Images from x1000 and AxioLab Re - is image quality as expected or crap and need to upgrade?

[FungusMan]

Hi, I have submitted some posts over my setup these last few days, including buying a new scope and some asked why and that I should keep my AxioLab. I wish to reply to the reason on a separate post here.

First of all, I take images in two ways. Either with my portable Canon GX5 through the eyepiece (works fast, versatile and I often go with this method) or by connecting my EOS 750d via a convertor tube/lens called NDPL-1 adaptor: https://www.aliexpress.com/i/33004421318.html



I tried to examine some spores and take photos with the AxioLab RE and x100 objective (Zeiss Plan-NeoFluar x100 - 1018-595) where in general I am not happy with the results, but my question, after all, is if this the max quality to be expected at x100 ?

Then comes the questions:
Will I get significantly better image quality if I buy a new scope and convertor tube?
Will I get significantly better image quality if I buy a trinocular for my old AxioLab (very difficult part to find!)

1. from a digital Gx5 camera through the eyepiece
2. from EOS 750d SLR using an adaptor that fixes instead of the eyepiece

IMG_9467s.jpg (109.5 KiB) Viewed 7122 times

So I publish the resulting images to have feedback and if my opinion to buy a new setup is justifiable. I can say that at x400 I get very nice images (with a bit of background artefacts) while my x63 obj is a bit crap.

GX5 Images

Gx5_x1000_s.jpg (106.21 KiB) Viewed 7122 times

750d + adapter Images

750D_x1000a_s.jpg (99.98 KiB) Viewed 7122 times

I place some cropped and non-resized images too in the following posts

[FungusMan]

Gx5 images

[FungusMan]

EOS 750d images on NPLD1 adapter (x100 obj, oil immersion)

[MichaelG.]

I can’t give you any example of what results you should be getting from that set-up, but I think there must be something seriously wrong … you have what should be a very fine objective, so either it’s faulty or there is something else [possibly trivial] spoiling the images.

MichaelG.

.

Price alone is not a reliable indicator … but here’s an ‘as-new’ one on ebay: https://www.ebay.co.uk/itm/194246305010
… and another [sold] from a dealer: https://www.fluorescencemicroscopes.com ... objective/

[Gatorengineer64]

What type condenser are you using?.... I get similar images out of my new to me eclipse and assumed that an Abbe wasnt up to the 1000x, like you my scope runs well at 200x and 500x... I have an Ebay Nikon Achromat in the mail.....

As an addenda I also dont have a 100x of the same cailber that you have.

[smollerthings]

The image doesn't look like it is in focus. Did you solve your cover slip thickness issue? I had the same struggle with my 90x.

[FungusMan]

Yes I agree,

trivial: at x100 I noticed that slight movement while holding camera on eyepiece results in shakes/vibrations that affects the image quality, while a timer option (usually 2sec) on the EOS standing on its own reduced that effect (=less blurry). Yet my images are not sharp/good. They are OK if I resize the image by 50% or more and crop and apply a bit of sharpness and contrast. I also have video showing the image while I raise and lower the condenser (very little effect on the image to be honest). I want to learn if others have similar image output at x1000 magnification (oil).

at x40 - I get decent images

IMG_9462s.jpg (135.03 KiB) Viewed 7098 times

Video show what I see (captured) at x100 while raising/lowering condensor
http://www.maltawildplants.com/!up/MVI_9470.MP4

[FungusMan]

What type condenser are you using?.... I get similar images out of my new to me eclipse and assumed that an Abbe wasnt up to the 1000x, like you my scope runs well at 200x and 500x... I have an Ebay Nikon Achromat in the mail.....

As an addenda I also dont have a 100x of the same cailber that you have.

It is the original condenser that came on the Zeiss AxioLab RE. Image attached. Hope it replies your question.
What Nikon model you bought? Why you chose that over the rest ?

Tnx!
S.

