I can't seem to focus

Here you can discuss topics such as focus stacking, stitching and other techniques that relate to the processing of micrographs.
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Finelld
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I can't seem to focus

#1 Post by Finelld » Fri Jul 29, 2022 6:20 am

Greetings,

I am new to micro photography but am extraordinary experienced and qualified in general photography. I have a Nikon Microphot-FX connected to a 6.4mp astronomy camera (ASI178MC). I have this placed on top of the trinocular photo tube of the microscope. I can't seem to get my images anywhere near as sharp as I can see them through the eyepieces. I can do some sharpening but I consider this largely to be fake focus. Can someone offer any suggestions to help clear my images.

I have attached two images of Vitamin C crystals taken under polarized light. They are the same image but I have added sharpening to one. They are taken with a 4x objective.

Best Regards,

David Finell
Vitamin C Normal.jpg
Vitamin C Normal.jpg (262.52 KiB) Viewed 5462 times
Vitamin C Sharpened.jpg
Vitamin C Sharpened.jpg (342.42 KiB) Viewed 5462 times

Scarodactyl
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Re: I can't seem to focus

#2 Post by Scarodactyl » Fri Jul 29, 2022 12:25 pm

How do you have it attached? Is it in focus at the same setting as the eyepieces?

Finelld
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Re: I can't seem to focus

#3 Post by Finelld » Fri Jul 29, 2022 12:50 pm

No, I must refocus when I use the camera. I display the image from the camera magnified 1:1 on my computer to focus. The camera is placed on top of the photo tube with the sensor directly exposed without any intermediate optics. I believe that the photo tube was originally designed to be used with a projection eyepiece on to a 35mm film camera. I don’t have one of these eyepieces or the associated camera adapter. I hope to solve that next month and switch to a full frame digital camera. Unfortunately I can’t find documentation or other reference material to inform me of exactly what I need.

Hobbyst46
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Re: I can't seem to focus

#4 Post by Hobbyst46 » Fri Jul 29, 2022 1:41 pm

I believe that without a reduction lens in between the camera and objective, you only catch a small fraction of the field of view.
That can be remedied with a ~0.5X reduction lens, if available for that microscope.
As for the sharpness issue - it might be better to judge sharpness with a flat thin specimen, a dark one over a bright background, for example, a stage micrometer.

apochronaut
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Re: I can't seem to focus

#5 Post by apochronaut » Fri Jul 29, 2022 3:02 pm

Is it possible you are experiencing vibration somewhere during camera activation? The shutter is a rolling shutter and it would see the vibration as motion . Rolling shutters will distort motion that is too fast, which might look blurred as in poor focus with busy samples such as the one you profile. Try shooting something very flat and uncomplicated as Hobbyst46 mentioned.


You don't mention whether you can focus on the monitor, just that you can through the eyepieces.

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blekenbleu
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Re: I can't seem to focus

#6 Post by blekenbleu » Fri Jul 29, 2022 5:48 pm

Finelld wrote:
Fri Jul 29, 2022 6:20 am
I have a Nikon Microphot-FX connected to a 6.4mp astronomy camera (ASI178MC).
I have this placed on top of the trinocular photo tube of the microscope.
A snapshot of your configuration could be helpful.
This image is from https://www.microscopyu.com/museum/micr ... microscope
Image
I have also seen tall chimneys on the front photo port...

Changing focus from that in eyepieces will compromise captured images.
Instead, using a frosted flat glass ( or if not, 50mm or so square of wax paper),
remove so much hardware as easily possible from any Microphot photo port,
then find the distance at which images come into focus on that flat square.
This is most easily done with room lights off and a simple object in focus,
e.g. something covering half the condenser beam ("half moon").
That establishes parfocal image sensor distance.
You might be able to fabricate something parfocal for that rear port
using the flange from a cheap old Nikon F teleconverter and some focusing helicoid.
Metaphot, Optiphot 1, 66; AO 10, 120, EPIStar, Cycloptic

deBult
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Re: I can't seem to focus

#7 Post by deBult » Fri Jul 29, 2022 7:07 pm

This may seem counter intuitive but the 4* objective (or any low magnification) is the most difficult one to achieve sharp pictures due to limited depth of focus plus the fact you require the most camera megapixels using low magnification objectives.

