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Lighting for macro photography of fern gametophytes
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Chris S.
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PostPosted: Wed Dec 06, 2017 1:10 am    Post subject: Reply with quote

jsp wrote:
Thanks so much for looking in. I'm full of curiosity to know what the better optics are now. [smile] I wondered if you would have a minute to tell me? I'm really chasing great optics with this, and keen to not do anything daft by buying something that turns out to be second best.

Jen,

I just did a quick test to see if the Mitty 2x covers full frame DSLR without vignetting. Unfortunately, I'd call the test inconclusive, as I did get vignetting--but it was a hard-edged vignette, which makes me think it was the converging lens I chose (micro-Nikkor 200mm IF-ED), rather than the objective. Though I use FF in the field, I shoot almost entirely AFS-C in the macro studio; hopefully, someone who routinely shoots FF in the macro studio can test coverage of the Mitty 2x for you on a converging lens whose FF behavior is well-understood.

In terms of chasing better options for 2x, ugh--it's a long conversation. I have some ideas, but since my opinions are mostly based on APS-C, I'm ill-equipped to answer the question of FF coverage. Also, I've done no rigorous parallel testing at 2x.

Ideas, though:

Rodenstock Apo-Rodagon-D 2x 75mm f/4.5: This lens is designed specifically for use at 2x. I use and like this lens. It's a bellows lens though--so you would need to remove your converging lens from your rig, and substitute in a bellows or tubes of the proper length.

Printing Nikkor 105mm f/2.8: This is an amazing lens--almost a legend, a bit hard to find, and rather expensive. It is optimized for 1x and is exquisite at 1x magnification. I've used it some outside this range, as well, with quite good results. Recent work by forum members suggests adding a 2x teleconverter to get 2x--something I want to try. Again, this is a bellows lens.

Some of the Canon macro lenses come to mind. I have the 35mm, and if I ever get around to doing a careful test at 2x, will definitely include it. This said, I tend to think of this as a 3x lens. Again, a bellows lens.

A lens I like at 2.5x is the Nikon M Plan 2.5x/0.08. Very nice, but you must crop the corners, as it vignettes on APS-C. If a bit of cropping is acceptable, though, this is a very nice optic. (Bellows lens.)

This quick list is far from complete--am speaking only of some of the optics I own and use. The 2-3x range is a magnification for which there are many lenses and many opinions. A careful tester could spend weeks attempting to clear these waters.

--Chris S.
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jsp



Joined: 28 Mar 2015
Posts: 354
Location: United Kingdom

PostPosted: Wed Dec 06, 2017 4:32 am    Post subject: Reply with quote

Hi ChrisS,

Thanks so much for your thoughts on the 2x options. The Printing-Nikkor 105mm f/2.8 sounds fantastic and I see online that the optics really are amazing. There is a description here:

http://coinimaging.com/printing-nikkor_105.html

I have a 2x teleconverter too, so that's a very interesting thought indeed. My funding isn't set up and working yet, but I will ask whether they stretch to ebay. :-) I'm not sure how to deal with bellows or attach a Nikor lens to a Canon camera but I will get reading. :-)

Do you think that the Mity 5x and 20x are worth a go?


Separately - I have checked with the MP-E and my Canon 5d MkII says "peripheral illumination correction data not available" so there's definitely something gone amiss there.
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jsp



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PostPosted: Wed Dec 06, 2017 6:28 am    Post subject: Reply with quote

This is an MP-E image on x5 with the peripheral illumination data now in the camera.

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jsp



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PostPosted: Wed Dec 06, 2017 7:01 am    Post subject: Reply with quote

Here is another one at 5x with the MP-E



Interestingly these are quite mature plants and I'm still managing fine on 5x.
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Chris S.
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PostPosted: Thu Dec 07, 2017 1:31 am    Post subject: Reply with quote

Jen,

I think that bellows lenses like the Printing Nikkor 105 are an unproductive tangent for your particular set of goals. Lenses are important, but beyond a certain point, small differences may cost more in money and trouble than they are worth to a particular photographer.

