40x SPlan woes

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ImperatorRex
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Re: 40x SPlan woes

#31 Post by ImperatorRex » Sun Apr 19, 2020 8:51 am

Thomas,
are you saying that only the video/camera performance is (was) poor? So observation through the eyepieces provides good performance?

deBult
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Re: 40x SPlan woes

#32 Post by deBult » Sun Apr 19, 2020 6:34 pm

Question: do you Kohler and adjust the condenser diaphragm for each objective: the SPlans are coated, so less sensitive to flare but my 40* image improves considerably after proper illumination setting.

And yes the image quality, particularly the image contrast Impression of the SPlan 40* is worse compared to my 20* SPlan.

Best, Maarten

Hobbyst46
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Re: 40x SPlan woes

#33 Post by Hobbyst46 » Sun Apr 19, 2020 7:21 pm

My experience with a BH-2 is that it is a superb microscope and that SPLANS (at least the 10X) are great objectives.

Having looked at the various videos from top to bottom, my personal feeling is that nothing is wrong with the microscope or objective.
The illumination seems less than optimal. Two much non-specimen detail is seen in the background (not the slide plane) in the latter videos, after the repair of the photo tube.

I echo others remarks about Kohler illumination. Possibly also the condenser setting.

A decent stage micrometer costs around 10-15$ on eBay and provides a permanent flat field with regular marks. Very convenient to evaluate images, check focus and calibrate the field-of-view dimensions.

Another flat, thin, easy preparation is the pappus (seed-parachute) of flowers - Sonchus, Dandelions and similar. They proliferate in Spring. Can be mounted dry in air. Place one (only the soft part - not the seed!) on the slide, cover and attach the coverslip to the slide by means of four drops of nail polish at the four corners. Lightly press on the coverslip to ensure a thin preaparation.

Brightfield is just fine to rectify the mentioned doubts. I find still images to be better than video clips.
Zeiss Standard GFL+Canon EOS-M10, Olympus VMZ stereo

thomas.schwarz
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Re: 40x SPlan woes

#34 Post by thomas.schwarz » Sun Apr 19, 2020 9:37 pm

...started making videos to explore/critique my current solution, based on your comments, and I find that I am more confused than ever, I cannot effectively demonstrate that the shorter tube (the MTV-3 tube without additional optics) improves resolution. But it does negatively effect parfocality (thanks Scarodactyl).


So in short, my calibration slide (stage micrometer) should come on April 28th. What is a good test that I can do with my calibration slide to prove that my BH-2 BHS is performing optimally (optimal resolution)?

...other responses...
Scarodyctyl: yes with the MTV-3 phototube (lens removed) my 4x and 10x loose parfocality.
Debult: I thought that I had my issue resolved, but I was wrong. So I will order some Zeis guarantied .17 cover slides (+- .005) and test
ImperatorRex: now, looking more closey at binocular, I am starting to think that the phototube is no better, no worse....
Hobbyst46: yes, I did clean the photo projection eyepiece after making the videos (that is were the dirt was). As I mentioned, on April 28th, I get my calibration slide. Please let me know what test I could do to prove the optimal resolution of my equipment. And I will do a bright field still image of a Dandilion Parachute as soon as the change (currently yellow bloom).

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Re: 40x SPlan woes

#35 Post by Hobbyst46 » Sun Apr 19, 2020 9:58 pm

thomas.schwarz wrote:
Sun Apr 19, 2020 9:37 pm
Hobbyst46: yes, I did clean the photo projection eyepiece after making the videos (that is were the dirt was). As I mentioned, on April 28th, I get my calibration slide. Please let me know what test I could do to prove the optimal resolution of my equipment. And I will do a bright field still image of a Dandilion Parachute as soon as the change (currently yellow bloom).
The pappus was suggested as a cheap and available option for checking the focus, until the stage micrometer arrives...
The stage micrometer is not a standard for resolution. Its chief purpose is to calibrate the size of the field of view. In your case, it also provides a good target to check the focusing, since the thin black lines on bright background afford contrast. There are resolution targets, for example prepared diatom slides and other, more "man-made" standards.
Zeiss Standard GFL+Canon EOS-M10, Olympus VMZ stereo

thomas.schwarz
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Re: 40x SPlan woes

#36 Post by thomas.schwarz » Tue Apr 21, 2020 7:06 pm

OK... here is calibration slide video with the OEM photo tube, GH4 Panasonic Camera:
https://www.youtube.com/watch?v=FXMqjLc ... e=youtu.be

My current theory is that I had dirt in my projection eyepiece (on and between the two lenses) and after cleaning it, my microscope is performing normally (40x resolution). Your opinion?

