Looking “inside” a silkworm using Frustrated Total Internal Reflection

[Ramon]

Hi Holger

3. If you have a surface coated half transparent mirror at hand it would be interesting what happens if such a surface narrows down to the cover slip during evaporation. (Silvered surface facing your device.)

Let me  know if this is what you mean.

Repeat the dry out with some "textured" transparent probe-> Diatoms.

Can you let me know where I can buy this "textured" Diatom specimens?

5.Repeat this with some fine sized fluorescend particles->pollen and/or tumeric powder

Regarding the Pollen; can I use pollen collected from the enviroment or we need Pollen prepared on a slide?

6. Use small glass beads suspendet in water.

Let me know if this will be OK for the experiment. Regarding the glass beads; how should I mount them? Should I use the immersion oil or water? If in oil or water the refraction index of the media and the glass beads will be pretty similar. We may have no reflection or reffraction. Will we see anything?

If you want, I could send you a small amount of each of them, as well as some strew diatom material, mentioning above.

Lets see if I can get all the material we need. Regarding the specimens on slides; when I use the small version of the FrTIR device (Diameter 86mm) I have to cut the slide for it to fit on top of the acrylic light guide.

If I cannot get the proper material I will ask you.

Ramon

[Spectrum]

Hi Holger

3. If you have a surface coated half transparent mirror at hand it would be interesting what happens if such a surface narrows down to the cover slip during evaporation. (Silvered surface facing your device.)

Let me  know if this is what you mean.

No, I mean a small semitransparent mirror made of thin glass.
They are used as beamsplitters in optical instruments.
You can buy them cheap online (send you a PM about that)

Repeat the dry out with some "textured" transparent probe-> Diatoms.

Can you let me know where I can buy this "textured" Diatom specimens?

Best is to ask the diatom community here in the forum.
You need a solution of them.
Best would be some plain marine probe with some big flat species with a grid pattern like Cosconidiscus, or similar. I hasn't got to be something fancy, but it must be unmounted and still diluted in water!
I have some selfmade freshwater species in water here, that will also do the job.
(I hope so)

Regarding the Pollen; can I use pollen collected from the enviroment or we need Pollen prepared on a slide?

Yes.
Some plain fresh collected pollen from a flower will do the job.
Dandelion or Lily, whatever.
Separate the pollen grains and dilute them in a drop of water on a slide, cover glass...done.
Try tumeric powder (kitchen) as well.
This test specimen should show fluorescence under the blue light included in the spektrum of your LED's. If there is some TIRF fluorescence going on with your device, it should show up here.
(And in a much more standardized way, than in your fixed mounted fern...)

6. Use small glass beads suspendet in water.

Let me know if this will be OK for the experiment.


[/quote]

No.
You need so called "microsphere" glass beads-> PM you about that.

Regarding the glass beads; how should I mount them? Should I use the immersion oil or water? If in oil or water the refraction index of the media and the glass beads will be pretty similar. We may have no reflection or reffraction. Will we see anything?

For all test specimen just use a clean plain glass cover slip.
The specimen (pollen, micro beads, diatoms) should be mixed with one drop of destilled water and covered with a cover slip.
That's all.
Mount this cover slip via immersion oil (under the cover slip) on your device and record what you see.
The water will evaporate after some time.
Then the gap between the cover glass and the cover slip will be getting thinner and thinner...
Pressing the specimen more and more on the slide.
If there is some "Frustrated Total Internal Reflection" happening, it will show up.
Wait till all the water is evaporated and show your results...
(For the first experiment just use water, and for the half silvered mirror test, use the mirror instead of the cover glass)

Got it?

Greetings Holger

[Spectrum]

Hello Ramon,
Found a very informative article about a very similar device, based on the same principle:

https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0058344

Definetely worth a look.
A lot of your questions concerning the properties of using this kind of lighning for biological specimen are answered there.
Greetings Holger

[Ramon]

Alo Holger

I saw this device before. Guys from Photomacrography.net showed it to me. Also, see similar devices made by amateur microscopists:





Thanks for pointing it out! Indeed this device works by the same principle than the Frustrated TIR device I developed. However, there are differences. In the FrTIR device the light guide is designed at will to provide the proper spatial distribution of light. And another difference is that with the with the FrTIR device you can investigate transparent and opaque samples.

I am going through your last post about the IC lithografies. Let me study it and I will come back to you.

Be well Holger, it is a pleasure to engage in these discussions

Ramon

[Ramon]

in my view, the complementary colours you see in brightfield vs. your glazing incidence illumination are caused by the relation between absorption coefficient and reflection coefficient. In physics, these relations are dealt with by the so-called Kramers-Kronig relation: If you know the absorbance of a material over a broad wavelength range, you can calculate the reflection from that.

In consequence, this leads to the fact that a strong absorbance in a given wavelength range leads to a weak reflection in this range. This means e.g. that gold is yellow in reflection, but blue in transmission. If you have a very thin gold layer, this becomes easily visible. You can also try that out by using a blue pen and drawing a 5x5mm^2 square. If you look at this square from a small, glazing angle, the square will reflect in reddish colour.

In daily life, this phenomenon is easily overlooked, e.g. leaves are green in transmission, but also in reflection. But actually this presumed reflection is caused by scattering of the light within the leave, and not by "physical reflection" at the surface.

Jürgen

Hello Jurgen

I think I finally figured it out. Below find a summary of all that is happening in four scenarios. Fresh leaf under ambient and FrTIR light and Fearn leaf under ambient and FrTIR light.



Be well
Ramon

[Ramon]

Hello Jurgen and all

I made a mistake. Pls., let me correct it. In Fresh Leaf/Extremely oblique lighting/ Energy balance says; Colour stays Green. It should say; if wax cuticle surface layer is not removed colour stays green

Sorry about that. I corrected it in the draw below:



Ramon