Experiments with afocal photomicrography: illuminations, spider threads...

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Hobbyst46
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Experiments with afocal photomicrography: illuminations, spider threads...

#1 Post by Hobbyst46 » Sat Feb 10, 2018 3:26 pm

Afocal photomicrography setup.jpg
Afocal photomicrography setup.jpg (72.43 KiB) Viewed 6412 times
Here are my first attempts of using the mirrorless Canon EOS-M10 for microscopy.

Although it is not the ideal camera for the job, I chose it because of it is featherweight, and because it is a Canon. Unfortunately, like other Canon EOS-M series, it has no Live View with PC (but can be remote controlled from phone/tablet). My setup consists of the trinocular photo tube, a spacer collar (ID 25mm, OD 34mm) fabricated from a PVC pipe, a 1.25" to 49x0.75mm telescope adapter, a Zeiss 8x18 or 10x16 KPL eyepiece (~7mm eyepoint) hidden inside the adapter, a Canon 15-45mm kit zoom lens and the camera. This zoom lens is very good according to some Internet reviews (e.g. by Rockwell). I fixed the focal length to 45 mm with a piece of adhesive tape, to prevent mechanical drifting caused by gravity. I set the camera to manual focus, since I only focus with the microscope. In fact, I have no idea how to preset the camera focus to infinity - it seems to be impossible.

The KPL photo eyepiece is very close to the front end of the camera lens, so a soft rubber annular spacer is placed in between, to protect.
The photos below were taken with the 8x18 KPL eyepiece. The light source is a 6500K 10W cool LED, attenuated with a 21KHz PWM dimmer. Objectives are Zeiss Plan 6.3x/0.16, Plan 16x/0.32 and Neofluar 40x/0.75 Ph2, and an Olympus SPlan 10x/0.30.

My test specimens and results:

(1) A Zeiss stage micrometer - transparent scale marks on an opaque background. With it I find, that the APS-C sensor sees about 90% of the theoretical
field of view through my 10x20 viewing eyepieces. Visually, the micrometer ticks are brightly lit, uniformly blue-white. However, the photo indicates
some chromatic aberrations with all objectives.

Note: Similar chromatic effects were obtained when a 50mm 1:1.7 Minolta MD fixed lens was used instead of the zoom.

(2) Threads from a locally collected spider web, obtained by touching a slide to the web and mounting with glycerol. Brightfield and darkfield images are
single photos. Darkfield either by means of the Ph3 phase ring of the condenser (as instructed by Zeiss) or by means of the D position of the
condenser. The latter method yielded a more uniform background but lower contrast in general.
The phase contrast is a stack of 5 images, spaced at 2 micrometer height steps.
Notice that the thicker and thinner threads are about ~3 and 0.7 micrometers thick, respectively. I am deeply impressed by the ability of the spider to
tie its threads so nicely.

BTW, I intend to try direct projection as well.

But, more important, I would appreciate very much your opinions. Can I get much better than these with my present gear?
Attachments
Spider web - 16x Ph3 DF.jpg
Spider web - 16x Ph3 DF.jpg (74.38 KiB) Viewed 6412 times
Spider web - 10x Ph3 DF.jpg
Spider web - 10x Ph3 DF.jpg (82.79 KiB) Viewed 6412 times
Spider web - 6.3x Ph3 DF.jpg
Spider web - 6.3x Ph3 DF.jpg (86.59 KiB) Viewed 6412 times
16x with 8x ocular 15-45mm zoom.jpg
16x with 8x ocular 15-45mm zoom.jpg (83.9 KiB) Viewed 6412 times
Last edited by Hobbyst46 on Sat Feb 10, 2018 4:13 pm, edited 1 time in total.
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Re: Experiments with afocal photomicrography: illuminations, spider threads...

#2 Post by Hobbyst46 » Sat Feb 10, 2018 3:30 pm

The brightfield, phase contrast and darkfield with the D position of the condenser of the spider web are attached.
Attachments
spider web 16x brightfield.jpg
spider web 16x brightfield.jpg (60.78 KiB) Viewed 6411 times
Spider web 40x phase contrast, stack of 5 images 2 micron apart.jpg
Spider web 40x phase contrast, stack of 5 images 2 micron apart.jpg (31.58 KiB) Viewed 6411 times
Spider web -16x D condenser DF.jpg
Spider web -16x D condenser DF.jpg (58.72 KiB) Viewed 6411 times
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Re: Experiments with afocal photomicrography: illuminations, spider threads...