[MicroBob]

Hi,
many but not all Canons have an optinal vibration free exposure by means of an electronic first shutter cuteain function (google efsc +Eos...). This function allows to expose without any movement out of live view and has to be switched on manually. Do you know and use this function? Is your camera vibration free in EFSC?
A good test might be to take photos at lower magnifications and compare them, still not sharp?
A 100x oil doesn't offer much image content, just a couple of Megapixels. So the 24? MP image has to be unsharp when viewed at 100%.

A trino tube would be more convenient and remove some glass out of the image path, but apart from that you would get the same quality. Is your bino in good condition? To check it you can take it off remove eyepieces and point to light source - hazy??

Bob

[FungusMan]

The image doesn't look like it is in focus. Did you solve your cover slip thickness issue? I had the same struggle with my 90x.

Yes I did solve it. I used new coverslips and fixed the stage more solidly in place. Still, I have lots of movement going on in the mount (spores turn around when I move the stage), but clipping the coverslip to the slide with a special paper clip helped to get things stationary.

I can't get better sharpness (making it look blurry and out of focus as u pointed out). On live examination (with my eyes) the image is a bit better... but I can't say wow

Maybe there is no wow at x1000 (that's part of my question!)
https://commons.wikimedia.org/wiki/File ... rmis-4.jpg
https://www.shroomery.org/forums/showfl ... r/11260819
https://www.wikiwand.com/en/Psilocybe_semilanceata

Half-wow:
https://www.uwyo.edu/virtual_edge/lab13 ... cota01.jpg

But this is WOW
http://nefsg.co.uk/portfolio (we don't know the gear and post-editing, etc)

[FungusMan]

Hi,
many but not all Canons have an optinal vibration free exposure by means of an electronic first shutter cuteain function (google efsc +Eos...). This function allows to expose without any movement out of live view and has to be switched on manually. Do you know and use this function? Is your camera vibration free in EFSC?
A good test might be to take photos at lower magnifications and compare them, still not sharp?
A 100x oil doesn't offer much image content, just a couple of Megapixels. So the 24? MP image has to be unsharp when viewed at 100%.

A trino tube would be more convenient and remove some glass out of the image path, but apart from that you would get the same quality. Is your bino in good condition? To check it you can take it off remove eyepieces and point to light source - hazy??

Bob

I have my camera for 5 years and I have not seen that option anywhere. I guess the timer option will solve any vibrations. To be honest, I am quite at happy at x400 and below. I wish the trino and my bino is good, at least at 400 its a joy to use. No haxy light without eyepieces.

[75RR]

.
Not sure where you are located but ideally you will be able to find either a microscopy club, a friendly university/college or a kind fellow microscopist in your area that would let you see what results they get with your samples on their equipment.

It may well just be a setup problem

Best images always come when all the ducks are in a row

[MichaelG.]

I found this page last night:
https://www.first-nature.com/fungi/index1binom.php

Most of the high-power images appear to be ‘useful but unexceptional’ … so they are probably a realistic benchmark.
[ not sure where you would place them on the WOW-scale ]

The whole site seems nicely organised, and well-worth visiting.

MichaelG.

[75RR]

.
This thread by Roldorf may be of interest: viewtopic.php?f=6&t=7685

[MicroBob]

Here you can find information on the electonic first shutter curtain menu setting:
https://www.digitalcameraworld.com/au/t ... t-shooting

In higher magnifications the shock of the shutter is enough to blur the image.

[Adam Long]

Your photos do not look to be affected bu shutter shock to me but I can see why you are not satisfied with the results.

My experience with Zeiss finite optics is that the old adage is true - 400x will show you 90% of what is possible to see. I have 100x oil objectives and both dry and oil 63x, but they do not offer a significant improvement in most circumstances over a Neofluar 40x. With a well-prepared flat specimen (e.g. bought histology sections) I can get nice images with the expected gain in image scale plus a slight increase in visible detail, but with 3D objects like protists or pollen the main effect is reduced depth of field and increased blur from the out-of-focus elements. Generally I find the faff of oil is rarely worth the resolution gain.

