What am I doing wrong - see my post on the Macro Forum

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NewForestTel
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What am I doing wrong - see my post on the Macro Forum

Post by NewForestTel »

Dear Photomicrographers – Can you help me please?
For some years I have been a member but in recent years I have not had the courage to post my images.
Until now I have concentrated on “extreme macro” work with insects and have done most of my work with either a Canon EOS 1D Mk4 (full frame) or a Canon M6 Mk2 (cropped frame)
The lenses I have used are Canon MPE 65mm or Canon 100mm Macro f2.8 with the 1D Mk4.
And Laowa 2.5-5x or the Canon 100mm Macro f2.8 with the Canon M6 Mk2.
Some of the work has been manual Stack and Stitch (SnS)
I have always used Zerene Stacker Pro for my stacking work and am used to Slabbing and the associated retouching.
For purposes of showing the level of my work I have posted three images on the Macro images forum today.

However, this week I thought that I would make a foray into working with a microscope objective - Nikon CFI Plan Achromat Microscope Objective, Infinity corrected 10X /0.25.WD10.5.
I have had disastrous results and need serious advice if I am going to progress.

My setup, as seen in the photo, is: -
Canon EOS 1D Mk4, set at 1/200sec exposure, fitted with bellows (the bellows are lined with black flocking) to form a tube with a reversed Raynox 150 and then the Nikon objective. The distance from the sensor mark on the camera body to the Raynox 150 is 208mm.
This is run on a Stackshot Rail set to move 6um per image on a very heavy and solid base.
For lighting I used two PixaPro CITI 600 TTL studio flashes (left hand 50cm distance at 1/8 power, right hand 50cm distance at 1/8 power). Diffused through white foam sheet.

I took 269 images at the interval of 6um using a seed head carrying slime mould with a 10 second time lag between shots. The idea was to finish with an interesting slime mould.

In retrospect – far too ambitious for a first try!!

The images were imported into Lightroom, and one image was fine tuned (minor exposure adjustment) and the whole set of images was synchronised and then exported to Zerene stacker.
In Zerene stacker the images were Slabbed in 10’s with an overlap of 3.
This produced 36 Pmax slabs which were than stacked to produce the awful image below. Individual images looked OK but each slabbed image showed signs of the banding.

I can see that the trail marks are due to a dirty sensor but a I don’t know what has caused the banding on the right-hand side or in the top left.
Also, the stacked image is not at all sharp and I would expect it to be pin sharp with 6um slices and with what I thought would be an excellent objective.

Any help would be greatly appreciated. The image is embarrassing but I cannot progress without your expert comments.
Many thanks for any comments - I am able to take criticism so don't hold back. I need to improve.
Attachments
Final image od 36 slabs stacked
Final image od 36 slabs stacked
One of the slabs of 10 images
One of the slabs of 10 images
A single image from the 269 images taken
A single image from the 269 images taken
Camera setup 1
Camera setup 1
Alternative view of camera setup
Alternative view of camera setup
Terry Turner

Pau
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Re: What am I doing wrong - see my post on the Macro Forum

Post by Pau »

Your extreme macro setup seems very adequate and your macros at the other thread are excellent, so there are just some aspects to improve

- One is evident: as you say the sensor dirt, and the solutions are clear: clean the sensor o use the other camera if its sensor is clean.

- The stripped bands are a classic artifact produced when the stacking software needs to deal with images shifting laterally, so the cause must be the lack of parallelism between the optical axis and the rail movement axis (this is why the bands are shorter in the partial stack). Please compare the position of the white things (spore clusters?) at the single image and at the final stack: their shift roughly equals to the length of the strips. You must check and correct the parallelism -and likely figure a solution for making the centering repeatable once found. Less likely, it also could be due to the subject shifting during the session.

