Diatom Brightfield/Darkfield/PC/DIC

NicoVB · Post by **NicoVB** » Mon Oct 10, 2016 1:50 pm

Just as an exercise to practice some different techniques.
Olympus PlanApo 10 0.32 0.17
Olympus PlanApo 40 0.95 0.11-0.23
Olympus A40 PL 0.65 160/0.17

Helio Focus 6.3.0

DarkField:
16 image stack

Brightfield:
15 image stack

DIC/NIC
14 image stack

Phase contrast
12 image stack

I removed some backgrounds, just to see all the differences in the techniques, because this was mainly a drill to get them right and see the stacking results.

I am getting a magnification of the Objective + 1.25x of the top Normanski intermediate tube + a FK 2.5x on m4/3 sensor, so...
Are these images even sharp enough?
Because i was a bit disappointed, it's on the edge i think.

Plz let me know what you think.
Thanks for positive criticism.

Pau · Post by **Pau** » Mon Oct 10, 2016 3:13 pm

1.25 X 2.5 = 3.13X This is too high relay magnification for a 4/3 sensor. For your camera the ideal relay mag. will be around 1.2X, so you are getting too cropped image compared with the visual field.

This is a problem, not only because the reduced field but also because you easily get empty magnification with high power objectives. In fact your first image looks much more sharp than the others and this may be the cause.

There aren't NFK photoeyepieces so low, the closer ones are 1.67X and they are very scarce and expensive. Another approach, if your phototube allows to use normal eyepieces, is afocal. Take a look at http://www.photomacrography.net/forum/v ... hp?t=15607 and the links inside, specially http://savazzi.net/photography/zeissadapter.html , the combo he uses, a 30mm lens over a 10X eyepiece, provides just 1.2X

About the images the first one looks very nice, it could be improved with a better centering of the dark field stop (or condenser) and with some retouching of the stack.

About the high magnification ones, a priori phase doesn't seem a good method for this subject but, despite the lower NA, it is resolving the same detail than the others. DIC, in principle the most desirable method may produce nasty stacking artifacts with high contrast subjects* , it seems that you used it at full extinction, try providing some shear displacing the upper prism with its rotatable knob, this is how DIC is intended to work.

* contrast in big part due to the very different RI of the mounting medium

NicoVB · Post by **NicoVB** » Mon Oct 10, 2016 4:56 pm

I should buy me a Hasselblad X1D, problems solved... hehe

NicoVB · Post by **NicoVB** » Mon Oct 10, 2016 5:04 pm

Yes indeed Pau, good idea, because the eyepieces fit in the phototube.
And i have some spare eyepieces with different magnifications laying around to make different setups.
I'll try that out.

The magnification now is just to much on a crop camera.
It looks sooooo good when looking in my eyepieces, but the photo result is very disappointing.
Although disappointing, it's also remarkable what resolution i still get
But the images get to blurry due to the 2 extra lenses.

Great forum here, you guys always know some kind of workaround to get the results what i want.
Respect for your insights.

Pau · Post by **Pau** » Tue Oct 11, 2016 8:58 am

BTW, they are not diatoms but silicoflagellates, and a very nice slide!

NicoVB · Post by **NicoVB** » Tue Oct 11, 2016 11:39 am

You are right... i feel ashamed, misread the label

Charles Krebs · Post by **Charles Krebs** » Tue Oct 11, 2016 5:18 pm

Yes indeed Pau, good idea, because the eyepieces fit in the phototube.

What trinocular head do you have? If it is the Olympus BH2-TR30 you may need to modify it to use the afocal method because a regular eyepiece is too wide to seat down into the tube at the required distance. (The Olympus photo-eyepieces are considerably narrower).

NicoVB · Post by **NicoVB** » Fri Oct 14, 2016 4:14 am

nono, it's a BHB so it uses the BH TR30 (not to BH2 TR version), this one:

So i can use normal eyepieces on the tube.

I already tried various setups, but no pleasing results.
Let's see what the combinations i tried:

I added my 50mm to my camera and positioned my camera above the phototube.
I replaced the PFK 2.5 with a 10x eyepiece.
But the image on the sensor was very small when avoiding vignetting. The 50mm wide angle is too narrow to display the whole image.
I have not tried it with wide angle eyepieces yet, maybe it's a bit better, but i doubt it.

In a second setup i removed the whole Trinocular head, and added a dovetail to T-mount directly on the scope. in the adapter i can add a photo eyepiece. On top of the T-mount i added my camera with om adapter, without objective.
I used a K5x, a PK6.3x and a PZ7x just to find the right sensor display, but the results were horrible.
The center displays a rather sharp image, but the fallout to the edged quickly deteriorates in chromatic aberration.
To that extend that the image is not usable.

The only option i have now is to try with K15 and K20 eyepieces.

Really scratching my head on this.
I know i have a good scope with a lot of features like NIC and PC, but the transfer to a sharp image is holding me back.

Pau · Post by **Pau** » Fri Oct 14, 2016 5:11 am

I added my 50mm to my camera and positioned my camera above the phototube.
I replaced the PFK 2.5 with a 10x eyepiece.
But the image on the sensor was very small when avoiding vignetting. The 50mm wide angle is to narrow to display the whole image.
This trinocular head seems adequate for afocal.

