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Lighting for macro photography of fern gametophytes
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jsp



Joined: 28 Mar 2015
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Location: United Kingdom

PostPosted: Thu Dec 07, 2017 11:21 pm    Post subject: Reply with quote

I take 1/160th second shots with flash. We speeded up the motor that drives the focus block a lot so that the whole series takes a few minutes rather than an hour. My tube lens has an aperture control inside it because it is a prime lens. I don't quite follow how changing the aperture helps. What does that do for it?
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Chris S.
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PostPosted: Fri Dec 08, 2017 12:29 am    Post subject: Reply with quote

jsp wrote:
My tube lens has an aperture control inside it because it is a prime lens. I don't quite follow how changing the aperture helps. What does that do for it?

You have it right, Jen. Your converging lens should generally be used wide open. Closing the iris mostly does nothing except introduce the possibility of vignetting.

The one exception I've noted is that stopping down the converging lens a little bit moderately improves contrast with one lens I've tried. I presume this is because the iris is acting as a flare-cut stop, preventing a bit of stray light frome bouncing around in the lens. But if I close this iris down too far, I get vignetting.

Edit to add:

ChrisR wrote:
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.

When ChrisR wrote this, I'm sure he referred to the objective, not the converging lens. By using an objective with a smaller aperture, you get more depth of field, and therefore need fewer shots to stack. The (very significant) trade-off is that as aperture goes down, so does resolution.

Most microscope objectives do not have irises. In that case, you can mount an iris immediately behind the objective, and usually get the same effect.

--Chris S.


Last edited by Chris S. on Fri Dec 08, 2017 12:55 am; edited 2 times in total
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jsp



Joined: 28 Mar 2015
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PostPosted: Fri Dec 08, 2017 12:33 am    Post subject: Reply with quote

Hi ChrisS, Thanks, that's good to know.

ChrisR I didn't realise that I hadn't described my system very well. It's written up on hackster and GitHub if you would like to see:

https://github.com/BioMakers/23_Focus-stacking-system-for-gametophyte-ferns/blob/master/README.md

https://www.hackster.io/jendeegan/focus-stacking-system-for-gametophyte-ferns-1bc883
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ChrisR
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PostPosted: Fri Dec 08, 2017 7:16 am    Post subject: Reply with quote

Yes - don't close the "tube" or "converging" lens diaphragm for this purpose.
The sort of diaphragm to use , if you want one, is like ebay item 142571689624.
I have seen versions with 52mm (filter thread) and M42mm threads. Put behind the objective it's nearer to the correct place for the objective, - it works.

If you stack with the steps too wide you will have OOF blur showing. That will me more than the diffraction blur you'd have got if you'd used (the correct ) reduced aperture to cover at the step used.
You'd be looking through OOF blur to try to see detail which would , much of the time, be missing, with only a proportion in focus Sad.

You didn't give your cycle time. If you want to be quick, lock the mirror up (if you're doing that) as soon as you've made one exposure, so those vibrations settle while you're moving focus. Some have had success not stopping the rail at all (!), with flash it doesn't need to be long. Maybe half a second. Keep the room dim so the 1/160th doesn't do anything.
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jsp



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PostPosted: Fri Dec 08, 2017 7:49 am    Post subject: Reply with quote

I have been fiddling around trying to lock the mirror up, but it doesn't seem to work with my setup. I do the focus on live view, which I think it also mirror lockup, but in order to switch to a mode where I can fire the shutter using an infra red remote control, I have to switch off live view, and then the mirror doesn't lock up any more. It then goes up and down with every shot.

I'm not exactly sure how long my cycle time is. I'd need to measure it.

I don't really understand what the diaphragm that you suggest is doing for the system. Is there any chance that you can explain in a bit more detail? I see it there on ebay and I understand what it looks like now.