In addition to that if your scope uses objectives marked “160” you will require the missing projection eyepiece (objectives marked oo often can be used without one).

Suggest to experiment with a flat subject and your 10* objective.

Note: the easiest way to reduce vibration is either high shutter speed (> 1/250) or low shutterspeed (0.5-2s).

https://www.microscopeclub.com/depth-of ... icroscope/

Finelld
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Re: I can't seem to focus

#8 Post by Finelld » Fri Jul 29, 2022 7:53 pm

Thanks for all of the comments. I agree that a reduction eyepiece would help. I didn’t know they even existed. As for my configuration I am using the tall chimney on the front of the scope. I am using an astronomy camera. This has the benefit of not having any moving parts as vibration limitation is extremely important in astrophotography as well. The biggest drawback to this is the camera lacks an ir blocking filter so everything appears orange.

Does anyone know where to obtain a reduction eyepiece?

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Re: I can't seem to focus

#9 Post by PeteM » Fri Jul 29, 2022 8:34 pm

In the Microphot-FXA and perhaps the FX, Nikon included reduction optics for full frame cameras within the scope. They also made a C-mount adapter to fit the bayonet mount at the top front - this should show up in FX or FXA documentation on the Web.

Chances are, with a bit of shop work, you could also adapt a generic C-mount reduction lens between your Microphot and your astro camera. As others have said, you need to get the spacings right to get the best resolution - those spacings most easily determined by having your eyepieces and astro camera parfocal at the same time.

One way you might quickly get a sort of cross check would be to get a cell phone camera adapter to fit one of your binocular eyepieces. You might already have one for astro use that will fit? If not, they're cheap. A good cell phone camera centered over an eyepiece and wirelessly triggered (another cheap adapter available for most phones) can take surprisingly good photos.

Finelld
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Re: I can't seem to focus

#10 Post by Finelld » Fri Jul 29, 2022 9:42 pm

I was able to find a reduction eyepiece on Amazon. It will arrive tomorrow. I forgot to mention that I can adjust the focus and see the immediate results on my computer. It appears that I didn’t make that clear before. Next month I intend to get a dedicated microscope camera. Can anyone recommend one with greater than 10mp, Mac compatible, that I won’t have to sacrifice body parts for. I am a bit embarrassed with how cluttered my lab currently is so I will delay posting images of my setup until it looks tidier.

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Re: I can't seem to focus

#11 Post by PeteM » Fri Jul 29, 2022 10:18 pm

Just to be clear, when your eyepieces are in focus they will be picking up the image at the correct distance for a 160mm finite tube. One could get a camera to focus with, say, an equivalent 150mm or 170mm tube length through various adapters - but the image will have aberrations.

If your eyepieces AND your camera show sharp images at the same time, same focus point, then you know that you're at or near the proper 160mm tube length (the image pickup point is actually around 10mm deeper into the tube).

Is your astro camera C-mount - and the new reduction lens C mount?

Finelld
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Re: I can't seem to focus

#12 Post by Finelld » Sat Jul 30, 2022 12:03 am

Both the camera and ordered reduction lens are C mount.

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Re: I can't seem to focus

#13 Post by PeteM » Sat Jul 30, 2022 4:15 am

Here's what the Nikon Microphot C-mount adapter looks like (this one priced way too high): https://www.ebay.com/itm/384145526536

You'll want to get your generic one with the C-mount positioned at the same height.

