the LEDs bulge a little from the strip itself. I used a double-sided mounting tape on each side of the LEDs (only one side is shown in the photo) to create a narrow platform along the LEDs, of the same height as the LEDs. On these adhesive platforms I affixed two parallel black paper strips, at a distance of ~0.5mm between each other, to form a slit and narrow the LED light beam.

Notes:
(1) warm-white LEDs are less bright than cool white LEDs of a similar strip. With neither type, however, are there any important heating effects.
(2) light from these tiny LEDs is extremely bright, take appropriate eye precaution measures.
(3) this adaptation of a research article idea can be done with simple hand tools.

Below are photos of the device and some examples. In the 3rd and 4th photos, the LED strip carrier is on the left of the slide and the mirror holder is on the right side.
Hope it may become useful for others as well - I drew a lot of fun from it.

Hobbyst46 · #60 Post by **Hobbyst46** » Thu May 31, 2018 6:02 pm

And here are some examples. Not ideal I am afraid, but I think they convey the idea. The low magnifications are of the white pappus seed of the local Sonchus plant, mounted in polystyrene-cinnamon bark oil. The high magnification are of something that might have been Monte Kristo cave of treasures or a pile of broken glass - actually a dense strew of marine diatoms. The 40x0.95 (dry) is a difficult objective even at brightfield, but I tried it anyway. Auto white balance was used throughout.