Down the rabbit hole… and some questions

Starting out in microscopy? Post images and ask questions relating to the microscope and get answers from our more advanced users on the subject.

Moderators: rjlittlefield, ChrisR, Chris S., Pau

mbf
Posts: 16
Joined: Fri Dec 29, 2023 7:31 am
Location: Germany

Down the rabbit hole… and some questions

Post by mbf »

Dear all,
I just found this forum and as usual for a newbie, there are some questions…

Well, at first, I am not new into photomacrography, as well as “just for fun”, but also for my beloved wife, who is doing some research and works with strange tools. Oh, btw, why can’t her “company” take the pictures? There is no “company”, it is a university. Therefore, there is no money for fancy things beyond basic funding, but that’s off topic.
However, here is a first impression (with a quite old and rather bad photo):
chip_old.jpg
It is a chip made out of glass with an etched inner structure for some microfluidics (it is the same chip as here https://lightfab.de/Microfluidics_Products.html - last picture, at the bottom).
Well, my technique improved and today I generate something like this (not finally retouched):
g9_oly60.jpg
The steps visible in the glass are resulting from the (layered) etching process.
I am sorry, I cannot show the rest of the chip, so it is just an impression. The horizontal “pipe” at the bottom (left side) has an inner diameter of approx. 50 µm, the first cylindrical flange at the right opens to approx. 250 µm before it widens up. This part is used to insert a capillary (I will come back to this ).

Excursion: my standard camera equipment for photomacrography is a Panasonic G9 in combination with an Olympus 60/2.8. My standard software is DXO PL, Helicon Focus and sometimes GIMP.

This works fine for these pictures.
But now I need pictures of the ending of the capillary itself. OD 250 µm, ID 50 µm.
Here is a try, quite unmotivated, I have to admit, because I do not really have a chance for a good picture. It is some kind of okay-ish, sufficient for a technical documentation, but not exactly what we need.
capillary.jpg
In combination with a Raynox 250 a FOV of approx. 7 mm * 9 mm is achievable. This is obviously not sufficient for the capillary mentioned above, so when I am asked by my beloved wife (you remember?), my answer is: “I have a camera, not a microscope.”

Well.

Well, I stumbled over this forum… aaaand… down the rabbit hole. Technically it seems to be easy (which usually makes things difficult later on): grab a finite lens and use a tubus or take an infinite lens and have a look for a nice tubus lens. Fine. There are quite a lot of hints in the forum to pick an adequate lens for both scenarios. I can deal with that.

Using a 10x objective would result in a FOV of 1.3 mm * 1.7 mm, which seems to be much better than what I can achieve now. Furthermore, it is a magnification I can deal with in terms of the size of the stacking steps. Currently I am using a manual micrometer-driven system, which allows steps of 10 µm, maybe a little bit less.

Using an mFT system, I am however deep into diffraction country. As far as I understood, this can be healed up to a certain grade by quite massive sharpening. OK, fine, I will try this. But – could using a camera with a larger sensor (APS-C, FF) help here? Of course, the magnification has to be adapted to keep the FOV. And I have to ensure to cover the full sensor with the objective.

But actually I want to carefully test the new terrain without too much technical and financial effort.

So, I think I am not totally wrong with my approach, but if someone has a valid point like “Ah, err… no…?” I would be glad to hear from you. Or did I miss an important aspect?
-- Matthias

rjlittlefield
Site Admin
Posts: 23508
Joined: Tue Aug 01, 2006 8:34 am
Location: Richland, Washington State, USA
Contact:

Re: Down the rabbit hole… and some questions

Post by rjlittlefield »

mbf, welcome aboard!

What you propose to do sounds fine.

Being deep into diffraction country simply means that your camera sensor will definitely capture all the detail that is present in the optical image created by the microscope objective.

To do any better -- as in capturing finer detail on subject -- you would have to use an objective with higher NA, which would also require a smaller step size to avoid obvious focus banding.

10X is a good place to start.