Finelld
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Re: I can't seem to focus

#14 Post by Finelld » Sat Jul 30, 2022 4:42 am

Is that adapter for the front or rear camera ports? Where can I get a generic one? What is a fair price? I have the chimney with the 42mm opening and and what I believe to be a place for a projection eyepiece inside the chimney. Eventually I want to have a dedicated microscope camera. Right now I am using the astronomy camera until I get the necessary adapters for a dslr and or a standard microscope camera. I want what will work best on a shoestring budget. Fortunately with this scope it can always be upgraded in the future. You just need to find the accessories and the money. I need to check if I won the lottery 😀

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Re: I can't seem to focus

#15 Post by PeteM » Sat Jul 30, 2022 6:23 am

David, the adapter is for the front port and fits a sort of bayonet mount. As you've already surmised, this position gives you the straightest shot to the objective, less glass in the way. There's another, taller, Nikon adapter for the same position for full frame cameras - originally for 35mm Nikon but any full frame mirrorless or DSLR camera should be able to be adapted. The full frame cameras can also directly attach to either side at the back in an FXA. Some of these research scopes might once have simultaneously hosted a 4x4 Polaroid, a full frame Nikon F, and a C-mount video or movie camera.

I suspect your astro camera can provide very good images and also that you may be able to cobble together some sort of adaptation so your generic C-mount ends up securely mounted and at the right height to be parfocal with your eyepieces.

The Nikon C-mounts, as shown earlier, might show up cheap (under $100) sometime.

If you decide to buy a full frame camera, a mirrorless body would be a better bet (and about the same cost) as a DSLR.

Depending upon what objectives your Microphot came with, you may have already "won the lottery"? It's a fine microscope.

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Re: I can't seem to focus

#16 Post by zzffnn » Sat Jul 30, 2022 7:30 am

Another remote possibility is that your focus gear of the microscope is broken, if you cannot (could not) obtain sharp image at visual eyepieces. That happens when the stage drops under its own weight and will not stay in focus. Some Nikon focus gears have plastic / nylon and are not very durable.

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Re: I can't seem to focus

#17 Post by Hobbyst46 » Sat Jul 30, 2022 7:39 am

Please note, that a "dedicated microscope camera" is not made or marketed for hobby use. Hence, a new microscope brand name camera will be expensive.
Also, its most important feature is not necessarily the number of MP, but the sensitivity and speed. Modern research microscopy calls for low-light sensitivity (for fluorescence, in particular) and high quality rapid videos.
You might page the Zeiss and Olympus (and others) sites to see what they boast with their modern microscope cameras.
Moreover, these brand name cameras are computer controlled, sometimes exclusively by means of proprietary software ($$$ !).

So, saving money on hardware and software dictates that a new dedicated microscope camera will be from a third party vendor, rather than one of the major microscope makers.
In turn, that may amplify the problem of compatibility of the camera with the scope optics.
So I would try and squeeze the best performance from your present camera (its advantage is indeed the absence of moving parts) or, as others have suggested, a DSLR/mirrorless combination, as relatively inexpensive learning steps.

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Re: I can't seem to focus

#18 Post by Finelld » Sat Jul 30, 2022 7:47 am

Thank you for all of the fantastic information, advice, and support.

I will keep my eyes out for the C mount adapter at a reasonable price. I have the taller chimney for the front port. I still need a camera adapter that will fit it and a suitable projection eyepiece. I have leads on both. The only mirrorless camera I have is the astronomy camera and it doesn’t have the best resolution. But I do have three DSLRs two of which are full frame. These will work well.

My microscope didn’t come with any objectives so I bought a few no name ones just to get going off of eBay. I will be upgrading in the future. I have a phase contrast condenser that also does darkfield. I look forward to getting a few phase contrast objectives to complement a few standard ones.

My focus knobs work but the coarse and medium focus are extremely stiff. I have scheduled a microscope tuneup and oil change with a local microscope service company on the 22 of August. I can’t wait. They will also be servicing a Bausch & Lomb Stereozoom that I use for other hobbies like electronics and horology.

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Re: I can't seem to focus

#19 Post by Finelld » Sat Jul 30, 2022 9:57 pm

Update.

