Going beyond 5x

Discussion about cameras, microscopes, stains, and gadgets, along with useful tips for preparation of fungi samples
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Pitufo
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Going beyond 5x

Post by Pitufo » Tue Jul 05, 2016 5:11 pm

Hi Dave (and anyone else who would like to pitch in)

Regarding our discussion in the Myxo of the Day thread, I was looking at your shots of Cribraria - I have a few to take myself. I just wondered what method you used as it looks like you have a higher than 5x magnification. I am awaiting an adapter to mount a 10x objective on a tube lens, so I am going to try that route first.

Thanks in advance for your help.

John

Waxcap
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Re: Going beyond 5x

Post by Waxcap » Tue Jul 05, 2016 6:52 pm

Hi,

I use two slightly different methods.

The simplest is:- camera, an old set of bellows, an adapter and a finite microscope objective. The old 210mm Nikon M Plan finite objectives work well. In this case you only have air between the objective and the sensor. The focus is at 210mm behind the objective - you adjust the bellows accordingly. You get the magnification of the objective.

The other method is using a more modern infinite objective and using a good quality prime lens as a "tube" lens or "focussing" lens. I use a Canon body, an adapter to a Old Nikon Nikkor 200mm f4 or Nikkor 105 mm f2.5 a further adapter and a Nikon CFI Plan 4X or 10X or 20X infinite objective. Here the Camera lens focusses the light on to the sensor. You can mix and match the tube lens and objective to get different levels of magnification. The 200mm lens gives the magnification on the objective and the 105mm gives half - if that makes sense.

Both methods have worked well for me. Method 2 has more glass in the light path and maybe a little more chromatic aberration but the objectives are slightly better.

You can pick up a Canon Body (40D is the oldest I use as it has mirror lock up and quiet mode) and the lovely Nikon primes cheap on Ebay, along with the objectives. The objectives need to be as "correction free" as possible and designed for reflected light with no coverslip - Like the Nikon M Plan range - this becomes more important as you go up in magnification (the "Correction Free" CFI range is good up to 20X but wasn't designed for reflected light).

Of course, as you know, you need a very stable base, a method for moving either the camera or the subject in as little as 1 micron intervals for stacking and some very good, variable position lighting (I use two full size Canon flash guns and as much diffusion as I can get between the flash and the subject).

Edit: Of course at the higher levels of magnification the DOF is so much smaller so you will be taking many more photos per stack. Maybe 80 just to stack a Cribraria :o
Dave

Pitufo
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Re: Going beyond 5x

Post by Pitufo » Wed Jul 06, 2016 7:19 am

Thanks Dave, that's very helpful. It sounds like I'm on the right track. I have a decent x10 objective and a telephoto zoom to mount it on. 80 shots for a Cribraria is a bit more than I was expecting and might be beyond my hand-cranked microscope stand. I'll see how it goes.

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Re: Going beyond 5x

Post by Waxcap » Wed Jul 06, 2016 11:23 am

The number of shots required is obviously down to the depth of field and the depth of the subject that you need in focus after the stack. You do need a reasonable overlap of focus on each shot. The depth of field you get from the objective is a factor of the wavelength of the light, the refractive index of the air, the numerical aperture and magnification of the objective and the resolution ability of the sensor on the camera. A ball park resolution for modern SLR sensors is around 5 microns. Crunching the numbers for the 0.25NA 10X Nikon gives a Depth of field of just over 10 microns. I would overlap by 3 microns each end so you get a per-shot depth of 4 microns.

The head of a Cribraria is around 200 microns and of course you want the stem in focus, which will often be outside of the head diameter at the base. Lets say (conservatively) that the base of the stem is outside the head by half the diameter of the head i.e. 100 microns. The total depth of focus you need is 300 microns. 300/4 is 75 photos. Of course you don't want to cut off the focus right at the edge of the subject so add a little each end and voila! 80 Photos. See how I did that :D

Using the 20X NA 0.4 objective will give a depth of field of 4 microns with other factors remaining the same, Overlap 1 micron each shot leaves 2 microns per shot that's 150 photos.

These numbers are in keeping with my experience. I use an ex-laboratory Newport Linear Translation Stage Micrometer, which is calibrated in 1 micron divisions, to move the camera between shots. Some use the "Stackshot" automated system from Cognisys. I would add that my system is horizontal while others use a vertical approach.

Dave

Pitufo
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Re: Going beyond 5x

Post by Pitufo » Thu Jul 07, 2016 8:46 am

:D Thanks Dave, I currently just guestimate the step size I need from experience. It will be a challenge to see if I can still overlap by hand at 10x.

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Re: Going beyond 5x

Post by Waxcap » Thu Jul 07, 2016 10:04 am

As you can see from the numbers above, if you only had an overlap of focus of 1micron with the 10X you would only need a 40 photo stack. That's probably OK on a StackShot controller but with my manual system I need a fair bit more room for error. In reality, like you say, you get experienced in how much to twist the knob between photo's. With the 10X the minimum would be 40 (but you may get the odd OOF stripe) and above 80 would be wasted effort. It's good to understand the theory and the numbers though, as it gives you a good base to work from.

I used to use a zoom as a tube lens but of course they have a fair amount more glass in them than the primes, something that can make a difference at the higher magnifications - I can't say I saw much difference at 10X though.

Dave

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