1st Photomacrography Image - Technical Queries

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ojd01
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1st Photomacrography Image - Technical Queries

Post by ojd01 »

Hello!

I have recently ventured into the world of photomicrography, especially using microscope objectives, attached to DSLR! I have completed my rough setup and taken a test run, 250 image stack, using an achromatic 4x objective (further setup details below). I'm going to be honest and say... the first image stinks... and I expected it.

This is my setup:

Tripod and Macro Rail: WeMacro Auto Rail (1mm screw turn)
Canon EOS 500D + M42 tubes (total: 160mm) + 4x achromatic objective (objective has not been counted in 160mm)
Specimen holder
1 LED light (ordered a twin gooseneck LED light for tomorrow)
Zerene Stacker - using PMAX align and stack
  • I feel as if lighting was the first major problem, since it was very dark and grainy after post-production in Lightroom. Lighting I can sort with LEDs or flashes.

    The second point I feel is wrong is the objective, its a simple achromatic and not very good quality. I have a Lomo 3.7x (No.3) and Lomo 8x on order.

    The third point, I feel I am massively amateurish about, is the stacking and steps. I have done loads of usual 1:1 and 2:1 magnification with macro lenses and rails, but this is very different (in my opinion). I actually ran 400 images at 5um steps, across 2000um distance. 150 of the images were useless, so this makes me feel I should have decreased the step distance. I think the rail can drop to 0.5um or 1um each, so lots of very fine steps if needed. I understand there are calculations that can be made - I just haven't got that far yet.
I am not disheartened at all, it is only day 1, and I know with more practice and learning great things can be achieved! Any advice would be amazing as all of you are superstars on here! :) If the image quality is bad on your screen, it could be the actual image (ahaha), or I can upload one which is better.

Kindest Regards

Owen

https://www.photomacrography.net/forum/ ... ew&id=3009
Attachments
2021-02-20-19.51.07 ZS PMax.jpg
Instagram: @ojd_photo

rjlittlefield
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Re: 1st Photomacrography Image - Technical Queries

Post by rjlittlefield »

Owen, congratulations on your first foray into using microscope objectives.

Regarding step size, see https://zerenesystems.com/cms/stacker/docs/tables/macromicrodof for tables that will simplify your estimates. Table 2-C is appropriate for a microscope objective. Your 4X objective is probably NA 0.1, which implies nominal DOF = 0.055 mm. I would typically shoot a little smaller than that, like 0.050 or 0.040 mm, but in no case approaching the 0.005 mm = 5 µm that you used. 0.005 mm step size is typical for a 10X objective.

Objective quality is seldom an issue in the center of the image. Good objectives have flatter fields that retain image quality farther into the corners. But in the center of the field, there is seldom much difference in sharpness between any two objectives that have the same magnification and NA. (NA, Numerical Aperture, is the microscopy equivalent of F-number, except that with NA bigger numbers mean wider aperture, while with F-numbers it's the other way 'round.)

Your image appears soft, but that's probably due to reasons other than lens quality. Unless the subject itself is lacking in sharp detail, softness is probably due to some issue with motion blur.

The most common cause is vibration introduced by mirror slap and/or shutter shock. You are fortunate in that your camera, the Canon EOS 500D, provides an excellent method to avoid those issues. All you have to do is shoot in Live View mode, and then the camera will use what is called EFSC, Electronic First Shutter Curtain, to totally avoid mechanical shock at beginning of exposure. See https://www.robertotoole.com/blog/2014/01/28/electronic-first-shutter-curtain for an extended discussion. One warning: if you are tethering your camera to a computer, then in some modes the camera will ignore shutter triggers from the rail controller. There is a simple workaround for that; see Step 9 of the recipe at https://zerenesystems.com/cms/stacker/docs/stackshot#canon_t1i_with_canon_eos_utility_software .

Another common cause is environmental vibration, for example due to people moving around the room, nearby traffic, nearby motors or fans, and so on. These issues can be exacerbated by any flexibility in the subject mounting. When you're shooting a flower at high magnification, it's best to clip onto it very near the head.

If you still can't get a sharp image, then the next thing to do is to switch to electronic flash illumination, preferably placed close to the subject so that you get nice short "low power" flashes. A flash that's operating at say 1/16 power will typically provide an effective exposure time in the range of 1/5000 second or shorter, which will do an excellent job of freezing out whatever motion blur you might get with a longer exposure.