Here it is just so I don't for get when the ebay item closes:

IRIS DIAPHRAGM aperture with male/female M42x1 thread ring activated

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Pau
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PostPosted: Fri Dec 08, 2017 12:20 pm    Post subject: Reply with quote

I see that you're using A Canon 5D II,III or IV. With that camera you have Live view with silent shutter that is better than mirror lock up, you just need to select Silent mode I at the camera menu. If your infrared remote control doesn't work in this mode (is the original one?) just switch to a cable release or even better connect the camera to a computer via USB and use the Canon Utility software (free provided with any Canon EOS). You can fire the camera, control some important camera settings and see the live image at the computer monitor (and also download directly the images to your hard disk). It's how I always work at the studio.
But I think you already know all this, maybe you're referring to your stacking device, but I think that a wired remote would be easy to implement.

The diaphragm placed just behind the objective acts much like an ordinary lens diaphragm: Closing it you get less light, more DOF and less resolution due to diffraction.
I have one like the posted but bough from a different chinese seller at lower price. Because those diaphragms are much wider than the objective when you start closing it you have no effect up to to the point where it's smaller than the objective rear aperture. Closing it too much it clearly kills resolution.
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jsp



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PostPosted: Fri Dec 08, 2017 1:54 pm    Post subject: Reply with quote

Hi Pau,

Thanks, that was what I thought about the diaphragm. I'm quite happy with the sharpness of my big fern photo actually. The smaller fern is a bit fuzzy but that was because something went bit wrong in the run. It was a one-off though.

The silent shutter thing is a bit tricky. I have it set up that the arduino drives the movement of the microscope focus block and then also fires the shutter via an infra red remote control. However to do that, I have to turn off live view and the mirror lockup doesn't seem to work then. At least I haven't worked out how to do it.

If I drive the camera shutter from the EOS software then it is no longer automatically synchronised with the movement of the focus block.

So at the moment I haven't figure out how to have the movement and the shutter automatically synchronised and the shutter locked up as well. It's a Canon 5d MkII, if that helps any.

Thanks!

Jen
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PostPosted: Fri Dec 08, 2017 2:42 pm    Post subject: Reply with quote

Hang on a minute! How are you firing the flash?

From the Arduino or from the camera?

Does a Canon 5Dn fire the flash without flipping the mirror?
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PostPosted: Fri Dec 08, 2017 11:16 pm    Post subject: Reply with quote

The arduino fires a canon infra red remote control, which triggers the camera, and the camera has a yongnuo infra red remote control on its hotshoe, which triggers the flash guns.

On this page https://www.hackster.io/jendeegan/lighting-and-vibration-in-photography-of-fern-gametophytes-72a6ce

you can just see the infra red remote control sitting next to the 200mm prime lens on the metal plate.
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Pau
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PostPosted: Sat Dec 09, 2017 1:37 am    Post subject: Reply with quote

Ah! I was thinking that you shot with continuous light. A Canon EOS (and most other cameras) can't fire the flash while in LV as they need to close and open the mechanical shutter to provide the sync signal.
While in LV it closes and opens two times the shutter and resumes LV, at least when fired from the camera or computer, so the only advantage is to maintain the mirror up.

Likely there is a mirror lock up set somewhere hidden in the menu, not sure.
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PostPosted: Sat Dec 09, 2017 1:50 am    Post subject: Reply with quote

Thanks. yes there is a mirror lock up in the menu but I haven't managed to get the magic combination of settings to make it work. I'll have another go.
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Chris S.
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PostPosted: Sat Dec 09, 2017 2:08 am    Post subject: Reply with quote

I'll ask a stupid question, just in case. In the Nikon world, "Mirror Lock Up" isn't for shooting, but for sensor cleaning. It's "Mirror-up mode" that's used for shooting. If Cannon has something similar, could it be causing confusion?

Also, does Canon have something like Nikon's "Shooting delay mode"? With this feature, one sets a delay between when the mirror goes up, and when the shutter opens. This delay can be varied by the user. It gives the same effect as Mirror up, but requires just a single, rather than double, button press. I've taken to using shooting delay rather than mirror up, most of the time.