#3 Post by zzffnn » Sat Feb 10, 2018 7:28 pm

Do you see less purple/blue color, when you set your camera's white balance to custom Kevin temperature of 6500k?
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Re: Experiments with afocal photomicrography: illuminations, spider threads...

#4 Post by Hobbyst46 » Sat Feb 10, 2018 7:54 pm

zzffnn: Thanks for viewing. I am still unfamiliar with setting the white balance correctly in photomicrography in general and in this camera in particular. There are far too many menu items... so the white balance was either Auto or unknown to me.
I will try to learn how to do what you suggest. Currently the camera is set to Av mode. White balance is "custom" and when I tap it, places a square on the screen, with a dot inside. Must wander inside the Canon Manual to find out.
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Re: Experiments with afocal photomicrography: illuminations, spider threads...

#5 Post by Hobbyst46 » Sat Feb 10, 2018 8:53 pm

zzffnn:

I am not quite clear how to set the custom WB. The Custom WB menu icon gives me a square marked BAGM, with a dot inside. I found out what that means, so I can change the color temperature. But do I do it on the image of the micrometer ticks prior to shooting, or do I apply it to a true white background as a constant correction ? If so, how do you define a true white image on the stage of the scope? a white translucent paper perhaps? and what is the starting point? I mean, what color temperature does the dot inside the square refer to ? Is this method of the square specific to Canon ?
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Re: Experiments with afocal photomicrography: illuminations, spider threads...

#6 Post by zzffnn » Sat Feb 10, 2018 9:25 pm

Sorry, I have not used a Canon; you may have to google/research it.

You Zeiss 6.3x should not produce too much CA with KPL eyepiece. Your 40x phase Neufluar changes color, because of light passes its phase ring. If you have a brightfield apo with the correct compens eyepiece and still get lots of purple, then chances are high that the LED is to blame. Maybe change a light source and see if color changes?

My Olympus E-M10 II let me set custom white color to Kevin temperature of 6500. Or I shoot a pure white image and use that as custom white.

This is what I found when searching "BAGM square and camera white balance"
https://snapshot.canon-asia.com/article ... te-balance

Sunny daylight is about 5000-5500 Kevin.
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Re: Experiments with afocal photomicrography: illuminations, spider threads...

#7 Post by Hobbyst46 » Sat Feb 10, 2018 9:42 pm

Thanks a lot, zzffnn. The link clarifies the square and dot method. In this case the Olympus tools are better than Canon's...
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Re: Experiments with afocal photomicrography: illuminations, spider threads...

#8 Post by MichaelG. » Sat Feb 10, 2018 10:27 pm

Hobbyst46 wrote:zzffnn:

I am not quite clear how to set the custom WB. The Custom WB menu icon gives me a square marked BAGM, with a dot inside. I found out what that means, so I can change the color temperature. But do I do it on the image of the micrometer ticks prior to shooting, or do I apply it to a true white background as a constant correction ? If so, how do you define a true white image on the stage of the scope? a white translucent paper perhaps? and what is the starting point? I mean, what color temperature does the dot inside the square refer to ? Is this method of the square specific to Canon ?
Some observations, if I may:
1. You mention using a "6500K 10W cool LED" ... You may therefore have difficulty with White Balance, because the spectrum will most probably have significant peaks in the Blue and Yellow bands. Getting familar with that BAGM setting will be very helpful, I think.
2. The page that zzffnn found provides a good introduction: Basically you have two 'sliders' controlling Blue--Amber & Green--Magenta and the dot is an indication of 'where you are' in that two dimensional space.
3. No, the square is not specific to Canon; my Lumix G1 has a similar feature.
4. Rather than paper, I would suggest using 'opal' glass or plastic ... but it shouldn't matter too much.
5. Remember that if you have a slide where the mountant is yellowed, you can adjust for that as well as for the light source.
6. Despite what I said about 'opal' ... If you are trying to adjust for an actual slide, it may be easier to just de-focus the image to 'average' the colour.