The last issue I see is that the center of your image has a broad area that appears to be a "hot spot" with reduced saturation and no good blacks. A common cause of that problem is stray light reflecting off the sides of extension tubes. All but the most expensive tubes, and even some of those, will have this problem. Extension tubes almost always need to be flocked or baffled to avoid this problem. A simple solution is to slip a cylinder of flocking paper, or even black construction paper, inside the tubes. The problem can be diagnosed clearly and easily by simply removing the camera and looking into the tube with your eye, placing it where the camera sensor would normally be. See https://www.photomacrography.net/forum/viewtopic.php?t=35350 for discussion and illustrations.

I hope this helps!

--Rik

JKT
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Re: 1st Photomacrography Image - Technical Queries

Post by JKT »

In addition to the tubes, the LOMO 3.7x is also prone to reflections. A very small black "hood" protruding a few mm past the end of the objective might help. I also painted the end of the objective body black, but that may be an overkill.

The nominal length for the tubes in the direct projection is roughly 150mm and that includes the distance from the sensor to the beginning of tubes. That distance is 45.46mm for M42. So the theoretical nominal setup would need about 100mm of tubes. Add the 45.5 and the RMS adapter (assuming flat type) to that and you are close to 150mm. If you meant that the total length with all those considered is 160mm, you are only slightly over the nominal and the objective should work well.

As far as the objective quality is concerned, I would not call LOMO 3.7x as "not very good quality". It is quite good and way way above what its price suggests. The LOMO 8x .20 (if that is what you are getting) is unfortunately not in the same league.

ojd01
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Location: Teesside, Middlesbrough, UK

Re: 1st Photomacrography Image - Technical Queries

Post by ojd01 »

rjlittlefield wrote:
Sat Feb 20, 2021 5:59 pm
Owen, congratulations on your first foray into using microscope objectives.

Regarding step size, see https://zerenesystems.com/cms/stacker/docs/tables/macromicrodof for tables that will simplify your estimates. Table 2-C is appropriate for a microscope objective. Your 4X objective is probably NA 0.1, which implies nominal DOF = 0.055 mm. I would typically shoot a little smaller than that, like 0.050 or 0.040 mm, but in no case approaching the 0.005 mm = 5 µm that you used. 0.005 mm step size is typical for a 10X objective.

Objective quality is seldom an issue in the center of the image. Good objectives have flatter fields that retain image quality farther into the corners. But in the center of the field, there is seldom much difference in sharpness between any two objectives that have the same magnification and NA. (NA, Numerical Aperture, is the microscopy equivalent of F-number, except that with NA bigger numbers mean wider aperture, while with F-numbers it's the other way 'round.)

Your image appears soft, but that's probably due to reasons other than lens quality. Unless the subject itself is lacking in sharp detail, softness is probably due to some issue with motion blur.

The most common cause is vibration introduced by mirror slap and/or shutter shock. You are fortunate in that your camera, the Canon EOS 500D, provides an excellent method to avoid those issues. All you have to do is shoot in Live View mode, and then the camera will use what is called EFSC, Electronic First Shutter Curtain, to totally avoid mechanical shock at beginning of exposure. See https://www.robertotoole.com/blog/2014/01/28/electronic-first-shutter-curtain for an extended discussion. One warning: if you are tethering your camera to a computer, then in some modes the camera will ignore shutter triggers from the rail controller. There is a simple workaround for that; see Step 9 of the recipe at https://zerenesystems.com/cms/stacker/docs/stackshot#canon_t1i_with_canon_eos_utility_software .

Another common cause is environmental vibration, for example due to people moving around the room, nearby traffic, nearby motors or fans, and so on. These issues can be exacerbated by any flexibility in the subject mounting. When you're shooting a flower at high magnification, it's best to clip onto it very near the head.

If you still can't get a sharp image, then the next thing to do is to switch to electronic flash illumination, preferably placed close to the subject so that you get nice short "low power" flashes. A flash that's operating at say 1/16 power will typically provide an effective exposure time in the range of 1/5000 second or shorter, which will do an excellent job of freezing out whatever motion blur you might get with a longer exposure.

The last issue I see is that the center of your image has a broad area that appears to be a "hot spot" with reduced saturation and no good blacks. A common cause of that problem is stray light reflecting off the sides of extension tubes. All but the most expensive tubes, and even some of those, will have this problem. Extension tubes almost always need to be flocked or baffled to avoid this problem. A simple solution is to slip a cylinder of flocking paper, or even black construction paper, inside the tubes. The problem can be diagnosed clearly and easily by simply removing the camera and looking into the tube with your eye, placing it where the camera sensor would normally be. See https://www.photomacrography.net/forum/viewtopic.php?t=35350 for discussion and illustrations.

I hope this helps!

--Rik
Hello Rik,

Thank you so much for your response - and apologies for the delay in replying (few reasons for that).