Also here is the dandilion hair from a blow-away seed, with entellan permanent mount. in other words, i took one of those things that flies away when you blow on a dandilion, i very carefully removed what it probably the seed from the hairs, and made a permenant mount with the hair like strands that help the seed to fly away from its parent plant. This is 40x bright field. I am using the original OEM phototube from olympus. Thanks for the suggestion to do this Hobbyst46, it was actually my first ever wet mount and was great fun to make. Had bought the entellan some time ago. Usually I work with microbes and take great care not to kill them....

So million dollar question, based on the dandilion hairs and the calibration side, is this as good as is gets (resolution) in bright field, 40x with my Olympus BH-2 BHS? Other realization about my set up? Is 4x suppose to be that much magnification???
rsz_p1000091.jpg
rsz_p1000091.jpg (85.32 KiB) Viewed 1940 times

thomas.schwarz
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Re: 40x SPlan woes

#37 Post by thomas.schwarz » Wed Apr 22, 2020 8:18 pm

Hello Friends,

Here is a comparison of a Zeiss .17 mm coverslip (guaranteed to within .005 mm) and a generic coverslip betweeen .17 and .13 mm thick.

https://www.youtube.com/watch?v=rGDbb5UH4gE

I created the two permanent mounts with the same dandelion seed hairs, with entellan mounting medium. At 40x the width of the image is about 1/10 mm across (11/100 mm). I also tried to do a lot of variations with condenser and kohler as well as focusing. I am looking for feedback on microscope performance as well as general tips because I am new to microscopy.

thanks!

MichaelG.
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Re: 40x SPlan woes

#38 Post by MichaelG. » Wed Apr 22, 2020 9:28 pm

An excellent comparative test, Thomas ... except for one failure in the ‘design‘ of the experiment

Zeiss covers are 0.17 +/- 0.005
Generic covers are 0.13 to 0.17

But, what is the thickness of the actual generic cover you are testing ?

... In any one box they could be all thick, all medium, all thin, or a mix of thicknesses
... Hopefully each cover has reasonably constant thickness though !!

I think you need to measure the test cover.


MichaelG.
Last edited by MichaelG. on Wed Apr 22, 2020 9:34 pm, edited 1 time in total.
Too many 'projects'

thomas.schwarz
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Re: 40x SPlan woes

#39 Post by thomas.schwarz » Wed Apr 22, 2020 9:31 pm

...just when you thought it was safe to put my mtv-3 on ebay.... ...here it is again, same slides, this time with mtv-3 phototube, no lense, 40x field of view becomes 2.3 mm across instead of .12 mm accross. But there is so much new and valuable resolution! Why? and yes there is loss of parfocality particularly between 4x and 10x, but that is not a show stopper....
- Does anyone agree that the resolution is improved/ new details are visible with mtv-3 tube without lens (shorter phototube)?
- Can anyone explain why?

https://www.youtube.com/watch?v=gxxHg9u_9nQ

Thanks!
Tom

MichaelG.
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Re: 40x SPlan woes

#40 Post by MichaelG. » Wed Apr 22, 2020 9:54 pm

Massively better result, Thomas !!
Now we need to work-out why that could be.

My guess is that the problem lies with your extension of the original photo-tube

Consider this:
The lensless MTV-3 is, I believe, too short and giving sub-optimal results
The extended original photo-tube is giving worse results
There’s a Goldilocks zone somewhere in between, which will give you what you need.
... The standard length of photo-tube, as specified by Olympus

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Scarodactyl
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Re: 40x SPlan woes

#41 Post by Scarodactyl » Wed Apr 22, 2020 10:08 pm

What does the longer adapter look like?
It should be totally parfocal at the right height.