Have fun ... The initial results look very promising.

MichaelG.
.
Regarding my first point: This explains it quite well
https://electronics.stackexchange.com/q ... l-spectrum
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Re: Experiments with afocal photomicrography: illuminations, spider threads...

#9 Post by Hobbyst46 » Sat Feb 10, 2018 11:12 pm

Thanks again zzffnn and MichaelG.

Owing to this post I have already learned about setting white balance - so it helped me already in photography!
However, I changed the white balance to the four maximum possible combinations (square corners). Only the hue of the color fringes in the image of the micrometer tick marks was modified not its intensity or width. So perhaps the WB is not effective.

I am familiar with LED's spectra. The blue component of the cool-white is indeed strong, relative to the spectrum of warm-white LEDs. But, many modern microscopes use LED light sources. Are they cool-white or warm-white? I do not know. They certainly are not monochromatic...

This LED replaced an old faulty Tungsten lamp that, even if it could be repaired, supplied more heat than light to the specimen. An amber filter (mine is amber, KR12) removes a lot of the blue from the cool-white LED and renders the light warmer, fairly similar to halogen light, which is visually pleasant. But as experts here have already warned me, the Camera should be pleased as well.

Actually, the stage micrometer ticks are so thin (~2-3 micrometers) that they behave as diffraction slits. When held up to direct sunlight, one can see separate color spots or fringes. They certainly diffract the LED's light as well. But the effects are enhanced in the camera. I had hoped that they could be minimized.

And I wonder if direct projection, instead of afocal photography, will make it better.
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Re: Experiments with afocal photomicrography: illuminations, spider threads...

#10 Post by Micro-Bob » Sat Feb 10, 2018 11:22 pm

I wouldn't care about white balance too much. If it is somewhat off in a certain image I would adjust it afterwards in the editing software. To me your camera adaptation looks good. Even lighting and no hotspot and a good use of the image circle. In your third image there are diagonal streaks over the whole image. This would be difficult to clean up so I would look for the source.

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Re: Experiments with afocal photomicrography: illuminations, spider threads...

#11 Post by zzffnn » Sat Feb 10, 2018 11:49 pm

I have a gut feeling that the BAGM square may produce better results than simply setting Kevin temperature, but I could be wrong.

My LED is warmer, but my camera does not get the color perfect either.

Darkfield and phase sometimes exaggerate CA, while brightfield and oblique show less.

Does your KPL or 8x eyepiece provide perfect compensation to your 6.3x objective? That low power objective is likely less prone to CA. The Olympus objective is a mismatch, most likely.

Another thing to try (though it is not likely at the main fault), is to close down lens aperture a bit to around F/4.0.
Last edited by zzffnn on Sun Feb 11, 2018 2:30 pm, edited 1 time in total.
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Re: Experiments with afocal photomicrography: illuminations, spider threads...

#12 Post by MichaelG. » Sun Feb 11, 2018 6:40 am

Hobbyst46 wrote:Thanks again zzffnn and MichaelG.

I am familiar with LED's spectra. The blue component of the cool-white is indeed strong, relative to the spectrum of warm-white LEDs. But, many modern microscopes use LED light sources. Are they cool-white or warm-white? I do not know. They certainly are not monochromatic...

This LED replaced an old faulty Tungsten lamp that, even if it could be repaired, supplied more heat than light to the specimen. An amber filter (mine is amber, KR12) removes a lot of the blue from the cool-white LED and renders the light warmer, fairly similar to halogen light, which is visually pleasant. But as experts here have already warned me, the Camera should be pleased as well.
That ^^^ is a good summary of the situation.
I don't know what 'modern microscopes' use; but probably every variation on the theme will be represented somewhere ...
I do believe, however, that warm-white LEDs are genreally recognised as having a better 'CRI' [Colour Rendering Index] than cool-white. That said; your amber filter will be softening the spectral peaks, to give much the same effect.

It is an unfortunate truth that the camera is more revealing of these errors than our eyes.

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Re: Experiments with afocal photomicrography: illuminations, spider threads...