I got the .5x lens in today. It has a much closer to parafocal distance than my previous method. Images still seem to be a bit fuzzier than through the eyepiece. I suspect some of this may be due to low megapixels. As funds increase I will be trying more things.

Thanks again everyone for the great assistance and advice.

David Finell

deBult
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Re: I can't seem to focus

#20 Post by deBult » Sun Jul 31, 2022 5:34 am

Finelld wrote:
Sat Jul 30, 2022 9:57 pm
I got the .5x lens in today. It has a much closer to parafocal distance than my previous method. Images still seem to be a bit fuzzier than through the eyepiece. I suspect some of this may be due to low megapixels. As funds increase I will be trying more things.
David, apart from the 4* objective the megapixels in your current camera should suffice.

Enclosed a video from our host on this subject: How many megapixels does you microscope camera need:

https://youtu.be/2JhWaMFBm4I

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Re: I can't seem to focus

#21 Post by deBult » Sun Jul 31, 2022 5:43 am

And a good read on why you need EFSC when you decide buying a DSLR camera

https://krebsmicro.com/Canon_EFSC/

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Re: I can't seem to focus

#22 Post by imkap » Mon Aug 01, 2022 10:14 pm

Finelld wrote:
Sat Jul 30, 2022 9:57 pm
Update.

I got the .5x lens in today. It has a much closer to parafocal distance than my previous method. Images still seem to be a bit fuzzier than through the eyepiece. I suspect some of this may be due to low megapixels. As funds increase I will be trying more things.

Thanks again everyone for the great assistance and advice.

David Finell
The image on my camera is also fuzzier than on the eyepieces. No matter what I did, I think that there just might be a limit, as when photographing you can zoom in and enlarge the image a lot on the screen. So actually it is not e.g 200x, like while looking through the eyepiece, but 4000x as the PC monitor is at least 20 times larger than the EP image, who knows how much when zooming in...

I did see some quite sharp online images here or elsewhere but never did pixel peeping on other peoples images and you can never find full (24mpx) resolution. Probably most of the good images are shot on better slides than I make, so that is an important thing in the chain too...
Does all this make sense?

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Re: I can't seem to focus

#23 Post by blekenbleu » Tue Aug 02, 2022 12:15 am

imkap wrote:
Mon Aug 01, 2022 10:14 pm
The image on my camera is also fuzzier than on the eyepieces.
No matter what I did, I think that there just might be a limit,
as when photographing you can zoom in and enlarge the image a lot on the screen.
...
Does all this make sense?
Yes. When Canon released their 20D digital SLR in 2004, photographers discovered that
expensive and highly regarded L series lenses were less sharp than supposed.
That 20D sensor was only 8 megapixels, but it was a crop sensor.
This led to Canon releasing Mk II and Mk III lens versions, but
Canon now offers 32 megapixel crop sensors,
and photographers are learning to accept
that so-called pixel peeping leads to disappointment,
and a main use for so many pixels is to apply more elaborate digital filtering
that e.g. suppresses noise and sharpens with fewer visible artifacts.

FWIW, displaying camera Live View on a very large monitor
can help can help obtain focus as sharp as possible
Metaphot, Optiphot 1, 66; AO 10, 120, EPIStar, Cycloptic

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Re: I can't seem to focus

#24 Post by Scarodactyl » Tue Aug 02, 2022 1:32 pm

There are certainly inherent limits in microscopy and we're never too far from diffraction. Better objectives can help combat that though. You should probably check out the photomacrography.net forums as well, dince the focus there is more strongly on photograohy (leaning more towards reflected light work but traditional microscopy gets some love too).

