Various bacteria 1000x

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Jcoogler
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Various bacteria 1000x

#1 Post by Jcoogler » Fri Feb 03, 2023 3:00 am

These were found in pond water samples that have been sitting in jars for months. I have added a blueberry that has began to decompose to one of the jars and others have decomposing leaves. Then blueberry sample was teeming with bacteria. Many types of bacteria were seen with all different shapes, some have an endospore visible on one end as well. There were so many more types seen but they were even smaller than these! These are heavily cropped from my Nikon D7500 using a 100x oil objective on an Amscope T340B. Lots of sharpening and editing, most if not all of these photos were processed by taking only the red or green channel and using that, this gave me a sharper result. I know this is kind of niche but does anyone have any tips or tricks to get a bit more detail? Any camera or processing techniques, lighting tricks, etc? I am uso oblique lighting in these images. Anyway, here are the images.
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PeteM
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Re: Various bacteria 1000x

#2 Post by PeteM » Fri Feb 03, 2023 3:54 am

A sharp (monochromatic) green filter over the field iris might help sharpen things up, perhaps more so than taking one channel in post processing.

Just this morning I was thinking blueberries with oatmeal (minus the one blueberry with fungus) were good for me . . . :-) A friendly microbiome one hopes.

Jcoogler
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Re: Various bacteria 1000x

#3 Post by Jcoogler » Fri Feb 03, 2023 5:22 am

Hmmm, I do have a green filter that came with a set of colorful filters. I’ll have to give it a shot. I have a feeling I’m nearing the limit of what is actually resolvable though haha!

MichaelG.
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Re: Various bacteria 1000x

#4 Post by MichaelG. » Fri Feb 03, 2023 7:36 am

Very impressive results … Thanks for sharing them

Using the Green channel only is probably a good move, and starting with Green light should sharpen things further.

Given your phenomenal success thus far … I would probably be looking for suitable Green LEDs

MichaelG.
.
Edit: __ I assume from this work https://www.dpreview.com/forums/thread/4390987
that 470nm would suit Nikon sensors nicely.

Edit: ___ Having read that ^^^ more thoroughly, forget the 470 and try to find the actual curve for the sensor !!
Last edited by MichaelG. on Fri Feb 03, 2023 10:39 am, edited 1 time in total.
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Hobbyst46
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Re: Various bacteria 1000x

#5 Post by Hobbyst46 » Fri Feb 03, 2023 8:54 am

Nice work. Blue light is somewhat better (by ~15%) than green light in terms of resolution, but green is supposed to be more pleasing for the eye.

In general, extreme oblique can introduce some artifacts into file details in images, by comparison with brightfield.
For example, tiny dots are smeared into lines. Not claiming that it happened here.

To better appreciate the resolution, you might include a scale mark in the image. This can be done via software such as ImageJ or other, after calibration
with a stage micrometer (which is pretty inexpensive these days).

Maybe the diameter of the field of view through the eyepiece was 2mm for the 10X objective (say) and 200um for the 100X objective,
yet the final size is determined by how much the camera catches, and the cropping.
Zeiss Standard GFL+Canon EOS-M10, Olympus VMZ stereo

apochronaut
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Re: Various bacteria 1000x

#6 Post by apochronaut » Fri Feb 03, 2023 10:56 am

These are very impressive. BF has it's limitations, especially when the objectives are achromats. I agree that blue filtering has an advantage over green, one being that there is less need to convert to bl/wh, so a degree of natural colour can be retained. You probably have the resolution about as fine as possible, with the equipment you have.
You didn't mention the condenser. It was immersed I would assume and needs to be.Condenser and objective N.A. need to be prime.
How did you achieve the oblique? A mask? There are several methods and microscopes made prior to the introduction of phase contrast were more frequently equipped with specialized condensers for oblique. The more common idea was a split condenser, which could move the collecting element against the upper element(s) via a geared track. A separate shutter with variable 1/2 moon shaped leaves below the iris was another system and it could rotate. Sometimes they were combined. The idea is to achieve a variable shift and an angle shift too with oblique Lomo versions of the former are often on ebay and at 1.4 N.A. Both B & L and Spencer research condensers equipped with oblique from time to time show up as well. Condenser conversions are not that difficult. Having access to a printer helps.
Another option is circular oblique, which overcomes some of the shadowing effects mentioned ahove.

MichaelG.
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Re: Various bacteria 1000x

#7 Post by MichaelG. » Fri Feb 03, 2023 1:29 pm

Just a note … and it may not be valid, so I am happy to be corrected

My reason for agreeing that Green would be good is more to do with the sensor than the optics.

MichaelG.
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Hobbyst46
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Re: Various bacteria 1000x

#8 Post by Hobbyst46 » Fri Feb 03, 2023 3:51 pm

MichaelG. wrote:
Fri Feb 03, 2023 1:29 pm
Just a note … and it may not be valid, so I am happy to be corrected

My reason for agreeing that Green would be good is more to do with the sensor than the optics.

MichaelG.
Michael, is the camera especially sensitive to green ? or is it more sensitive in general to longer wavelengths (my guess), i.e. would be even more sensitive to red ?
Zeiss Standard GFL+Canon EOS-M10, Olympus VMZ stereo

MichaelG.
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Re: Various bacteria 1000x

#9 Post by MichaelG. » Fri Feb 03, 2023 4:30 pm

Michael, is the camera especially sensitive to green ? or is it more sensitive in general to longer wavelengths (my guess), i.e. would be even more sensitive to red ?
.