#13 Post by MichaelG. » Sun Feb 11, 2018 6:51 am

zzffnn wrote:I have a gut feeling that the BAGM square may produce better results than simply setting Kevin temperature, but I could be wrong.

My LED is warmer, but my camera does not get the color perfect either.
I'm sure your gut feeling is correct.
Although the wavelengths are different, the problem is akin to what we suffered with fluorescent tubes: where the green peak needed to be selectively filtered.
Simple adjustment of the Kelvin temperature assumes 'black body radiation' ... which is provided by tungsten-filament lamps, but not by LEDs or Fluorescent tubes.

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Re: Experiments with afocal photomicrography: illuminations, spider threads...

#14 Post by MichaelG. » Sun Feb 11, 2018 7:19 am

Hobbyst46 wrote:However, I changed the white balance to the four maximum possible combinations (square corners). Only the hue of the color fringes in the image of the micrometer tick marks was modified not its intensity or width. So perhaps the WB is not effective.
Apologies for submitting three posts in a row, but it seemed simpler to address the points separately.

That finding is a clear indication that you have two issues:
White Balance is relatively simple to adjust, either in camera or by post-processing.
But you also have Chromatic Aberration; which really needs to be optimised optically [although Photoshop does offer some helpful tools].

The 'BAGM square' is effectively a colour compensating filter, similar in function to the filter packs that we used in photographic printing from film.
Chromatic Aberration is a 'Garbage in >> Garbage out' sort of problem, and it's therefore best to minimise it at source.

MichaelG.
.
P.S. The good news is that most of your CA seems to be 'purple fringing' which is evidence of longitudial aberration, and might be relatively easy to reduce by filtering the light source.
... Lateral CA [or Chromatic Difference of Magnification] is much more troublesome.
Last edited by MichaelG. on Sun Feb 11, 2018 7:54 am, edited 1 time in total.
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Re: Experiments with afocal photomicrography: illuminations, spider threads...

#15 Post by Hobbyst46 » Sun Feb 11, 2018 7:49 am

zzffnn and MichaelG - thanks again for elaborating on these points. Great help.
I expected that the Zeiss objectives 6,3x and 16x will be compensted by the KPL eyepieces, although they are only Planachromats. Perhaps the (expensive) solution is Neofluar equivalents.
And to verify or negate that the light source is not the issue, I will attempt to borrow a fiber optic halogen source, for comparison.
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Re: Experiments with afocal photomicrography: illuminations, spider threads...

#16 Post by Hobbyst46 » Sun Feb 11, 2018 10:01 am

MichaelG
P.S. The good news is that most of your CA seems to be 'purple fringing' which is evidence of longitudial aberration, and might be relatively easy to reduce by filtering the light source.
... Lateral CA [or Chromatic Difference of Magnification] is much more troublesome.
Just noticed this important P.S. I was really in doubt about the type of CA. Will invest more time with the light source.
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Re: Experiments with afocal photomicrography: illuminations, spider threads...

#17 Post by Hobbyst46 » Sun Feb 11, 2018 9:14 pm

Update:

1. I Borrowed a Zeiss 25x0.8 Neofluar (MI) - and even when used dry, no purple fringes.
2. I Tried various amber filters in the light path - the purple fringes are there, no difference.
3. I tried a primitive direct projection, using the 10x KPL eyepiece but no camera lens - same result, purple fringes (although now radially worsening - the
image center is better).

It appears that the only minimization of the CA is indeed Neofluars or Apochromats.
I would prefer a 16x Neofluar Ph2 if anyone has it.
On eBay there are very few of these, either very expensive ($189+$53 shipping) or less expensive ($80) but "sold as is - no means to test"...
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Re: Experiments with afocal photomicrography: illuminations, spider threads...

#18 Post by MichaelG. » Sun Feb 11, 2018 9:40 pm

Hobbyst46 wrote:Update:

2. I Tried various amber filters in the light path - the purple fringes are there, no difference.
That's a little disappointing

Are you still using the LED ?
I may be 'clutching at straws' but I suspect that you might still be getting too much short-wavelength contribution.
A "dichroic long pass filter" would probably work wonders; but they are not cheap!
See Figure_2 here: https://www.olympus-lifescience.com/es/ ... e/filters/
... There is no absorption that filter can match that performance.