--Chris S.
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jsp



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PostPosted: Sat Dec 09, 2017 2:26 am    Post subject: Reply with quote

I just fiddled about with the live view and silent shutter and the remote control and all I can say is that it's a bit flaky and unintelligible.

I'm not using canon flashes, but yongnuo ones, so the canon menu entries refuse to control the flash, and the buttons in the EOS Remote software refuse to communicate with them. Only actually firing the shutter with the remote or the camera button causes the flashes to fire.

When I set the camera on timer mode to use the infra red remote control with EOS Remote connected, there is no way to predict whether it is going to count to 2 or to 10 or not at all before the shutter is fired. It just makes its own decision and gets on with it. As luck would have it, my arduino and remote control set up causes instant firing with no timed wait. I'm not sure why. I'm sure there must be some rationale behind it, but I do not know what that is.

If I have the camera on live view, controlled by EOS Remote, then the camera can take a shot but cannot fire the flashes, so that's no good. I have to turn off live view in order to be able to fire the flashes. There's no point detaching the camera entirely from EOS Remote as I can't then turn on live view with the big screen to find out what's going on with the subject (is it in focus etc.)

So the bottom line is:

I have to be able use EOS Remote to see the subject in live view on the big screen.

I have to be able to turn it off while shooting in order to fire the flashes. This also turns off mirror lockup and I can't get it to turn on again.

I have to use the infra red remote in order to synchronise the shots with the movement of the focus block.

I have no idea how to get the mirror to lock up. None of the mirror lock up settings seem to have any effect on the mirror at all, though at least one of them seems to stop the flashes from firing. At this point I can't even remember which. In case you are wondering - Yes I have read the manual.

I see ChrisR's point that I could just go with continuous movement and take shots every x seconds. I do have a gadget to fire the camera every x parts of a second, but it goes on the hot shoe of the camera, which is currently occupied by the flash remote control.

It's very complicated. On balance, I think we're doing quite well getting any photos at all.



Very Happy Very Happy Very Happy



-----------------------

Later:

Ooo! For further fun, if I use a Canon brand infra red remote, it has three positions on the switch: Off, immediate fire, and two second delay fire.

That explains some of the random delay.

I still can't fire the flash with live view turned on though. If I could do that then I would have mirror lock up, and flash, and live view at the same time, which would perfect.

I wonder if this is because I'm on Yongnuo flashes? The EOS software has whole menu sections for flash firing but they refuse to recognise the Yongnuo flashes.


Last edited by jsp on Sat Dec 09, 2017 2:34 am; edited 1 time in total
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jsp



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PostPosted: Sat Dec 09, 2017 2:40 am    Post subject: Reply with quote

This is the system I'm using:

https://www.amazon.co.uk/YONGNUO-Speedlite-Transmitter-EACHSHOT%C2%AE-Diffuser/dp/B017CWKSVY/ref=sr_1_7?s=electronics&ie=UTF8&qid=1512812238&sr=1-7&keywords=YONGNUO+YN560+IV

YONGNUO 2pcs YN-560 IV Flash Speedlite With 560TX-C Transmitter for Canon EOS 5D,5D25D Mark II


It costs £138 for two flashes and remote control unit instead of £200+ for one Canon brand flash.
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jsp



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PostPosted: Sat Dec 09, 2017 2:45 am    Post subject: Reply with quote

Hi ChrisS,

This is what I have in my camera:

https://www.manualslib.com/manual/275471/Canon-Eos-5d-Mark-Ii.html?page=101

It's one press for mirror up and one more press for the shutter to fire, just as you say.

I kind of thought that since my ferns tend to explode after a short time though, and since I see no fuzziness in my big fern image, that I ought to speed things up rather than slow them down. Do you see vibration issues in the image?

My smaller fern image is a bit fuzzy but that was because of a technical problem I was having on that one shot.
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