The other limit is your condenser - what is it's NA limit and are you oiling it to the slide? Again, without oil and optimum settings it will limit resolution.

If you are getting good results at 400x I'd guess a microscope change would not achieve much. Have you checked the glass of the 100x objective is clear and clean?

[Gatorengineer64]

I thought I had hit submit on this earlier, from Wikipedia


The Abbe condenser is named for its inventor Ernst Abbe, who developed it in 1870. The Abbe condenser, which was originally designed for Zeiss, is mounted below the stage of the microscope. The condenser concentrates and controls the light that passes through the specimen prior to entering the objective. It has two controls, one which moves the Abbe condenser closer to or further from the stage, and another, the iris diaphragm, which controls the diameter of the beam of light. The controls can be used to optimize brightness, evenness of illumination, and contrast. Abbe condensers are difficult to use for magnifications of above 400X, as the aplanatic cone is only representative of a numerical aperture (NA) of 0.6.

This condenser is composed of two lenses, a plano-convex lens somewhat larger than a hemisphere and a large bi-convex lens serving as a collecting lens to the first. The focus of the first lens is traditionally about 2mm away from the plane face coinciding with the sample plane. A pinhole cap can be used to align the optical axis of the condenser with that of the microscope. The Abbe condenser is still the basis for most modern light microscope condenser designs, even though its optical performance is poor.[1][2][3]

On my new scope also with an Abbe but with phase etc 400 looks great 500 and 1000 are mush. will post back when the achromatic condenser arrives. Willing to bet its mostly the condenser.... Do you have an image to share of what its supposed to look like? or what your expectations are?

[apochronaut]

Whether a condenser is abbe or achromat does not affect the resulting image very much. The resulting difference is subtle. It certainly will not cause your image to be mush. If the N.A. of the objective exceeds the functional N.A. of the condenser, then the condenser will impose a limitation upon the objective. Even with a dry condenser working at an N.A. of .90, a 1.30 objective will be working at about 1.15 N.A. , if properly immersed.

It seems that, in your case, sample preparation may be overlooked as a cause of poor imaging?
With all samples, there is a condition referred to as homogeneous immersion where the refractive index of the media through which the illumination beam and image travel through is quite even . The light goes through the condenser, immersion oil,slide,sample,coverglass,oil and into the objective lens, with all being close to a 1.52 refractive index. The closer all of those media are to an r of 1.52 the better the imaging will be. This is also true for high N.A. dry objectives where the objective is corrected to accomodate an air immersion, rather than.an oil immersion., so the air gap between the cover and objective front lens is accounted for in the calculations of the objective's spherical aberration corrections.It is often overlooked that ALL of the conditions for homogeneity must be met and if the sample itself is not homogeneous, that can throw everything off. Water as a sample medium can cause s.a. , thus a mounting medium such as balsam or one of the more modern ones is preferred. Air can be deadly bad. I just wonder if you are making a spore print on a slide and then covering it dry, with a coverslip?

[jimur]

xx

[FungusMan]

I found this page last night:
https://www.first-nature.com/fungi/index1binom.php

Most of the high-power images appear to be ‘useful but unexceptional’ … so they are probably a realistic benchmark.
[ not sure where you would place them on the WOW-scale ]

The whole site seems nicely organised, and well-worth visiting.

MichaelG.

I start thinking that images of spores or mycolgical tissue at x1000 are mostly same standard or close to my images (straight from the camera without post processing). Yet, I master a bit more with the condenser setup.

[FungusMan]

I found this page last night:
https://www.first-nature.com/fungi/index1binom.php

Most of the high-power images appear to be ‘useful but unexceptional’ … so they are probably a realistic benchmark.
[ not sure where you would place them on the WOW-scale ]

The whole site seems nicely organised, and well-worth visiting.

MichaelG.

