Peacock feathers

[hkv]

Posting some details from a Peacock feather I had in a drawer for a couple of years, waiting to be photographed.
Peacocks are known for their bright colors. However, when their feathers are wet they appear brown which indicates that the bright colors aren’t a result of pigmentation. Something else is going on! Peacocks achieve its stunning plumage display through structural coloration, more commonly known as iridescence. Structural coloration results from a lightwave interaction with the surface. The light can be reflected, refracted, or sometimes both, and the effect is angle-dependent. Depending on the angle and the wavelength, lightwaves may constructively or destructively interfere with each other resulting in different colors and brightness.

It turned out to be very difficult to photograph these feathers. Reflections were very difficult to eliminate which made focus stacking impossible as white spots do not have sufficient contrast and was confusing the focus stacking software. I ended up using three levels of diffusers. 4 external light sources, all equipped with double diffusers. The first one a small test tube in plastic surrounded by a ping-pong ball. Then a half ping-pong ball was used over the objective to cover the specimen. The extreme level of diffusion ended up in an almost painterly style. In some images, I used a very faint red backlight to enhance the contrast of the feather. The objective used was an Olympus UPLXAPO4X.

Peacock feather by Håkan Kvarnström, on Flickr


Peacock feather by Håkan Kvarnström, on Flickr


Peacock feather by Håkan Kvarnström, on Flickr


Peacock feather by Håkan Kvarnström, on Flickr


Peacock feather by Håkan Kvarnström, on Flickr


Peacock feather by Håkan Kvarnström, on Flickr


Peacock feather by Håkan Kvarnström, on Flickr

Peacock feather by Håkan Kvarnström, on Flickr

[Lou Jost]

Very nice, but personally I prefer the ones without the bright orange backlight though (for example, the third from the end). That orange color and high brightness does not go well with the darker greens and blues for me.

[Dalantech]

Those are some really beautiful abstracts -worth the effort!

[Macro_Cosmos]

These are lovely, it's like abstract art!

[hkv]

Very nice, but personally I prefer the ones without the bright orange backlight though (for example, the third from the end). That orange color and high brightness does not go well with the darker greens and blues for me.

Thanks Lou, I understand. Good then that I have most of them also without the backlight. I shot double stacks. ;-)

[hkv]

These are lovely, it's like abstract art!

Those are some really beautiful abstracts -worth the effort!

Thanks!

[Lou Jost]

I have most of them also without the backlight. I shot double stacks. ;-)

Excellent foresight!

[enricosavazzi]

Very good pictures. A couple of references about structural feather color and hydration:

https://www.sciencedirect.com/science/a ... 061100016X
https://www.osapublishing.org/josa/abst ... 50-10-1005
https://journals.aps.org/pre/abstract/1 ... .74.051916

[hkv]

Very good pictures. A couple of references about structural feather color and hydration:

https://www.sciencedirect.com/science/a ... 061100016X
https://www.osapublishing.org/josa/abst ... 50-10-1005
https://journals.aps.org/pre/abstract/1 ... .74.051916

Thank you! Great articles about an amazing specimen. I see that you are Swedish as well! Not too many microscopists in Sweden being active nowadays.

[Macro_Cosmos]

Accidentally clicked an image. Nice, you have a new follower.

How did you get backgrounds that are so clean? Incredible work in your gallery.
That copepod eggsack, simply amazing. The 1-65535mm Canon lens (according to exif) is sure outstanding... haha.

Your exif indicates a Sony a9. That's a very odd choice imo. If I had objectives in mind, I'd personally go with the a7m3. A speed machine like the a9 won't even cross my mind...
Are you using what's available, or are there other reasons? If so, please enlighten me.
My guess is fluorescence and darkfield work, and you work with live subjects. Usually a sufficient shutterspeed and higher ISO is needed, the little critters do move. I could be wrong though.

Either way, I'll look at some more photos of yours. They are amazing. To hold the urge of dumping another $2000, I'll gloss over the dic ones.

[hkv]

Accidentally clicked an image. Nice, you have a new follower.

How did you get backgrounds that are so clean? Incredible work in your gallery.
That copepod eggsack, simply amazing. The 1-65535mm Canon lens (according to exif) is sure outstanding... haha.

Your exif indicates a Sony a9. That's a very odd choice imo. If I had objectives in mind, I'd personally go with the a7m3. A speed machine like the a9 won't even cross my mind...
Are you using what's available, or are there other reasons? If so, please enlighten me.
My guess is fluorescence and darkfield work, and you work with live subjects. Usually a sufficient shutterspeed and higher ISO is needed, the little critters do move. I could be wrong though.

Either way, I'll look at some more photos of yours. They are amazing. To hold the urge of dumping another $2000, I'll gloss over the dic ones.