--Rik

mbf
Posts: 16
Joined: Fri Dec 29, 2023 7:31 am
Location: Germany

Re: Down the rabbit hole… and some questions

Post by mbf »

Hi Rik,
thanks for the warm welcome. Now I need some time to pick up the (optical) equipment.
And, of course, physics is everywhere (and sometimes quite nasty). Everything comes with (physical) price tag.
-- Matthias

mbf
Posts: 16
Joined: Fri Dec 29, 2023 7:31 am
Location: Germany

Re: Down the rabbit hole… and some questions

Post by mbf »

Ok, sometimes things envolve faster than you expected... and, yes, I know "learn to walk before you run."

But I could borrow a Nikon M PLAN 20 0.4 LWD 210/0. (I think I will keep it, but please, don't tell anybody... :twisted: )

After grabbing some M42 parts I got this proof of principle:

Capillary, OD approx. 250 µm.
20240107_124051_P1173406_STACK_61_C,S2_1000.jpg
Manual stack, approx 5 µm steps (should be smaller), full picture, resized and sharpened for forum. And using a lot of information from this forum.

Yes, technically it is not perfect, but it is in the range of what we need. At least now I know I'm on the right way. Now it is up to me. Thanks to everybody posting here!
-- Matthias

Chris S.
Site Admin
Posts: 4036
Joined: Sun Apr 05, 2009 9:55 pm
Location: Ohio, USA

Re: Down the rabbit hole… and some questions

Post by Chris S. »

Matthias, welcome to the forum! :D

You are moving fast. This is a very nice "proof of concept." I particularly admire the way you use light.

With the objective you're using, my standard step increment is 2 µm. Am pretty sure 4 µm would work with only a moderate reduction in fine detail contrast. Agreed that 5 µm is a bit of a stretch, particularly if your mechanics only permit approximate repeatability.

As I'm sure you're noticing, dust is everywhere! And much of it, as here, is flakes of skin. Every time I need to retouch dust out of an image (sadly often), I ask myself why I didn't clean the subject more before shooting. (I take it this capillary is made of glass? One time I cleaned something similar in size with canned air, only to watch the subject melt as I photographed it. Really should have asked the owner of the subject if it were water soluble. . . .)

Microfluidics is very cool. I hope someday you can tell us how some of your subjects are being used, and how they are fabricated.

--Chris S.

mbf
Posts: 16
Joined: Fri Dec 29, 2023 7:31 am
Location: Germany

Re: Down the rabbit hole… and some questions

Post by mbf »

Hi Chris,

yepp, the step size is my biggest concern. I have to find a solution - or reduce the magnification. Now the otimization process will start. But, however, I am very glad that this magnification range is within reach now. Even 10x in high quality should do the job.

And the dust, oh yes. It is indeed a surprise how much dust accumulates - every time :roll: . This is especially true for the glass chips. I usually use a quite harsh lighting to emphasize the corners and to get a "technical" look (some kind of low key photography). But under these circumstances even the smallest particle is visible. Wiping, gloves, blowing... but at the end, I often give up and leave some of the dust. On the other hand side, it gives a "realistic" element so it is obviously a picture and not a drawing. It's not a bug, it's a feature. :wink:

The capillary itself is made out of glass with a polymer coating which is good visible now. Having a rugged end like the one shown (I just breaked the capillary), it can generate troubles under certain circumstances, therefore a more defined end is needed in these cases. This is surprisingly quite easy to achive, easy to control with a magnifying glass, but harder to visualize for the public.

These systems shown here are used in separation science (no big secret about the general aspects, there are quite some publications available). They are used to couple electromigrative separation techniques in different combinations (please don't ask for details, I do not fully understand the science behind it). The chip from the cropped picture has some kind of detection system integrated, therefore the quite complex inner structure. Oh, I did not mention the size of the chips: the edge lenghts are in the order of 0.5 - 1.5 cm.

And for the process of making the chips, it is also not a secret, please refer to the manufacturer: https://lightfab.de/Technology.html
-- Matthias

lothman
Posts: 935
Joined: Sat Feb 14, 2009 7:00 am
Location: Stuttgart/Germany

Re: Down the rabbit hole… and some questions

Post by lothman »

mbf wrote:
Mon Jan 08, 2024 1:30 am
And the dust, oh yes. It is indeed a surprise how much dust accumulates - every time :roll: . This is especially true for the glass chips. I usually use a quite harsh lighting to emphasize the corners and to get a "technical" look (some kind of low key photography).
Hello Matthias,
nice to see fellows from German Universities here in this forum.