MichaelG.
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Re: 40x SPlan woes

#42 Post by MichaelG. » Wed Apr 22, 2020 10:45 pm

I believe this is it :
viewtopic.php?p=73981#p73981

MichaelG.
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deBult
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Re: 40x SPlan woes

#43 Post by deBult » Thu Apr 23, 2020 1:21 am

3 points for further exploration:
* the MTv-3 is designed to be used WITH the internal lens (and designed for TV cameras: so will project a small Image on your camera sensor)

* the L-Tube has the correct length for the original Olympus OM series of cameras. The only way to use this with a NIKON camera is to REMOVE the original Oly bayonet with a conversion kit to Nikon bayonet. The conversion bayonet is VERY thin as the total resulting length should be slightly SHORTER as per the difference in mount-to-sensor distance between Oly and Nikon.
The adapter shown in the link appears to EXTEND the length so no good

* I will give the supplied videos a better look, when on a computer with sound capabilty :oops: but agree on the observation on the “unknown” coverglass thickness. With the Zeiss coverglasses you have removed one possible source of error, BUT the resulting slide you build should have the dandelion hair VERY close to the coverglass: the layer of mounting medium should be as thin as possible. The (expensive) test slides from diatom lab as advertised on ebay meet this requirement.

Hobbyst46
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Re: 40x SPlan woes

#44 Post by Hobbyst46 » Thu Apr 23, 2020 6:24 am

thomas.schwarz wrote:
Tue Apr 21, 2020 7:06 pm
OK... here is calibration slide video with the OEM photo tube, GH4 Panasonic Camera:
https://www.youtube.com/watch?v=FXMqjLc ... e=youtu.be

My current theory is that I had dirt in my projection eyepiece (on and between the two lenses) and after cleaning it, my microscope is performing normally (40x resolution). Your opinion?

Also here is the dandilion hair from a blow-away seed, with entellan permanent mount. in other words, i took one of those things that flies away when you blow on a dandilion, i very carefully removed what it probably the seed from the hairs, and made a permenant mount with the hair like strands that help the seed to fly away from its parent plant. This is 40x bright field. I am using the original OEM phototube from olympus. Thanks for the suggestion to do this Hobbyst46, it was actually my first ever wet mount and was great fun to make. Had bought the entellan some time ago. Usually I work with microbes and take great care not to kill them....

So million dollar question, based on the dandilion hairs and the calibration side, is this as good as is gets (resolution) in bright field, 40x with my Olympus BH-2 BHS? Other realization about my set up? Is 4x suppose to be that much magnification???

rsz_p1000091.jpg
Excellent demonstrations of the problems. What I see is:
From the micrometer images - lack of focus at all magnifications, not specifically the 40X. Strangely, the focus in the first seconds (the numbers 0,1,2 etc) of the video is OK, later all images become unsharp. In addition, a hot spot is visible in the center. In addition, I see vibrations, as if the setup is shaky at high frequency.
The fiber image seems OK though.
My feeling is that the issues are not due to the microscope itself, neither the coverslip; maybe related to the camera adapter and parfocality.
Zeiss Standard GFL+Canon EOS-M10, Olympus VMZ stereo

deBult
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Re: 40x SPlan woes

#45 Post by deBult » Thu Apr 23, 2020 7:02 pm

In German: how to build a proper microscope table :roll:

https://www.mikroskopie-forum.de/index. ... ic=33816.0

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75RR
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Re: 40x SPlan woes

#46 Post by 75RR » Fri Apr 24, 2020 6:17 am

My feeling is that the issues are not due to the microscope itself, neither the coverslip; maybe related to the camera adapter and parfocality.
There are many potential variables which you need to check and confirm before you cross them off your suspect list.

Parfocality (camera is in exact focus with the eyepieces) is of paramount importance.

Set up Köhler every time you change objectives.
Zeiss Standard WL (somewhat fashion challenged) & Wild M8
Olympus E-P2 (Micro Four Thirds Camera)

thomas.schwarz
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Re: 40x SPlan woes

#47 Post by thomas.schwarz » Sat Apr 25, 2020 1:58 pm

OK firends, the grand experiment.... Variations for distance between upper projection eyepiece lens and camera sensor are A) 83mm, B) 105mm or c) 150mm. Please let me know your choice A, B or C, or in between?

Experiment Overview - https://www.youtube.com/watch?v=FO2QEB9RNEQ
A 40x - https://www.youtube.com/watch?v=k8oeLsYFgIs&t=9s
B 40x - https://www.youtube.com/watch?v=IKLJECqdhKQ&t=13s
C 40x - https://www.youtube.com/watch?v=qyLPO_0kedQ
A 100X - https://www.youtube.com/watch?v=zDVGr1aEUEE
B 100x - https://youtu.be/w9ieJ1UsVKU

Thanks!