I started to think that images of spores or mycological tissue at x1000 shown on the net are mostly same standard or close to my images (straight from the camera without post processing). Hence maybe One should not expect much more at x1000 oil. Yet, I master a bit more with the condenser setup.

[FungusMan]

Here you can find information on the electonic first shutter curtain menu setting:
https://www.digitalcameraworld.com/au/t ... t-shooting

In higher magnifications the shock of the shutter is enough to blur the image.

Thanks for helping me about this, But the feature do not exist on the EOS 750d

[FungusMan]

Your photos do not look to be affected bu shutter shock to me but I can see why you are not satisfied with the results.

My experience with Zeiss finite optics is that the old adage is true - 400x will show you 90% of what is possible to see. I have 100x oil objectives and both dry and oil 63x, but they do not offer a significant improvement in most circumstances over a Neofluar 40x. With a well-prepared flat specimen (e.g. bought histology sections) I can get nice images with the expected gain in image scale plus a slight increase in visible detail, but with 3D objects like protists or pollen the main effect is reduced depth of field and increased blur from the out-of-focus elements. Generally I find the faff of oil is rarely worth the resolution gain.

The other limit is your condenser - what is it's NA limit and are you oiling it to the slide? Again, without oil and optimum settings it will limit resolution.

If you are getting good results at 400x I'd guess a microscope change would not achieve much. Have you checked the glass of the 100x objective is clear and clean?

I agree with you and (sub-)spherical objects have like an own-lens effect causing blurriness. DOF might be the issue here, yet when I see life with my eyes, I am not really disappointed, yet I have never compared how it looks from another x1000 microscope. I have to examine a fungus today, and I will repost using some post-editing. Resizing to 800px and a little sharp and contrast makes a big deal of enhancement.

I put oil yes. Regards the condenser, I lack practice. I see a great deal of improvement on the x40, x100 and somewhat on the x400 when I apply Kohler illumination, but when I am on higher magnifications, I kinda see no big difference unless on the extreme settings (iris is fully open/close or condenser fully up/down). The image remains the same to my eyes, just the lines and particles gets a bit darker when very down and smoothened and bright when at the top. I might post again about this. There is 0,9 written on the condenser lens.

[FungusMan]

Whether a condenser is abbe or achromat does not affect the resulting image very much. The resulting difference is subtle. It certainly will not cause your image to be mush. If the N.A. of the objective exceeds the functional N.A. of the condenser, then the condenser will impose a limitation upon the objective. Even with a dry condenser working at an N.A. of .90, a 1.30 objective will be working at about 1.15 N.A. , if properly immersed.

It seems that, in your case, sample preparation may be overlooked as a cause of poor imaging?
With all samples, there is a condition referred to as homogeneous immersion where the refractive index of the media through which the illumination beam and image travel through is quite even . The light goes through the condenser, immersion oil, slide, sample, coverglass, oil and into the objective lens, with all being close to a 1.52 refractive index. The closer all of those media are to an r of 1.52 the better the imaging will be. This is also true for high N.A. dry objectives where the objective is corrected to accomodate an air immersion, rather than. an oil immersion., so the air gap between the cover and objective front lens is accounted for in the calculations of the objective's spherical aberration corrections.It is often overlooked that ALL of the conditions for homogeneity must be met and if the sample itself is not homogeneous, that can throw everything off. Water as a sample medium can cause s.a. , thus a mounting medium such as balsam or one of the more modern ones is preferred. Air can be deadly bad. I just wonder if you are making a spore print on a slide and then covering it dry, with a coverslip?

Thanks for your explanation. I recall that the image of the stage micrometre is not that blurred actually, so what you and few others said might be the main concern. Yet, if I can't have a decent photo of 10 um spores, I am disappointed. For these photos, I powdered some spores over the slide, added a drop of water, mixed /agitated to get homogenousity, placed a coverslip, pressed down, drop of oil and mounted - standard procedure.