I have attached a new version, of the same subject, taken earlier today using some of the advice you gave me :D Already I feel the image has got better! But needs some work still, but this is only one subject.

Firstly, I opted for around 0.040mm steps, much higher than the 5um I used before. Already the subject seems a bit more in-focus overall.

Secondly, I tried to minimise the motion blur by increasing ISO to 200, unlike 100 before (I am not sure how the ISO will play higher than 400). This meant a rough shutter speed of 1/80, with the new LEDs that arrived today. It is still not enough, so maybe a single flash, or dual flash, setup may be useful for some subject freezing. I would love shots at 1/4000+ SS, as that would really reduce the motion blur - I have some manual Yongnuo's somewhere I could rig up.

I got the live view shutter EFSC to work on the camera - thank you! I had read mixed reviews about using live view vs standard eyepiece view, so wasn't too sure. It definitely helped in reducing the shutter. I am going to try and obtain some special feet for my stand and tripod, to try and reduce vibrations a little (I know it won't be perfect at all).

I will look into the flocking / construction paper and see how I get on with that - seems like a simple solution! I have a list of things I want to learn and cover about my setup, and the photography, so I will scour the forum to see what I can get.

I have already learned so much in a short space of time, small tricks and tactics mainly, but all good learning! :D

Owen

https://www.photomacrography.net/forum/ ... ew&id=3015
Attachments
2021-02-21-19.56.59 ZS PMax-3.jpg
Instagram: @ojd_photo

ojd01
Posts: 49
Joined: Sun Nov 15, 2020 10:56 am
Location: Teesside, Middlesbrough, UK

Re: 1st Photomacrography Image - Technical Queries

Post by ojd01 »

JKT wrote:
Sun Feb 21, 2021 1:09 am
In addition to the tubes, the LOMO 3.7x is also prone to reflections. A very small black "hood" protruding a few mm past the end of the objective might help. I also painted the end of the objective body black, but that may be an overkill.

The nominal length for the tubes in the direct projection is roughly 150mm and that includes the distance from the sensor to the beginning of tubes. That distance is 45.46mm for M42. So the theoretical nominal setup would need about 100mm of tubes. Add the 45.5 and the RMS adapter (assuming flat type) to that and you are close to 150mm. If you meant that the total length with all those considered is 160mm, you are only slightly over the nominal and the objective should work well.

As far as the objective quality is concerned, I would not call LOMO 3.7x as "not very good quality". It is quite good and way way above what its price suggests. The LOMO 8x .20 (if that is what you are getting) is unfortunately not in the same league.
Hello!

Thank you for your response - it is very appreciated.

Firstly, I may have miscommunicated one point - I didn't mean to call the LOMO 3.7x bad quality - I have purchased it because of the amazing reviews. The current objective I have (unbranded) is a poor achromatic objective. I am very excited to use the LOMO 3.7x once it arrives! :) I will definitely take on your advice about the hood at the end of the objective, as it is always worth trying these things.

Yes, I meant the 160mm is the distance from camera sensor + EOS-M42 adapter + M42 tubes + M42-RMS adapter (not including objective). Although, I can easily drop the distance to 150mm, since the tubes come apart with different lengths.

Kindest Regards

Owen
Instagram: @ojd_photo

rjlittlefield
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Re: 1st Photomacrography Image - Technical Queries

Post by rjlittlefield »

The second image looks much better. Nice progress.
so maybe a single flash, or dual flash, setup may be useful for some subject freezing. I would love shots at 1/4000+ SS, as that would really reduce the motion blur - I have some manual Yongnuo's somewhere I could rig up.
If you do switch to flash, then be aware that when the camera is running in EFSC mode, it cannot provide a trigger signal to the flash. Depending on the exact model of flash, the camera may trigger flash from Live View mode, but if it does, then that's a carefully choreographed high speed dance that avoids EFSC. What the camera actually does in that case, in rapid succession, is to stop Live View by closing the shutter and dropping the mirror, then it raises the mirror again, opens the shutter, triggers the flash, closes the shutter, then raises the mirror and opens the shutter again to resume Live View. Of course this generates an enormous amount of vibration, which nonetheless is often hidden by the motion-stopping power of the short flash exposure.

With flash, a better approach is to shoot with the camera not in Live View, but with mirror lockup, with a couple of seconds delay between mirror-up and start of exposure. Check your camera manual for how to set mirror lockup mode, and remember to program your rail controller to issue two shutter pulses per step, with the couple of seconds delay between pulses. The first pulse will raise the mirror; second pulse will take the picture.

--Rik

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