If your subjects are solid and do not loose parts (like wings of butterflys) you can collect dust with so called cleaning putty. Take the putty roll it around a tooth pick and use this sticky roller to collect the dust particles. This is widely used in watch making, product is called Rodico, even picks off finger prints.
https://www.amazon.de/1x-Rodico-Reinigu ... B008YMGYBU

And since we scientist are always on tight budget :( you can use typecleaner (I use "Typutz" from Läufer) from the good old days:
https://www.kaufland.de/product/7950047 ... C_lowPrice

best regards
Lothar

Edit: an Illustration how to use the cleaning putty (only in German sorry, but I think pictures demonstrate as well)
dust.jpg

rjlittlefield
Site Admin
Posts: 23508
Joined: Tue Aug 01, 2006 8:34 am
Location: Richland, Washington State, USA
Contact:

Re: Down the rabbit hole… and some questions

Post by rjlittlefield »

lothman wrote:
Wed Jan 10, 2024 1:37 am
an Illustration how to use the cleaning putty (only in German sorry, but I think pictures demonstrate as well)
As explained at https://support.google.com/translate/an ... %3DDesktop , Google Translate can now do images!

Here is its English version:
e5107e33-4522-46e8-8cb6-5da8a289ddc3.jpg
I was working all within Chrome, so I just opened Translate in a separate window, and dragged the image directly from lothman's posting into the Translate window.

--Rik

mbf
Posts: 16
Joined: Fri Dec 29, 2023 7:31 am
Location: Germany

Re: Down the rabbit hole… and some questions

Post by mbf »

lothman wrote:
Wed Jan 10, 2024 1:37 am
Hello Matthias,
nice to see fellows from German Universities here in this forum.
Well, my wife is. But if your partner is working there, you are a part of the university, too...
If your subjects are solid and do not loose parts (like wings of butterflys) you can collect dust with so called cleaning putty. [...]
And since we scientist are always on tight budget :( you can use typecleaner (I use "Typutz" from Läufer) from the good old days:
Thanks! There is a Kaufland (for non-germans: it is a supermarket) nearby, but they do not have this great stuff in the market. I don't understand, is no one using typewriters anymore...? :-k
However, I will try to get some of this sticky stuff. Seems to be a must-have.
-- Matthias

mbf
Posts: 16
Joined: Fri Dec 29, 2023 7:31 am
Location: Germany

Re: Down the rabbit hole… and some questions

Post by mbf »

OK, back to topic.

After some experiments I decided not to buy an automatic rail. This would be quite perfect, I know, especially if I have access to the 20x Nikon mentioned above. But stacking 20x by hand in 2-µm-steps... with up to 200 steps... no. And I was deeply pondering, if I really need an additional piece of equipment. No. OK, at least, no for now... :wink:

So I decided to stay with 10x. And to do things right, I got a Nikon CFI Plan Achro 10x/0.25 infinity. I put it in front of my 100-300 Panasonic lens (f = 200 mm) and let the focus braceting funktion of my G9 do the work. The focus range is (barely) sufficient to cover the capillary and the image quality is more than acceptable.

Random capillary from the front:
20240218_124050_P1223397_STACK_212_C,S2_1000.jpg
Sideways:
20240218_130146_P1223881_STACK_240_C,S2_1000.jpg
And even with a focal length of 100 mm (5x) it works better than everything before:
20240218_130833_P1224123_STACK_142_C,S2_1000.jpg
Pictures cropped and resized.

So I can reach 10x without problems, I get what I need and I am quite flexible now. And in case of emergency, I have to dial 5-µm-steps by hand. Check.

Now I will have to play around with some subjects...

Once again: thanks to all members of the forum for contributing such a hell of knowledge!
-- Matthias

rjlittlefield
Site Admin
Posts: 23508
Joined: Tue Aug 01, 2006 8:34 am
Location: Richland, Washington State, USA
Contact:

Re: Down the rabbit hole… and some questions

Post by rjlittlefield »

Those look great to my eye -- nice work!

Thanks for letting us know.

--Rik

Post Reply Previous topicNext topic