[viktor j nilsson]

Here you can find information on the electonic first shutter curtain menu setting:
https://www.digitalcameraworld.com/au/t ... t-shooting

In higher magnifications the shock of the shutter is enough to blur the image.

Thanks for helping me about this, But the feature do not exist on the EOS 750d

According to http://www.alanwood.net/olympus/digital.html the 750D does have EFSC, aka Silent mode.

He notes:

The 3-digit (e.g. EOS 600D) and 4-digit (e.g. EOS 1200D) Canon EOS camera do not have a selectable silent mode, but this feature is included and is permanently on when Live View is selected.

So shoot in live view and you should see significant improvement in vibration control.

I still think sample prep is the main culprit here. Try putting the spore print directly on the cover slip instead.

[FungusMan]

These other folks are much more advanced than I, but my curiousity demands a few questions. I'm not familiar with the
AxioLab but I do know a Canon/Zeiss combo should give better images unless there is a problem with your 100x objective.
1) Are you focusing via the camera screen or the other eyepiece? (try each way and note the difference if any)
2) Are you tethering via the EOS utility and focusing via the computer monitor?
(if not, try it, you'll like it much better than the camera timer and screen)
3) Have you made different adjustments to the WB? That will improve the photo lighting and contrast at least.

Again, I'm just a curious rookie.

1. First from the eyepiece, then when I take the photo from the live view screen, which usually incurs just a bit of fine tuning.
2. No, but I magnify the view image on the camera screen and it works OK.
3. Sometimes I post-process and yes, it helps, but in this post - straight from the camera

[FungusMan]

Here you can find information on the electonic first shutter curtain menu setting:
https://www.digitalcameraworld.com/au/t ... t-shooting

In higher magnifications the shock of the shutter is enough to blur the image.

Thanks for helping me about this, But the feature do not exist on the EOS 750d

According to http://www.alanwood.net/olympus/digital.html the 750D does have EFSC, aka Silent mode.

He notes:

The 3-digit (e.g. EOS 600D) and 4-digit (e.g. EOS 1200D) Canon EOS camera do not have a selectable silent mode, but this feature is included and is permanently on when Live View is selected.

So shoot in live view and you should see significant improvement in vibration control.

I still think sample prep is the main culprit here. Try putting the spore print directly on the cover slip instead.

Oh yes I found the silent mode and I try it. It was buried in another menu, not in the Q function button. I am going to examine and test another fungus shortly, and I hope it has good spores. (a Gasteromycete). THANK YOU

[Alexander]

Here you can find information on the electonic first shutter curtain menu setting:
https://www.digitalcameraworld.com/au/t ... t-shooting

In higher magnifications the shock of the shutter is enough to blur the image.

Thanks for helping me about this, But the feature do not exist on the EOS 750d

Your camera has that feature for sure. Every Canon with LiveView has ist.

[MichaelG.]

I have been browsing through that first-nature website, in search of a good spore to use as a test specimen:

This looks promising : https://www.first-nature.com/fungi/aleuria-aurantia.php

MichaelG.

.
Edit: __ See also Fig.2 here: http://www.leifgoodwin.co.uk/General/Microscopy.html

[MicroBob]

Hi together,

apparently not all Canon DSLRs work equally well on the microscope, some models still produce some vibration. Here is an older thread from the german forum with links to tests of different models (many in english).
https://www.mikroskopie-forum.de/index. ... ic=25992.0

The 750D is said to have a goood EFSC mode but some banding issues created by AF sensors on the chip.

I don't use Canon DSLRs so I can't report out of my own experience. Once I tried a SONY NEX 5 (not "N") and the shutter shock blurred the image consiederably even when ridgidliy mounted on a photo tube - I wouldn't have expected this. The vibration is of cause most problematic with fresh slides with objects floating in water.

I think Canon was first to come out with EFSC and it earned them a good reputation in the microscopy scene.

As far as I know the combination of electronic first shutter curtain and mechanical last shutter curtain is best, no vibration and no artifacts which could result from a fully electronic shutter.

Bob