If you were a photo geek pursuing the ultimate at any cost (nothing wrong with this! Very Happy), then sure, look at Printing Nikkors, macro-Varons, etc. But my sense is that you are a scientist more intent on studying and photographically documenting fern gametophytes, who also wishes to teach other people how to photograph such things. If so, your emphasis is probably better served with a solid, replicable system that produces excellent results with items anyone can buy off the shelf, and usable with a minimum of hardware juggling.

You already have the Canon MP-E 65. While I've no personal experience with this lens, it's widely regarded as excellent in the 1x-3x range. Above this, I'd definitely agree with the Mitutoyo 5x, 10x, and 20x, if your subject fields match these magnifications. These are very solid optical choices; gains from more exotic options, where real, fall within the realm of diminishing returns.

If you don't want to switch hardware when you go below 5x (definitely possible, with your fast-drying subjects), it's reasonable to substitute the Mitutoyo 2x, and crop the corners if necessary.

Cheers,

--Chris S.
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jsp



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PostPosted: Thu Dec 07, 2017 2:51 am    Post subject: Reply with quote

Hi Chris,

Thanks, that really makes a lot of sense. If I get my report right at the end of the six months then they may give me another £1000. If I don't buy the 2x then with the extra money I think I could stretch to a 50x. That could be very interesting indeed. I'll aim for that I think, and start with the 20x and 5x.

These are the prices I've been quoted:

2X M Plan Apo Objective £592.92
5X M Plan Apo Objective £632
20X M Plan Apo SL (30.5mm WD) £1544
50x M Plan Apo £1933
50x M Plan Apo SL £2300
50x M Plan Apo HR £3758


So on a budget of £4000, buying the 5x and 20x = £1824 left.

With the extra £1000 I would have £2824 which is enough for a x50 as well.

That might get me a photo of an asymmetric cell division in a filamentous cell that I think is possibly the most exciting thing ever. Woohoo!

Thanks for your advice. :-) This is all very exciting.
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enricosavazzi



Joined: 21 Nov 2009
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Location: Stockholm, Sweden

PostPosted: Thu Dec 07, 2017 3:01 am    Post subject: Reply with quote

jsp wrote:
...
50x M Plan Apo £1933
50x M Plan Apo SL £2300
50x M Plan Apo HR £3758
...

Probably you already know this, but it may be useful for some readers:

The SL series increases the working distance with respect to the M Plan Apo series, at the cost of a lower NA (and therefore a lower resolution). The difference is not very big, but it is there. Also, mixing ordinary M Plan Apos with SL on the same microscope turret may force you to refocus substantially when switching between the two series. Unless you really need the increased working distance of the SL, there is no point in paying more for an SL.

The HR series goes in the opposite direction, it increases NA with respect to the M Plan Apos at the cost of DOF (I don't remember if the HR also decrease the working distance, but they might). The HR make sense when coupled to a zoom tube lens (so that you can push the 50 x well above 50 x, for example), or if you need the extra resolution at the expense of a bigger image stack to post-process.
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jsp



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PostPosted: Thu Dec 07, 2017 4:32 am    Post subject: Reply with quote

Hi Enrico,

Thanks, that really is extremely useful to know. I hadn't figured that out.

My minimum step size in focus stacking is 1 micron, and I probably need about 1cm working distance if I can get it. Do you think the cheapest 50x would get me that?

I'm am working on 17 micron step sizes with the 10x and that is working fine. I was able to measure the depth of field with a stage micrometer:

https://www.hackster.io/jendeegan/measuring-depth-of-field-with-a-stage-micrometer-cea0b3

Thanks!
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enricosavazzi



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PostPosted: Thu Dec 07, 2017 5:05 am    Post subject: Reply with quote

The Mitutoyo M Plan Apo 50x (non-SL, non-HR) has a working distance of 13 mm according to specifications. It has NA=0.55 and a resolving power of 0.5 micron, also according to specifications.