- Another issue often found with high magnification work is blur due to camera vibrations (I'm nor sure if your 1D has quiet EFSC). Although shooting with flash can avoid it, testing with your M6 II with full electronic shutter and continuous illumination is a good idea

-also likely you will need more light diffusion, not only for avoiding some reflections but also for improving resolution
Pau

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Re: What am I doing wrong - see my post on the Macro Forum

Post by Pau »

Just a small point: at our community images taken with microscope objectives like with your setup but outside the frame of a conventional microscope belong to the macro galleries forums and the present thread will be better placed at the Equipment Discussions forum. I'll move it later, after you had time to see this post.
Pau

Adalbert
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Re: What am I doing wrong - see my post on the Macro Forum

Post by Adalbert »

Hi Terry,
When I hear about the M6 Mk2, I take action :-)
I also use this camera and like it a lot.
I also use a Canon 100mm L macro lens.
You write about a Macro-Forum, but do you happen to have a link to one?
Best, ADi

rjlittlefield
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Re: What am I doing wrong - see my post on the Macro Forum

Post by rjlittlefield »

Terry, welcome to posting images!

I'm the fellow who wrote & supports Zerene Stacker, so I've seen quite a few problems. Many of them were worse than you're showing.

As Pau says, the biggest problem is apparent motion of the subject across the frame. It's not possible to tell from the images exactly what has gone wrong. Having the camera pointed slightly sideways is one possibility. But it could also be that the subject moved while the stack was being shot. Typically this happens because of drying, but it can also be due to temperature changes or to slumping of the specimen holder due to plastic deformation or tape slipping, or even to physical pressure from something in the camera rig, if any moving parts touch the specimen holder. In rare cases the appearance of subject movement can itself be an artifact caused by the way that various parts of the subject go in and out of focus.

The first thing you should do is whatever's needed to make sure that the subject does not move and that nothing on the specimen holder is touched by any part of the camera rig.

Then, I suggest to make a couple of changes in your Zerene Stacker configuration: at Options > Preferences > Alignment, remove the checkmarks on Scale and Rotation. The general rule is that you should not do "corrections" for whatever aspects your rig has physically controlled. With microscope objectives Scale does not need to be adjusted, and it looks like your rig prevents rotation. However, you do still need to correct for Shift X and Shift Y, because StackShot rails are likely to shift slightly on each step.

I agree that the image is not as sharp as it should be. I do not see anything in your post about mirror lockup. That, combined with 1/200 second exposure time, leads me to suspect that you're getting camera-induced motion blur. That can easily be bad enough that not even a fast strobe will freeze out the motion. A good approach is to use mirror lockup and second-curtain flash sync, with a shutter time of 1/2 second or more, shooting in a darkened room so that nothing but the flash registers an image. (Check with flash turned off; you should get a black frame.)

BTW, I very much like your "heavy and solid base". We don't see a lot of milling tables used for photo bases, but that's what I started with so I have a soft spot for them.

I'm looking forward to seeing the next round of photos.

--Rik

Marcepstein
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Re: What am I doing wrong - see my post on the Macro Forum

Post by Marcepstein »

I have the 1DM4 too. Mirror lockup and continuous shooting is in the custom functions. You cant see live view with the mirror locked up but it helps. I see the little dots in pics 1-2 in some of my photos and have to clean the sensor to remove them, or spend time in PS. I see them more with the MP-e65. They may show the movement -

@Rik
suggest to make a couple of changes in your Zerene Stacker configuration: at Options > Preferences > Alignment
Would you suggest this for macro too?
A good approach is to use mirror lockup and second-curtain flash sync
Why second-curtain?

Marc

rjlittlefield
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Re: What am I doing wrong - see my post on the Macro Forum

Post by rjlittlefield »

Marcepstein wrote:
Fri Feb 17, 2023 8:23 pm
@Rik
suggest to make a couple of changes in your Zerene Stacker configuration: at Options > Preferences > Alignment
Would you suggest this for macro too?
Only the general advice that "you should not do "corrections" for whatever aspects your rig has physically controlled". For most macro stuff you do need scale correction plus X and Y shifts. But for most solid rigs with a subject that does not sag you can turn off Rotation. If you're using continuous illumination with long enough exposures to avoid mains-frequency flickering, then you can also turn off brightness correction.

A good approach is to use mirror lockup and second-curtain flash sync
Why second-curtain?
To avoid possible vibration from the first curtain opening. Shutter vibration is order of magnitude less than mirror vibration, but when you're still in the debug stages it's best to avoid any issues that you possibly can. For continuous illumination EFSC takes care of first curtain vibration, but flash requires mechanical shutter.