Alternatively; have you tried the Fibre Optic system that you mentioned ?

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Re: Experiments with afocal photomicrography: illuminations, spider threads...

#19 Post by Hobbyst46 » Sun Feb 11, 2018 10:45 pm

MichaelG. wrote:
Hobbyst46 wrote:Update:

2. I Tried various amber filters in the light path - the purple fringes are there, no difference.
That's a little disappointing

Are you still using the LED ?
I may be 'clutching at straws' but I suspect that you might still be getting too much short-wavelength contribution.
A "dichroic long pass filter" would probably work wonders; but they are not cheap!
See Figure_2 here: https://www.olympus-lifescience.com/es/ ... e/filters/
... There is no absorption that filter can match that performance.

Alternatively; have you tried the Fibre Optic system that you mentioned ?
Hopefully tomorrow I can borrow either a halogen-fiber optic (goose-neck) lamp, or a power supply for the tungsten lamp. I do not give it up already.
I had considered the long pass filters, from Schott (they are absorption filters) and dichroic mirrors. But transmission-wise,it appears that all of them have a steep rise. That is an advantage for spectroscopy, especially fluorescence of course, but I wanted to reduce the blue component, not to remove it completely; to convert the cool-white light to a warmer light as closely as possible. The transmission of the filter should rise gradually from 400 to 500nm. From literature (brochures) T% spectra I concluded that the Hoya LA-120 warmup filter is appropriate, so I bought its equivalent, the KR-12. As well as some cheap plastic amber pieces. Perhaps there is a dichroic mirror that will fit, but as you said, they are very expensive. But it is worth further thought.

Just to illustrate my point, here https://www.chroma.com/products/parts/at455dc
is the transm. spectrum of a typical (very good quality!) dichroic mirror that serves as a long-pass filter. It cuts off anything below about 455nm and equalizes everything above it. Great for excitation/emission but will remove all light at 380-455 from the LED. If I can borrow such thing, I will try it.
Thanks for the stimulating response!
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Re: Experiments with afocal photomicrography: illuminations, spider threads...

#20 Post by MichaelG. » Sun Feb 11, 2018 11:40 pm

I wish you joy with your tests ...
Everything you learn will probably be useful to us all some-day; so do please share the results !!

It may not be representative of the LED that you are using, but this specification sheet from Nichia is worth a look:
http://www.nichia.co.jp/specification/p ... (2619).pdf
The Spectrum plot on page_9 [page_10 of the PDF file] clearly demonstrates the strong 'Blue' peak.
I don't know the Canon's spectral response; but I'm sure it doesn't see this lamp as "White".

MichaelG.
.
.
P.S. ... I've just found a plot for the Canon 5D sensor:
http://i.stack.imgur.com/zVg2G.png
Compare that with the Nichia LED spectrum and the difficulties become obvious.
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Re: Experiments with afocal photomicrography: illuminations, spider threads...

#21 Post by MichaelG. » Mon Feb 12, 2018 12:34 am

.
.
Here is a rough & ready overlay of the two plots with X-axes matched:
.
IMG_1793.PNG
LED and Sensor, Spectra
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Re: Experiments with afocal photomicrography: illuminations, spider threads...

#22 Post by Hobbyst46 » Mon Feb 12, 2018 1:51 pm

Processing...
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Re: Experiments with afocal photomicrography: illuminations, spider threads...

#23 Post by Hobbyst46 » Mon Feb 12, 2018 2:41 pm

Done.
I attempt to continue your line of thought, by guessing - I have no background in digital sensors.

I calculated the total intensity per pixel per wavelength, as the some of contributions - source intensity times quantum yield. I adopted one of the two Canon sensors (the non-dashed lines), and the Nichia relative spectrum. From the nice graph you posted I roughly estimated at each of the following wavelengths: 425,450,475,500,550,600nm, the sum from the three channels (RGB). Plotted them and fitted a cubic spline for a better looking representation of the observed and calculated curves.
The calculated total is the dashed thick black line. On the right Y axis like the quantum efficiencies.
Arrows mark which trace belongs to the left or right Y scale. The thin black line is the LED intensity, on the left Y axis.
So I think that on the whole, the response is indeed fairly dominated by the blue region. i hope this is a correct conclusion (I am not happy but this is the situation).