Although the wavelength numbers are clearly wrong, the general shape of the spectral response curves in my earlier link is probably about right … add to that the fact that a Bayer matrix includes twice as many Green sub-pixels as Red or Blue and I think it worthy of consideration. [signal to noise ratio and all that]

I have discovered that the sensor in the D7500 is actually made by Sony … so there is a fighting chance that a proper data-sheet will be available [sorry, I haven’t found it yet] … if I find it, I will post the link here.

MichaelG.
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PeteM
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Re: Various bacteria 1000x

#10 Post by PeteM » Fri Feb 03, 2023 4:51 pm

Another factor is how sharply either a blue or green filter cuts off. Some filters that look blue or green can have very wide ranges, others will have sharp cutoffs. Consider, for example, these color balancing filters: https://hoyafilterusa.com/products/c2-color-correction

I don't know the cutoffs in the post-processing software.

FWIW, our eyes are most sensitive to green light. Could be, as Michael points out, many camera sensors are as well. The sharpness of the image will also depend on things like vibration in the scope/camera setup. Between choosing a bit of the spectrum, imaging at 1000x, camera sensitivity, and going darkfield I'd expect fairly long exposures are needed? Might even be that a flash would sharpen things up.

MichaelG.
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Re: Various bacteria 1000x

#11 Post by MichaelG. » Fri Feb 03, 2023 6:10 pm

The sensor in the D500 has been identified as: Sony IMX321 Exmor CMOS APS-C Sensor
https://camspex.com/comparison.php?came ... Nikon+D500
… and Nikon advertises the fact that the D7500 uses the same sensor as the D500

So far, so good … but there are several incarnations of Exmor

I am still failing to find a data-sheet for IMX321 … but Fig.3 in this paper provides a spectral response curve [from Sony] for the IMX117 : https://www.researchgate.net/publicatio ... n_Fraction

In a leap of faith, I am prepared to accept that the curve for IMX321 will be remarkably similar

I happily second Pete’s very valid comment about filters … which is why I was suggesting Green LED [520nm being common] as the optimum light-source.

MichaelG.
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Hobbyst46
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Re: Various bacteria 1000x

#12 Post by Hobbyst46 » Fri Feb 03, 2023 6:33 pm

MichaelG. wrote:
Fri Feb 03, 2023 6:10 pm
The sensor in the D500 has been identified as: Sony IMX321 Exmor CMOS APS-C Sensor
https://camspex.com/comparison.php?came ... Nikon+D500
… and Nikon advertises the fact that the D7500 uses the same sensor as the D500

So far, so good … but there are several incarnations of Exmor

I am still failing to find a data-sheet for IMX321 … but Fig.3 in this paper provides a spectral response curve [from Sony] for the IMX117 : https://www.researchgate.net/publicatio ... n_Fraction

In a leap of faith, I am prepared to accept that the curve for IMX321 will be remarkably similar

I happily second Pete’s very valid comment about filters … which is why I was suggesting Green LED [520nm being common] as the optimum light-source.

MichaelG.
Hmm. Or 546nm maybe...Well the top Exmor link answered my question. Thanks.
In retrospective, I was thinking about photodiodes instead of camera sensors...
Zeiss Standard GFL+Canon EOS-M10, Olympus VMZ stereo

MichaelG.
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Re: Various bacteria 1000x

#13 Post by MichaelG. » Fri Feb 03, 2023 7:03 pm

Hobbyst46 wrote:
Fri Feb 03, 2023 6:33 pm
MichaelG. wrote:
Fri Feb 03, 2023 6:10 pm
… which is why I was suggesting Green LED [520nm being common] as the optimum light-source.
Hmm. Or 546nm maybe...
.

I would be happy with either
… If we get to the point where it matters, I think we will have won already.

MichaelG.
.
Ref. __ From Toshiba:
.
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charlie g
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Re: Various bacteria 1000x

#14 Post by charlie g » Fri Feb 03, 2023 8:02 pm

Fantastic work.

Thiomargarita magnifica are naturally approx' 1.0 cm in length..and with a diameter making them visible to naked eye.

I mention this group of bacteria to suggest..watch for huge bacteria (relatively huge) for higher detail in your image captures. Thanks for posting these terrific images. charlie g

Jcoogler
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Re: Various bacteria 1000x

#15 Post by Jcoogler » Mon Feb 06, 2023 11:29 pm

Thanks everyone for the comments and the ideas! I used the stock condenser that came with my Amscope T340B, and the top of the condenser was immersed with oil as well. I experimented using a green filter but didn’t find much difference, but it’s not one with a specific cut off point so I’d probably need something more specialized, or the green led lights that were suggested. The oblique illumination was accomplished using a filter that looks like it has a pizza slice taken out of it. Here are a few more images I’ve gotten of some bacteria since
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Jcoogler
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Re: Various bacteria 1000x

#16 Post by Jcoogler » Wed Feb 08, 2023 5:02 am

Update today. I experimented with the filters I had and found that a different shaped filter was able to help me resolve a bit finer details. I actually was able to (easily!) see the flagella of many bacteria! They showed up even better in the images. Here are some images I took today, some have flagella, others like the group of cocci(?) show different stages of division. The first one, on the right is a bacterium with a very long tail! These images are downsized from original to “large” so I can post them here but the full sizes can be found on my Flickr. Here is a link to one of the images and the others can be found from there if interested. Thanks! https://flic.kr/p/2ofPUZo
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BramHuntingNematodes
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Re: Various bacteria 1000x

#17 Post by BramHuntingNematodes » Wed Feb 08, 2023 5:14 am

These are most excellent
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macnmotion
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Re: Various bacteria 1000x

#18 Post by macnmotion » Wed Feb 08, 2023 5:14 am

Very nice -- your persistence has paid off

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