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Re: I can't seem to focus

#25 Post by imkap » Tue Aug 02, 2022 1:34 pm

Scarodactyl wrote:
Tue Aug 02, 2022 1:32 pm
There are certainly inherent limits in microscopy and we're never too far from diffraction. Better objectives can help combat that though. You should probably check out the photomacrography.net forums as well, dince the focus there is more strongly on photograohy (leaning more towards reflected light work but traditional microscopy gets some love too).
Thanks, I've never seen that forum... I'll register

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Re: I can't seem to focus

#26 Post by Finelld » Tue Aug 02, 2022 3:08 pm

For now I have ditched the astronomy camera in favor of a canon 7d coupled to a 0.5x eyepiece. This has made a very substantial improvement. But the objectives I initially purchased to get me up and going are definitely not the best. About their only redeeming value is the spring loading on the 60x and 100x. But they all suffer from fuzzy focus around the edges and horrendous chromatic aberration. I know what causes these issues and how to fix them.

This is my first start at fixing these issues. I just purchased Zeiss Neofluar Plan Apo Ph2 Ph3 25x 40x 63x 100x Microscope Objectives at a very good price. The seller claims they are defective and that is why the cheap price. The only thing I can see going bad in these is possible moisture or mildew growth on the inside. I can fix that. These should be great objectives except for dark field. I do have a phase contrast condenser and am looking forward to trying phase contrast microscopy. I have also ordered some other camera adapters to try.

I will keep everyone posted. Thanks for all of the great advice and encouragement.

Best regards,

David Finell

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Re: I can't seem to focus

#27 Post by Scarodactyl » Tue Aug 02, 2022 3:50 pm

Oooh, that's probably a suboptimal pick for a few reasons.
-Zeiss 160 objectives require eyepiece compensations, the strongest of their era. Without the correct eyepieces you will get extreme chromatic aberrations.
-zeiss 160 objectives like to delaminate, and that's not a trivial fix (or a possible fix). Even mold growth inside objectives isn't really very fixable--theyre not really meant to be assembled without particular jigs etc, and that's assuming the glass isn't permanently damaged which it often is.

Finelld
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Re: I can't seem to focus

#28 Post by Finelld » Tue Aug 02, 2022 4:25 pm

Fortunately I am a retired chief broadcast studio engineer that maintained the camera gear and optics for the headquarters of an international news agency. I have rebuilt lenses in the past. These are just a bit smaller than I normally work on. They also came with Zeiss KPL 10x W eyepieces. I would expect that as these objectives are apochromatic, not achromatic they would have nearly no chromatic aberration. As the objectives are tiny I have a Bausch & Lomb Stereozoom to work on them. I normally use it for working on watches and electronics. If I can make the objectives work then I will have some very nice objectives. If I can’t it will have been a fun learning experience. Either way I win.😁. But I do hope that I can make them work. I’ll let everyone know how my money was spent.

I also ordered a 4x plan achromatic from Amazon that I get today. It won’t fix the chromatic aberration but it should solve the very fuzzy edges of my images.
Last edited by Finelld on Tue Aug 02, 2022 8:30 pm, edited 1 time in total.

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Re: I can't seem to focus

#29 Post by Scarodactyl » Tue Aug 02, 2022 7:22 pm

They will have little CA if viewed through the right eyepieces which will cancel it out. Without them the image will be bad. Nikon CF objectives have the advantage of not requiring particular eyepiece compensations so high quality generic adapters or even putting the image directly onto the sensor doesn't cause issues. With the Zeiss objectives you'll need to rig your camera up to shoot through one of the kpl objectives (or better, one of their better corrected photo eyepieces which are pricey). If you can get it all working Zeiss or course made excellent optics and the ciew will be very nice indeed.

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Re: I can't seem to focus

#30 Post by Finelld » Tue Aug 02, 2022 8:27 pm

I just tried the inexpensive 4x Plano objective from Amazon. Going through the.5x projection eyepiece the edges are still very blurry. I removed this lens from the equation and gust held the DSLR in place. The image was sharp nearly edge to edge. I hope to get a proper camera tube later this week. I couldn’t believe how difficult it was to find a 42mm od tube with m42 threads on one end. They seem to be made out of a material similar to unobtanium.

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