The specifications say 0.9 micron DOF, but Mitutoyo is known to specify DOF in an odd way. It must have been discussed on this site, but I cannot find a thread (actually, the problem is that I find too many). Working from memory, Mitutoyo specifies as DOF what others would call half-DOF, so the actual DOF as defined on this site should be 1.8 micron. Verify this independently, because I may remember it wrong. Sensor size and resolution also affect somewhat the calculation of DOF. The best thing would probably be to compute the DOF based on the rest of your system.

Based on the above, 1 micron steps are a bit small, while 2 micron are probably a bit too much. If you can get 1.5-1.8 micron steps, that should be about right.
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jsp



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PostPosted: Thu Dec 07, 2017 6:02 am    Post subject: Reply with quote

Hi Enrico,

Thanks, that's brilliant. I just thought about it and though the knob on my microscope focus block only goes down to 1 micron steps, the arduino can drive it in far smaller parts of a micron, down to 1/128th of a micron. So it should be able to do half microns quite easily I think.

The limiting factor at that point is probably the plastic gear wheels that allow the arduino to drive the focus block and I'd just have to try them and see what they can manage. Maybe if they won't do it I could try to get some better ones, or a belt.

Thanks for explaining. I will put the cheapest 50x objective into my budget plan.

Thanks :-)
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jsp



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PostPosted: Thu Dec 07, 2017 6:06 am    Post subject: Reply with quote

I just worked out and the three objectives together come to £4109 which is great for my £4000. I can add in a bit myself to make that up and have all three for the 6 months of my practical work. Yay!

Thanks!

Jen
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ChrisR
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PostPosted: Thu Dec 07, 2017 7:41 am    Post subject: Reply with quote

If it wasn't clear from Enrico's post don't buy that 20x in your list. You don't want the HR or SL versions of any of them.

17 micron steps at 10x is losing you resolution. If you look close enough you will see it.
I started at 10 µm and saw that it was too coarse.
I'd be using
6 µm at 10x with a 10x NA 0.28
2.5 µm at 20x with a 20x NA 0.42
1.5 µm at 50x with a 50x NA 0.55
Those give you about 20% overlap.

At 5x with your MP-E, at f/2.8 you shouldn't be using more than 20 µm steps. Maybe that was what was "wrong" with your MP-E?
What steps were you using? Does that account for the blurred parts in the image you showed? (Focus banding)

Proving it is easy. DO a stack with say 3µ steps with your 10x.
Then in Zerene you align the lot, but can choose to use only say every second, or every third, or every fourth frame. Then go pixel peeping.
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Last edited by ChrisR on Thu Dec 07, 2017 8:00 am; edited 1 time in total
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PostPosted: Thu Dec 07, 2017 7:52 am    Post subject: Reply with quote

Gosh! A new post just appeared before this one that I hadn't seen before.

I will try doing shorter steps. The tricky part is that if I go to short steps with a deep subject, the fern tend to overheat and fold up before I am finished. The 17 micron steps get me enough resolution to have a good image, but not such a long step that the fern implodes. I will see how far I can push them and report back. :-) Thanks!

I see what you mean now about the 20x SL. I will ask about a non-SL one. They must have quoted me for the wrong one. Thanks!


Last edited by jsp on Thu Dec 07, 2017 8:43 am; edited 2 times in total
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ChrisR
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PostPosted: Thu Dec 07, 2017 8:05 am    Post subject: Reply with quote

It's in Preprocessing - stack every Nth frame.
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ChrisR
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PostPosted: Thu Dec 07, 2017 6:00 pm    Post subject: Reply with quote

Quote:
the fern tend to overheat and fold up before I am finished.
Hmm, in that case you'd be better off using a lens with a smaller aperture. Unless you add a diaphragm to your adapter (they are available)you could use a lower mag objective.
You could use your 5x 0.14 with a converter behind your tube lens, instead of your 10x 0.28?

Or speed up our workflow if there's any scope. What's your exposure rate? Using flash?
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