Thanks for the follow! I will try to answer your questions as best as I can. The first question was regarding the clean backgrounds. I spend a lot of effort in making the backgrounds clean already when preparing my slides. In many cases, it is not perfectly clean, but during focus stacking, I carefully select the source pixels from the cleanest areas. Also using DIC helps as DIC cuts through layers like a laser knife and has very shallow depth of field (unlike darkfield) where every piece of dirt and debris is visible throughout the stack. A secret trick I also use is to always shoot a few images of the subject which is way out of focus so I more or less get a clean background, but with the same light conditions and colors. That one is then included in my stack so I can use it as a source image when doing retouching in Zerene or Helicon.

Regarding the choice of camera. The A9 is actually almost perfect for microphotography. The A9 uses a stacked sensor with built-in memory which makes it perfect for capturing moving cilia or vorticella and other stuff without a seriously warped image that slower sensors may produce. Almost liek a global shutter, even though it is not. Shooting moving subjects under the microscope is actually a bit like sports photography, for which the A9 is particularly good at. Overall the A9 is a better camera with bigger buffer, exposure times down to 1/32000 second, and a higher frame rate up to 20 images per second. The bigger buffer is a blessing for sure as the A9 (and A7 III) only has normal USB and not the higher speed USB-C. I use Capture One for tethering the camera and the software downloads all the images in the background from the buffer. Meaning I can shoot a burst of images at high speed and let the download go on in the background. However, the USB is slow, so in many cases, I fill up the buffer (245 images) and have to wait for the download over USB to clear some images before I can shoot the next burst. Also, until the buffer is empty, you cannot start to record videos, which also is a huge drawback. Emptying the buffer may take a minute or two and that is a long wait especially if your sample is moving or even starting to dry out. I am almost considering an upgrade to A9 Mark II just to get the USB-C speed... Then emptying the buffer takes seconds.

[Macro_Cosmos]

Thanks for the follow! I will try to answer your questions as best as I can. The first question was regarding the clean backgrounds. I spend a lot of effort in making the backgrounds clean already when preparing my slides. In many cases, it is not perfectly clean, but during focus stacking, I carefully select the source pixels from the cleanest areas. Also using DIC helps as DIC cuts through layers like a laser knife and has very shallow depth of field (unlike darkfield) where every piece of dirt and debris is visible throughout the stack. A secret trick I also use is to always shoot a few images of the subject which is way out of focus so I more or less get a clean background, but with the same light conditions and colors. That one is then included in my stack so I can use it as a source image when doing retouching in Zerene or Helicon.

Regarding the choice of camera. The A9 is actually almost perfect for microphotography. The A9 uses a stacked sensor with built-in memory which makes it perfect for capturing moving cilia or vorticella and other stuff without a seriously warped image that slower sensors may produce. Almost liek a global shutter, even though it is not. Shooting moving subjects under the microscope is actually a bit like sports photography, for which the A9 is particularly good at. Overall the A9 is a better camera with bigger buffer, exposure times down to 1/32000 second, and a higher frame rate up to 20 images per second. The bigger buffer is a blessing for sure as the A9 (and A7 III) only has normal USB and not the higher speed USB-C. I use Capture One for tethering the camera and the software downloads all the images in the background from the buffer. Meaning I can shoot a burst of images at high speed and let the download go on in the background. However, the USB is slow, so in many cases, I fill up the buffer (245 images) and have to wait for the download over USB to clear some images before I can shoot the next burst. Also, until the buffer is empty, you cannot start to record videos, which also is a huge drawback. Emptying the buffer may take a minute or two and that is a long wait especially if your sample is moving or even starting to dry out. I am almost considering an upgrade to A9 Mark II just to get the USB-C speed... Then emptying the buffer takes seconds.

Oh wow, thanks for the detailed explanation! Yeah it makes a lot of sense now, especially the "live action" aspect which I didn't think about. Maybe you can even get your a9 modified, remove the OLPF and use a thinner sensor cover glass, it will give better image quality. Didn't know the a9 exposes for 1/32k seconds, I just assumed they are all at 1/8000. I do however know that a9's sensor is terrific, there's a 16-bit version! Sony as we all expect now blocked the output on a hardware level to 14bit. If they want a bang, a 16-bit a9-mk3 would be an absolute winner. (And it will trickle down since I'm a Nikon user)

Your gallery is motivating I think I'm gonna go out a bit and collect pond water. I can see the drastic improvements and lovely "live creatures" in your gallery. Oh and congrats on the Olympus contest win, I recognised that shell shot and it connected instantly.

[hkv]

Oh wow, thanks for the detailed explanation! Yeah it makes a lot of sense now, especially the "live action" aspect which I didn't think about. Maybe you can even get your a9 modified, remove the OLPF and use a thinner sensor cover glass, it will give better image quality. Didn't know the a9 exposes for 1/32k seconds, I just assumed they are all at 1/8000. I do however know that a9's sensor is terrific, there's a 16-bit version! Sony as we all expect now blocked the output on a hardware level to 14bit. If they want a bang, a 16-bit a9-mk3 would be an absolute winner. (And it will trickle down since I'm a Nikon user)

Your gallery is motivating I think I'm gonna go out a bit and collect pond water. I can see the drastic improvements and lovely "live creatures" in your gallery. Oh and congrats on the Olympus contest win, I recognised that shell shot and it connected instantly.