I have not tried it with wide angle eyepieces yet, maybe it's a bit better, but i doubt it.
This setup will provide 2X, added your intermediate piece it will provide 2.5X, still too high for 4/3 sensors but a bit better covering. As I formerly said, a 30mm lens will be much better matched

What is working wrongly?
- Is the camera lens focused to infinite and well placed over the eyepiece (well centered and very close to it)?
- Is the 10X eyepiece at the phototube parfocal with the viewing ones?
A picture of the lens and of the full setup could help.

Other points to be aware of:
- projective eyepieces don't work well for afocal, I tested it with a 3.3X Oly NFK
- High eyepoint eyepieces (maybe what you tell wide angle) usually work better to avoid vignette and because optically they are better coupled with the camera lens entrance pupil but in many cases this is not necessary, in my setup the one I most use because its low magnification (6.3X) is not of high eyepoint type.
- there are other causes of unsharpness, more critical when magnification rises like
+ vibrations produced by the camera or by the environment
+ suboptimal microscope regulation or optical components, like too thick or thin coverglass, dirt, condenser aperture too closed or condenser not at its right height...
+ with the 40/0.95 you must regulate the correction collar to match the cover+medium thickness to get a good image, this is critical and not so easy to do at the beginning. A fine rich detail slide (diatoms, some histological preparations...) will be a better resolution test subject than your nice silicofagellates, if the subject has fine enough details and the slide thickness is adequate you must be able to get better resolution than with your 40/0.65

And a general tough: At high magnification even when using high NA objectives the effective F number is high and the image is affected by diffraction, so the sensor easily outresolves the optics and all must be optimized. If you want a sharp image you need to avoid empty magnification. Cropping the image as you're doing with too high relay magnification is very prone to get that empty magnification.

NicoVB · Post by **NicoVB** » Fri Oct 14, 2016 8:04 am

Indeed, avoiding empty magnification.
See the difference between with and without the DIC top condenser tube (1.25x). 1e with and 2e without.

Both images are 20 stack with 20x PlanApo 0.65, No corrections, straight from the camera.

Pau · Post by **Pau** » Fri Oct 14, 2016 8:40 am

They look pretty good, what camera adaptation was used?
Resolution at the subject size (what microscopists call resolution) will be the same. How the pictured field compares with the visual field through the eyepieces?
The 20X planapo looks like a very desirable lens
Illumination is not enough even, try to get Kölher

NicoVB · Post by **NicoVB** » Fri Oct 14, 2016 9:09 am

I really want to stick to the 2.5 photo-eyepiece.
So the only thing i did in the previous post was removing the top DIC tube.

But now I replaced the L-adapter with a bellows, so i could control the height of the camera sensor.

Ofcourse by lowering the sensor, the magnification lowers drastically.
And this image now is almost the same dimentions with what i see though the eypieces.

This is the result now. (without whitebalans correction or stack)
Same objective 20x PlanApo 0.65

Although i still think there's something is wrong, dunno, maybe i'm too demanding in search for perfect image quality on this old equiment.

Pau · Post by **Pau** » Fri Oct 14, 2016 9:55 am

Ofcourse by lowering the sensor, the magnification lowers drastically.
And this image now is almost the same dimentions with what i see though the eypieces.

This will alter the optical configuration of the microscope forcing to refocus the objective outside its design point. This will work quite well with low magnification objectives but could induce noticeable unsharpness because spherical aberration with high NA objectives.
In general you want the camera to be parfocal with the viewing eyepieces, is it?

In the other hand, the best value of your microscope is the DIC equipment, I wouldn't want to remove it

In the last image I see some lateral CA towards the image borders, likely the NFK is not perfectly matched with your old style objectives, in fact NFKs are recommended for the more modern LB 45mm parfocal objectives for the BH2 series, take a look to Olympus literature at Alan Wood's site

rjlittlefield · Post by **rjlittlefield** » Fri Oct 14, 2016 10:51 am

Pau wrote:
Ofcourse by lowering the sensor, the magnification lowers drastically.
This will alter the optical configuration of the microscope forcing to refocus the objective outside its design point.

That problem can be avoided by simultaneously raising the photo eyepiece so as to maintain parfocality with the regular eyepieces.

But all of this does not avoid the problem that the NFK 2.5 does not like to be used outside its designed focus arrangement either. See http://www.krebsmicro.com/NFK_lowermag/index.html for discussion.

--Rik

Alan Wood · Post by **Alan Wood** » Fri Oct 14, 2016 2:22 pm

The proper photo eyepiece for your objectives is the FK 2.5x, not the NFK.

You appear to be using an Olympus camera. Sadly, there does not seem to be any way to overcome the narrow field of view that results from the small sensor without affecting image quality.

A few Photomicro Adapter L have been modified to a T-mount instead of the original OM mount. This adds 10-11 mm to the height and makes it impossible to achieve parfocality between the camera and the binocular eyepieces. Yours looks like a modified one, but I can't be sure.

Alan Wood