Made my own laser cut darkfield & oblique filters

[Horseflesh]

When I realized I could use my laser cutter to make filter inserts I had to give it a shot. I don't have any thin clear acrylic for Rheinberg illumination yet, but I did have some 1.5 mm opaque black acrylic on hand so I tried making darkfield and oblique filters. If anyone has ideas for new shapes to try I would love to hear it!

https://i.imgur.com/hYqe8Oc.jpg (1.25" filters in 1.5 mm black cast acrylic)

Here are some sample photos. They are cropped in different ways so the magnification may not seem to match the label.

The C-shaped oblique insert definitely improved contrast if you got everything set just right. If you didn't, it might as well not be there.

https://i.imgur.com/hzjkDQI.jpg (aquarium water 400x)
https://i.imgur.com/EemH5Ka.jpeg (aquarium water 1000x)

The other obliques didn't work well at all ... but I am just making guesses here, going off shapes I saw online.

The darkfield inserts were a mixed bag. With the smallest one (5 mm center circle) I had some success but it seemed like the 10 mm and 15 mm versions just blocked too much light. I had to find just the right setting for the condenser height and iris to get a usable image at all. The colors look wild here but it didn't look this garish to the eye... the camera saw it this way and I didn't fix it.

https://i.imgur.com/WXmTyzF.jpeg (aquarium water 400x)
https://i.imgur.com/Qj3ezGY.jpg (aquarium water 1000x)

The darkfield images were just not as sharp as the obliques but I learned after doing this that darkfield is much much harder at high magnification, and not nailing it at 40x is expected. This is day 2 with my own scope! I was pretty happy with the contrast in a few views, though.

I think I can make some good multicolor Rheinberg filters. I'll be ordering clear acrylic in a variety of colors soon.

Scope: Swift 380T with 40x objective and 10x or 25x eyepiece.
Camera: Pixel 3 phone camera, just held up to the eyepiece... no proper camera mount yet. But it's a pretty good camera for a phone!

[crb5]

I assume your scope has a 1.25NA condenser. To achieve this maximum NA you need to have immersion oil contact between the condenser and bottom of slide. In normal dry use in air, the effective NA of the condenser = 1.25/1.51 = 0.82. So with a 40x 0.65 NA dry objective lens, you can achieve dark field by using a large stop diameter and a narrow annulus (corresponding to an NA between 0.65 and 0.82). Maybe using immersion oil on the condenser will give a bit more illumination intensity for dark field. As far as oblique illumination is concerned, I have played around with various shapes but at low magnification (10x objective) I found quite a strong gradient of illumination intensity across the field. One way to improve this was to put the oblique mask closer to the light source and put a diffuse disk in the condenser filter holder (made from translucent scotch tape stuck to some clear plastic cut from a food container). This trick (a form of Dodt contrast) gave more even illumination but retained the 3D effect of oblique illumination.

[Horseflesh]

Yes, 1.25 NA condenser.

I too noticed a strong visible gradient with the oblique insert at lower magnification.

Thanks for the tips! Lots to think about. This makes regular photography look trivial in comparison.

[hans]

I assume your scope has a 1.25NA condenser. To achieve this maximum NA you need to have immersion oil contact between the condenser and bottom of slide. In normal dry use in air, the effective NA of the condenser = 1.25/1.51 = 0.82.

Where does this formula come from? I was trying to figure this out recently but as far as I could understand the NA limit based on internal reflection should be the same independent of the specification for immersed NA. So for example a 1.25 and 1.4 condenser would both have the same NA limit when used dry?

[MicroBob]

Hi,
your laser cut filters or stops look great! Compared to milk carton cut outs they also don't get lost so easily. A good base for these stops are round cover slips that are available in sizes of e.g. 32mm.
The best contrast/resolution combination will probably be available form circular oblique lighting , so with a round center stop of the right diameter. These stops can also be combined with polarisation. I have made a set with interchangeable center stops that you could easily (and better) copy with your laser cutter. Strong oblique lighting sucks up a lot of the available light so the lamp might need an upgrade if you would like to use this intensively or for photography of quickly moving zooplancton. It alsways has to be kept in mind that oblique (and many other contrast techniques) generate contrast at the cost of added artifacts. Some images in this thread: viewtopic.php?f=6&t=11894 show some kind of black protruding matter in the middle of the diatom that in fact isn't there - it is more of a gap.
When using photo stacking all these contrast techniques have to be evaluated from new. A slight oblique lighting scres very well here.

Bob

[apochronaut]

There is an effect in DF , a kind of N.A. dependent flare that tends to obscure details. Much of it is caused by the illumination picking up haze and debris in the sample reflecting and re-reflecting, throwing it into contrast against the background. Even debris on the slide or coverslip becomes part of the illuminated sample and obscures detail. The effect is similar to a shaft of light showing up dust or smoke in the air, when it can barely be seen otherwise.
The camera highlights this.

This flare is enhanced when the minimum N.A. of the hollow cone of light is too close to the N.A. of the objective, yielding a relatively bright background : more of a dusk field than a dark field. Your mask needs to be larger. The best masks I have seen were caps that fitted over the condenser top lens but as with all things DF, precision is paramount.

[Horseflesh]

I was able to get a better dark field in a 400x view with a bigger patch, but that isn't pictured. I didn't realize when I started goofing around that good DF with the 40x objective is so much harder.

It is pretty easy to make patches so I am going to make a whole series where the patch size increases by 1 or 2 mm, so I can find the optimum for each objective.

[crb5]

Where does this formula come from? I was trying to figure this out recently but as far as I could understand the NA limit based on internal reflection should be the same independent of the specification for immersed NA. So for example a 1.25 and 1.4 condenser would both have the same NA limit when used dry?

Hans, you are right to question - I was trying to estimate the stop diameter relative to the maximum condenser iris diameter for a dark field annulus which is about 0.82 for a 40x 0.65 NA i.e. 23 mm for a 28 mm maximum iris and this "off the top of my head formula" was close by coincidence. Several factors contribute to this calculation and a good discussion for practical implementation is given at https://www.olympus-lifescience.com/en/ ... darkfield/. With my Swift 350 scope I found 22 mm stop worked with the 40x 0.65NA objective and an 18 mm stop with the 10x 0.25NA objective.

[Horseflesh]

> With my Swift 350 scope I found 22 mm stop worked with the 40x 0.65NA objective and an 18 mm stop with the 10x 0.25NA objective.

Interesting! With my Swift 380 and the same 40x objective, I have no light left for observation if I use even a 10 mm darkfield patch. It's just black. The bluish image I posted was with a 5mm patch and that was as good as I could get

I'm likely doing something wrong but I wonder what... there are only a few things to adjust for illumination and I turned all the dials I have!

Edit to add: next time I play around I will make a 23 mm patch and try it with an open iris, as you listed.

[Horseflesh]

Darkfield patches version 2, now with numbers and radiused corners.

https://i.imgur.com/Hilijm5.png