I want to understand more how the OLPF affect image quality and resolution. I was assuming that at high magnification, e.g. 20X+ the Abbes diffraction limit would be more limiting than the OLPF. I may be wrong, but I made an excel trying to calculate the resolution needed of the sensor at different magnifications. At low magnification, I would need a sensor resolution closr to the A9 (which has 24 megapixels), but at higher magnification, it drops dramatically. At 60X and the N.A 1.2 (water immersion), the sensor pixel density must be only 5,25 megapixels to capture all the details. I would assume the OLPF then makes little difference. Here is my list at different magnifications and assuming a Nyquist theorem factor of 3 (2 is normally enough, so figures should be even lower).

4X - 20,99 MP
10X - 20,99 MP
20X - 18,44 MP
40X -7,40 MP
60X (Oil) - 6,64 MP
60X (water) - 5,25 MP
100X (Oil) - 2,57 MP

So my question is how the OLPF really can limit the sharpness and resolution using e.g. a 60X objective when a sensor resolution of 6 MP should be enough and it is being captured using 24 MP? Help me to understand this! If I can improve resolution and sharpness I will for sure remove the OLPF on my A9!

[rjlittlefield]

I made an excel trying to calculate the resolution needed of the sensor at different magnifications.

Your pixel counts look small to me.

For comparison, https://www.microscopyu.com/tutorials/matching-camera-to-microscope-resolution gives 3660x2740 = 10 megapixels, for 60X NA 1.2 with video coupler 0.5X onto a nominal 1" sensor having size 12.8 x 9.6 mm (so, intermediate image size at eyepiece = nominal 25.6 x 19.2 mm). That calculation is with 2 pixels per cycle at diffraction cutoff with lambda 550nm. 3 pixels per cycle would be a factor of 2.25 higher over the same area.

Can you spell out the assumptions and details of your calculation?

--Rik

[Macro_Cosmos]

I want to understand more how the OLPF affect image quality and resolution. I was assuming that at high magnification, e.g. 20X+ the Abbes diffraction limit would be more limiting than the OLPF. I may be wrong, but I made an excel trying to calculate the resolution needed of the sensor at different magnifications. At low magnification, I would need a sensor resolution closr to the A9 (which has 24 megapixels), but at higher magnification, it drops dramatically. At 60X and the N.A 1.2 (water immersion), the sensor pixel density must be only 5,25 megapixels to capture all the details. I would assume the OLPF then makes little difference. Here is my list at different magnifications and assuming a Nyquist theorem factor of 3 (2 is normally enough, so figures should be even lower).

4X - 20,99 MP
10X - 20,99 MP
20X - 18,44 MP
40X -7,40 MP
60X (Oil) - 6,64 MP
60X (water) - 5,25 MP
100X (Oil) - 2,57 MP

So my question is how the OLPF really can limit the sharpness and resolution using e.g. a 60X objective when a sensor resolution of 6 MP should be enough and it is being captured using 24 MP? Help me to understand this! If I can improve resolution and sharpness I will for sure remove the OLPF on my A9!

Interesting thought, I think you're on the right track. I need to consult someone with more knowledge.
What I know is, the OLPF is a net negative to our stuff no matter which way it is sliced. That said, we are usually more limited by the objective's power, especially with Mitutoyo lenses that most use for reflected light.
It's hard to say which matters more. Nikon's entire sensor stack for the Z-mount is only 0.7mm. Sony's presumably older models has thick sensor coverglass, which is detrimental to UWA lenses. Examples can be seen here:
https://www.youtube.com/watch?v=NJBGAbcZc7s
https://www.youtube.com/watch?v=PMfshUJGZow
(Unfortunately the author seems to deny the existence of this problem, despite it being well known)
Here's why: https://www.lensrentals.com/blog/2014/0 ... it-matter/
Leica engineers seems to be fully aware of this problem, same as the ones that engineered the Z-mount cameras, as the sensor glass is very thin. For Nikon, it's a requirement as many of us would like to adapt the 14-24mm.

As for your MP calculations, would you mind sharing the formula? They all seem off to me, by a huge margin actually.

Assumptions: 550nm, monochrome
60x water, NA 1.2: 18.1529MP
100x oil, NA 1.25: 7.091MP
Since we are using colour sensors, the results should be multiplied by a liberal amount to account for debayering errors and other "esoteric" stuff. I usually toss in a factor of 2. This means an Oly 20x Sapo would work incredibly well with a 65MP monochrome fullframe sensor, which translates to above 100MP in colour as losses are accounted for. Assuming your 10x is the UPlanFL with an NA of 0.3, it gives ~41MP, so easily suitable for 60MP FF sensors.

Here's the formula I use: https://www.microscopyu.com/tutorials/m ... resolution