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Episcopic TTL microscopy compared Bio vs Industrial

 
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JohnyM



Joined: 24 Dec 2013
Posts: 448

PostPosted: Tue Feb 12, 2019 1:08 am    Post subject: Episcopic TTL microscopy compared Bio vs Industrial Reply with quote

First of all - images are very soft. Magnifications tend to be higher, while NA's tend to be smaller than bio objectives. But working distances are longer
Test subjects are: Bio - semi transparent, semi reflective - Pinus leaf. Industrial - completly opaque - coin.
Now results:
1) Darkfield (Top row) - might be useful in some bio aplications. Images are vibrant and contrasty, but produce lots of specular higlights.
Works briliantly with opaque subject.
This technique fall apart with more complex 3D subjects as light path is obscured by subject itself. Very light inefficient.

Brightfield (Bottom row) - Bio images are hazy, contrast is medicore, they absolutely lack color, highlights are terrible. Unusable.
Works briliantly with opaque subject.
This technique works well with any kind of subject structure, best light efficiency of them all (50% of light that stuck the subject).


2) Polarizer in (Top row) - very slight improvement over brightfield in both aplications at cost of 1 stop of ight.
Crossed polars (Bottom row) - That's the way to go with Bio
Vibrant, contrasty, very light inefficient.


3) Tint plate... (Top row) tints everything to user selected color... with some parts of the subject being slightly different color. Special technique, might work well with some subjects. Exposure is actually faster than crossed polars.

DIC (bottom row) - absolute disaster with bio subjects.
Works well with opaque subjects, but lack that "wow" effect seen with bio dic.


4) Fluorescence - usually used to detect contamination in industrial subjects. This time, it produced no image with coin (was cleaned before imaging) while bio subject shines nicely as supposed. Used cube was B-2A.

DIC + tint plate - AFAIK in this configuration it's used to detect how subject vary in height by color change.





TL DR: if interested in biology only, stick with brightfield illuminator with polarizers. Rest of techniques are costly and next to unusable.
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abednego1995



Joined: 01 Nov 2016
Posts: 37

PostPosted: Thu Feb 14, 2019 1:23 am    Post subject: Reply with quote

Splendid comparisons!
What are the magnifications you worked with here? I tend to see low mags in brightfield with lower contrast (probably internal reflections of the objective masking the sample reflections), but that improves with higher mags (higher NA).

Cheers,
John
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ChrisR
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Joined: 14 Mar 2009
Posts: 8161
Location: Near London, UK

PostPosted: Thu Feb 14, 2019 3:47 am    Post subject: Reply with quote

Interesting:smt023 . Impressed by Crossed polar bio. I daresay epi DIC will appeal to coin folk.
Thanks.
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JohnyM



Joined: 24 Dec 2013
Posts: 448

PostPosted: Thu Feb 14, 2019 5:19 am    Post subject: Reply with quote

Thanks for input. Thought nobody is interested with it.
First comprarision was done with Nikon BD Plan DIC 5x/0.1 through 1,5x teleconverter on APS-C A6500 sensor.

Indeed, things look better with higher magnifications, but i have a different theory behind why it's like that.

Here's comparision with Nikon BD Plan ELWD 50x/0.5 through 1,5x teleconverter on APS-C A6500 sensor. Subjects are:
Bio: fern leaf, bottom side.
Industrial: precision m5 shim

Darfkfield and brightfield


Polarization and DIC


DIC with tint plate and Fluorescence*
This time industrial subject shines... with bio contamination. AFAIK that's main use of Fluo in wafer industry, to identify if it's and error or contamination.


My observations are identical as with 5x, but indeed everything does looks slightly better imo.
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JohnyM



Joined: 24 Dec 2013
Posts: 448

PostPosted: Thu Feb 14, 2019 5:43 am    Post subject: Reply with quote

Subjects respond VERY well to sharpening and "dehaze" feature of CameraRAW. Here are 100% crops from bio parts, that were optimally (usually heavy) processed with those techniques.

Reason why bio is problematic with epi TTL is because it's partially reflective and partially transmitting. Why we usually get 2 competing images. One of surface and one of inside of cells. "Inside" usually provides the color, and "outside" texture. Sadly, those are usually mutually exclusive.

Of course, this isn't strict rule. I suppose arthropods, with their reflective armor will respond very well to this technique and will be next follow-up.

Darkfield

Here we can see, there is some nice diffused light that create image of the internal parts, while ignoring cell surface. But "proper" darfkield light, as indended, creates enormous higlights in every surface dent. And those are everywhere in bio subjects. Should be usefull in some subjects. I imagine insect eyes or hairs should work well. To be tested.

Brightfield

Here we can see, that we got very little of "inside", and very much of "outside". In this case outside is rather flat and uninteresting.

Crossed polars

Basically a brightfield with reflections killed. Very nice image of the "inside" with "outside" practically invisible.

DIC

Now, in contrary to test with 5x, DIC does seem like an improvement over plain brightfield. I need to take a closer look at it in the future.
It seems like it's reversed cross-polar (which is quite funny actually...)
Killed all the "inside", while making "outside" more pronounced. I can imagine, it might be usefull with some subjects. Could be a "killer" with some insects and as Chris pointed, coins.

Tint plate DIC

Only if one loves colors.
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