--Rik

Adalbert
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Re: What am I doing wrong - see my post on the Macro Forum

Post by Adalbert »

Hello Rik,
For continuous illumination EFSC takes care of first curtain vibration, but flash requires mechanical shutter.
But Terry has the Canon EOS M6 Mark II as well.
This camera has a fully electronic shutter.
So, I would recommend using this shutter.
And flash works with the full electronic shutter without any problem.
I have been doing this for years :-)

Best, ADi

Marcepstein
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Re: What am I doing wrong - see my post on the Macro Forum

Post by Marcepstein »

@Pau
so the cause must be the lack of parallelism between the optical axis and the rail movement axis (this is why the bands are shorter in the partial stack).
I notice this movement also, so are there good methods to improve the parallelism in composing the shot. I try to set the camera/lens as level as possible and then adjust the subject to center focus. I also see plants/flower sag on long stacks and try setups that reduce that.

Marc

rjlittlefield
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Re: What am I doing wrong - see my post on the Macro Forum

Post by rjlittlefield »

Adalbert wrote:
Sat Feb 18, 2023 2:59 am
Hello Rik,
For continuous illumination EFSC takes care of first curtain vibration, but flash requires mechanical shutter.
But Terry has the Canon EOS M6 Mark II as well.
This camera has a fully electronic shutter.
So, I would recommend using this shutter.
And flash works with the full electronic shutter without any problem.
I have been doing this for years :-)
Perhaps I am confused. At viewtopic.php?p=283200#p283200 you describe "I generate the signal from the controller about in the middle of the shutter speed. E.g. shutter speed = 1/10s, flash signal at 1/20s after triggering the camera."

This sounds like you are using a custom controller to generate a mid-curtain flash trigger, which the camera will not generate by itself.

Have I misunderstood? Or are you suggesting that Terry should use such a controller also?

--Rik

Adalbert
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Re: What am I doing wrong - see my post on the Macro Forum

Post by Adalbert »

Hi Rik,
Yes my controller generates such signal but you need only a simple time-delay.
AFAIK, such delay-triggers are offered by the producers of the rails.
Best, ADi

rjlittlefield
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Re: What am I doing wrong - see my post on the Macro Forum

Post by rjlittlefield »

Adalbert wrote:
Sat Feb 18, 2023 4:32 pm
Yes my controller generates such signal but you need only a simple time-delay.
AFAIK, such delay-triggers are offered by the producers of the rails.
Thanks for the clarification.

Some rail controllers do that. But the OP here says that he is using StackShot, which AFAIK does not.

Also I am not aware of anybody who sells a flash delay circuit that can be added to the StackShot.

If anybody does, I would be interested to know also, because this issue comes up pretty often.

--Rik

Adalbert
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Re: What am I doing wrong - see my post on the Macro Forum

Post by Adalbert »

Hi Rik,
maybe something like that ( with output frequency 1Hz ) ?

https://www.ebay.com/itm/225032503224?h ... BMnM6Ek81h

https://www.ebay.com/itm/193715091225?h ... R5zOhJPNYQ

Best, ADi

NewForestTel
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Re: What am I doing wrong - see my post on the Macro Forum

Post by NewForestTel »

Many thanks to everyone that has replied, I can see that I am on a steep learning curve.
However, onward and upward. I will apply all of the advice given and report back.

To Rik Littlefield and Pau many thanks for the full critique and suggested changes. I knew that you would have the answers.

Once again thanks to EVERYONE
Terry Turner

dolmadis
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Re: What am I doing wrong - see my post on the Macro Forum

Post by dolmadis »

Adalbert wrote:
Sun Feb 19, 2023 3:28 am
Hi Rik,
maybe something like that ( with output frequency 1Hz ) ?

https://www.ebay.com/itm/225032503224?h ... BMnM6Ek81h

https://www.ebay.com/itm/193715091225?h ... R5zOhJPNYQ

Best, ADi
Hi ADi

Using your m6mii as the subject could you show me please in detail how to deploy these components to sync flash and the electronic shutter.

Thank you so much, John

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