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Re: 40x SPlan woes

#48 Post by thomas.schwarz » Sat Apr 25, 2020 2:35 pm

hmmm.....just was thinking about 75RR's comment that paramount is parfocality between the eyepieces and the camera sensor..... so that is an easy experiment, and the winner hands down is "C", the 150 mm L Tube (with very thick adapter).

So maybe the answer here is that I am just so unfamiliar with the form of a Dandelion hair, that I dont know one, when I see one. And so back to the drawing board, but this time with a much cleaner/significantly cleaner projection eyepiece. Maybe that was my focusing problem.... So in that case, do others like what they see in the "C" variant above? I am starting to warm up to it.... ...again, back to 75RR's point, the manufacturer first and foremost set up the optics to work in binocular viewing. So the idea that when the binocular view is in focus, then the camera should be in focus also, seems a very logical "level set". Yes?

deBult
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Re: 40x SPlan woes

#49 Post by deBult » Sat Apr 25, 2020 4:53 pm

To achieve parfocality the correct distance from the baseplate of the L-tube to the sensor plane is 151.2 mm

http://www.alanwood.net/olympus/photomi ... ter-l.html

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Re: 40x SPlan woes

#50 Post by Scarodactyl » Sat Apr 25, 2020 6:35 pm

Video is not a great way to compare these. It really needs to be static images.

deBult
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Re: 40x SPlan woes

#51 Post by deBult » Sun Apr 26, 2020 8:46 am

Tried a similar setup: BH2, SPlan 40, L-tube, NFK 2.5, Abbe type Phaco condenser though,
2 different chinese OM-2-Micro-four-thirds adapter and an Olympus E-PL6 camera.

Was not able to achieve 100% parfocality between the viewing eyepieces and the sensor projection with BOTH adapters.
(Will try and find an original Olympus or an adjustable one).
A very slight adjustment of the fine focus was always required.

* object micro meter, diatom test slide from Klaus Kemp, some of my own botanical cuts
* initial precise tuning of the both the eyepieces used for viewing
* Kohler for EACH change of objective
* iteration in re-tuning the visual eyepieces to give give a sharp camera sensor sharpness projection (using the magnifying glass setting on the camera display)
* reached the end of the of the eyepiece diopter setting and would have required a fraction of mm more though to achieve 100% parfocality (note my inter-eyepiece/pupil distance is 71 mm: so lost a bit off the adjustment range here).

So maybe final tuning sharpness via the camera display may help to achieve ultimate sharpness in your case as well?
Last edited by deBult on Sun Apr 26, 2020 2:05 pm, edited 2 times in total.

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Re: 40x SPlan woes

#52 Post by deBult » Sun Apr 26, 2020 1:20 pm

MVS 2020_04_26 E-PL6 4260272.jpg
MVS 2020_04_26 E-PL6 4260272.jpg (53.91 KiB) Viewed 1763 times
Pretty new to taking pictures with a microscope myself: so for what it is worth a "comparison" sample here of a Diatom slide.

Set-Up as described in previous mail:
BH2, SPlan 40, L-tube, NFK 2.5, Abbe type Phaco condenser though,
Chinese OM-2-Micro-four-thirds adapter and an Olympus E-PL6 camera

Feed Back appreciated, next step is practice stacking.
Last edited by deBult on Sun Apr 26, 2020 2:03 pm, edited 2 times in total.

Hobbyst46
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Re: 40x SPlan woes

#53 Post by Hobbyst46 » Sun Apr 26, 2020 1:45 pm

deBult wrote:
Sun Apr 26, 2020 1:20 pm
Pretty new to taking pictures with a microscope myself: so for what it is worth a "comparison" sample here of a Diatom slide.
Set-Up as described in previous mail:
BH2, SPlan 40, L-tube, NFK 2.5, standard condensor though,
Chinese OM-2-Micro-four-thirds adapter and an Olympus E-PL6 camera
Feed Back appreciated, next step is practice stacking.
Very good image!
Zeiss Standard GFL+Canon EOS-M10, Olympus VMZ stereo

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Re: 40x SPlan woes

#54 Post by ImperatorRex » Sun Apr 26, 2020 2:24 pm

Very good de Bult. Photo exacetly proves the below:
"Stauroneis phoenicenteron: The shell has 10-20 transapical stripes by 10µm that are clearly punctured. With a lens n.A. 0.65 this structure must be clearly recognized."

Did you try also the diatoma Pleurosigma angulatum? This will be quite a challange with the n.A. 0,65 obejctive and with in Bright field.

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