Looking for advice to improve sharpness

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Mycobiont
Posts: 7
Joined: Tue May 29, 2018 10:55 pm

Looking for advice to improve sharpness

Post by Mycobiont »

I just got some microscope objectives and am beginning to play. I think my technique will need some work and would like a little advice in a direction that may help others who are just getting started too.
  • Image for critique is the first below or on flickr where you can zoom in further, at https://www.flickr.com/photos/116444714@N04/50834594418. This is a fungus that has spores piled over the black surface.
    • The image is a stack of 55 photos spanning about a 0.3 mm distance.
    • Each photo was 1/250 sec, ISO 100, with flash.
    • Objective requires 160mm distance; I should be to within a mm or two of that (from base of objective by threads to sensor plane)
    • Black felt has been added to interior of tube to cover a shiny metal flange where the conical objective adapter meets the extension tube.
    • Zooming in on them reveals that the image is not very sharp.
  • Image of setup is second below.
Since I'm just getting started with using microscope objectives, I am starting a bit cheap with the $37 Amscope objective,"10X Plan Achromatic Objective Lens with Knurled Ring". So lens quality may be one factor. But I'm thinking the direct flash may be the biggest issue? There seems to be a lot of glare when I zoom in. My camera sensor also needs a cleaning, but I think that should just affect localized spots within the image. Stacking is with Helicon Focus, method C (pyramid).

Any advise? Thanks!

Image

Image

Lou Jost
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Re: Looking for advice to improve sharpness

Post by Lou Jost »

One thing many forum members have noticed in the past is that those cone-shaped adapters can cause flare and should be flocked. Additional diffusion would also help, as you noted. But I suspect the main problem here is objective quality. There is no way anyone can make a good 10x 0.25 objective for $37.

rjlittlefield
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Re: Looking for advice to improve sharpness

Post by rjlittlefield »

Viewing your image at full resolution (5496 x 3864), I see a lot of lateral chromatic aberration -- color fringes away from center on light vs dark transitions. That is a property of the objective. You can eliminate the obvious fringing with post-processing in Photoshop and similar tools, but fringing is always accompanied by radial blurring.

At center, there may be a tiny smidgeon of motion blur. Electronic flash will freeze out most blur, but it is best used with second-curtain sync, with a shutter-open time of 1 second or more, in a room that is dark enough for no image to record except for the flash. You want time for the shutter shock to dissipate, before the flash fires.

Other than that, the image looks about as sharp as I would expect for a 10X NA 0.25 objective. If you're new to objectives, you may not realize that 10X NA 0.25 implies an effective aperture of f/20, which puts you well into diffraction territory on a 21 megapixel sensor. To partially counter the diffraction blurring, standard practice is to apply strong sharpening in post-processing. See for example https://www.photomacrography.net/forum/ ... 96#p253196 .

--Rik

Mycobiont
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Joined: Tue May 29, 2018 10:55 pm

Re: Looking for advice to improve sharpness

Post by Mycobiont »

Rik, wow, that is a neat idea I hadn't thought of with rear-curtain sync to wait for shutter vibration to end!

Mycobiont
Posts: 7
Joined: Tue May 29, 2018 10:55 pm

Re: Looking for advice to improve sharpness

Post by Mycobiont »

Thanks for the tips thus far! An quick test of flocking that conical adapter really made a difference! The stack below has some other problems... looks like I jumped to far with the focal plane a couple times. But I can already see a major reduction in the glare, allowing the true texture of the spores to be visible. When I get the time, I'll post a demo series with (1) the flocking of the cone, (2) rear curtain sync, (3) diffusing the flash, (4) chromatic aberration cleaned up to the extent I can in Adobe Lightroom.

Testing higher quality objectives will have to wait. Yes, a $37 lens is cheap. But I've got 2 teenagers and a wife going back to grad school. I'll keep my eyes open for a good deal, but it may take a while.
2021-01-16 22-50-40 (C,Smoothing2).jpg

Mycobiont
Posts: 7
Joined: Tue May 29, 2018 10:55 pm

Re: Looking for advice to improve sharpness

Post by Mycobiont »

Each step makes a difference (although the rear-curtain flash is pretty minor in my opinion). The following images add improvements one at a time.
A = original
B = flocking of cone
C = Rear Curtin Flash
D = Flash diffuser (and bounce) - used a slice of milk jug to create a band that goes all the way around the specimen with a single flash going through it. By going all the way around, there is some light being reflected back from the far side.
E = Same images as D, but the source images were first altered in Lightroom to reduce glare with a major reduction of the "highlights" control. Also a tad of sharpening and popping up the darkness of the spores. I'm still at version 6 - I think newer versions have some additional capabilities that may be relevant (defringe).

A.jpg
B.jpg
C.jpg
D.jpg
E.jpg

Chris S.
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Re: Looking for advice to improve sharpness

Post by Chris S. »

Mycobiont,

Moving from images A through E, it appears that noise is increasing. Any idea why this is? Are you increasing your ISO? Or is your histogram moving toward the left?

Plastic from most milk jugs, in my experience, is too close to transparent to be a good diffuser. To test it, try looking at a light through it; if the light is not broadly diffuse, try another material. For a very good, cheap diffuser, try a double thickness of tracing paper—you could line your milk-jug ring with it. Rik often uses facial tissue--a layer or two of this, taped loosely inside you the ring, might also work well.

This said, images with a single, diffused light source are often very sub-optimal in the macro studio. To make better use of the single flash, I’d suggest trying only bounced light from it. A neat trick for this to cut a ring from a polystyrene coffee cup, remove about a 45-degree section out of it, and place the remaining ring on the opposite side of your subject from the flash, so that your flash is pointed into the missing section. Then fold, trim, and place the scrap of polystyrene to completely block any direct flash light from hitting your subject. What you’re left with is entirely bounced light, which can be quite nice. (Also: If you like this light but want to "sculpt" its modeling a bit, you can flock inner portions of the bounce ring to subtract light here and there.)

My sense is that your lens needs to go. For a better objective, can your budget cover $100? There are some nice Nikon N Plan 10x/0.30 160/0.17 objectives on eBay for this price or less. This is a classic objective that many of us cut our teeth on, and that is capable of fully professional images. I paid $350 (second hand) for my first specimen of this lens, and was glad to get it at that. To see them being ignored now makes me shake my head. (I have a spare--PM me if you'd like to try it in your rig to see if it improves things.)

BTW, your shelf of stains and reagents makes me smile. I’ve studied lichens/fungi a bit, myself. :D

--Chris S.

Mycobiont
Posts: 7
Joined: Tue May 29, 2018 10:55 pm

Re: Looking for advice to improve sharpness

Post by Mycobiont »

Thanks Chris S.! Next on my agenda is indeed trying better diffusing methods. Your styrofoam cup idea sounds great. And I'm glad to hear that there are 'good' lenses available for around $100 - that should be possible in the near future.

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