I hope to show the calculated LED spectrum with an amber filter. Later on.
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Canon APSC sensor response vs white LED.jpg
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Re: Experiments with afocal photomicrography: illuminations, spider threads...

#24 Post by Hobbyst46 » Mon Feb 12, 2018 5:25 pm

MichaelG,

So I overlapped the Nichia cool LED spectrum with the transmission spectrum of a KR12 filter. A rough calculation, based on a few discrete wavelengths in the range 400-600nm and cubic spline fitting. The graph shows that the "problematic" blue constituent is trimmed.

Here is a separate reference to the Hoya LA-120/KR-12 amber filter: http://midopt.com/filters/la120/

I borrowed a power supply for my original 6V, 15W Zeiss tungsten lamp, installed it in the scope, added an LBD filter and recorded some photos, to be posted later. Setting up uniform illumination in the FOV was less convenient than with the LED. However, tungsten illumination eliminated most of the purple fringes. Green fringes appeared instead, but in the image recorded by the camera they are less disturbing than the strong purple ones. So, there is a chance to attenuate the CA effects by filtration.
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Re: Experiments with afocal photomicrography: illuminations, spider threads...

#25 Post by MichaelG. » Mon Feb 12, 2018 6:41 pm

Excellent work with those graphs !!

Glad to hear that the tungsten light is friendlier
... that supports the logic, so I think we must be on the right track.

My feeling regarding filtration of the LED is that although your amber filter helps greatly, it would be better to have a "brick-wall filter" to block wavelengths shorter than [say] 450nm ... This would halve the "Blue" content by removing the more troublesome violet region.

Those short wavelengths are essential to the operation of the White LED, because they excite the yellow fluorescence; but for anything less than an apochromatic objective they spell trouble.

I look forward to seeing your images.

MichaelG.
.
Incidentally: There is an alternative style of "White LED" which uses Red, Green, and Blue LEDs clustered together in a single package ... I haven't tried them yet, but these may prove very useful.
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Re: Experiments with afocal photomicrography: illuminations, spider threads...

#26 Post by Hobbyst46 » Mon Feb 12, 2018 10:37 pm

Some more images for comparison. These were just resized, to comply with the posting limitations.

Zooming shows the differences - fringes etc. I have no 10x/16x neofluars or planapos, but I think that the 25x and 40x neofluars create less CA than the planachromats (not surprising). LED is my only practical illumination.
I agree that filtration removes the purple fringe, however I think that CA should be eliminated with better corrected objectives - when I find ones... Anyway, I would like to test the illumination with other, more colorful specimens: Stained cells, flowers, algae...

But I will look for long-pass filters, as you suggest, though not dichroic - absorption filters from Schott or Corning may fit my budget. Perhaps there are used long-pass filters on eBay. They belong in the scientific community rather than the photographic community but maybe..

Notes: (1) tungsten illumination included an LBD filter. (2) One great advantage of the 10W LED is the high intensity, very useful for darkfield for example.
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Img 3.jpg
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Img 2.jpg
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img 1.jpg
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Re: Experiments with afocal photomicrography: illuminations, spider threads...

#27 Post by Crater Eddie » Tue Feb 13, 2018 2:17 am

Can you not change to a "warm white" or "neutral white" LED?
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Re: Experiments with afocal photomicrography: illuminations, spider threads...

#28 Post by Hobbyst46 » Tue Feb 13, 2018 7:21 am

Crater Eddie wrote:Can you not change to a "warm white" or "neutral white" LED?
CE
Yes, perhaps it is better - I do not know apriori. It is not a quick DIY job.
Zeiss Standard GFL+Canon EOS-M10, Olympus VMZ stereo

MichaelG.
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Re: Experiments with afocal photomicrography: illuminations, spider threads...

#29 Post by MichaelG. » Tue Feb 13, 2018 8:07 am

Hobbyst46 wrote:Some more images for comparison. ...
Thanks for posting those.

Here is a useful article [4 web-pages] from Lowel Tiffen, to add to 'the knowledge'
http://lowel.tiffen.com/edu/color_tempe ... ified.html

MichaelG.
Too many 'projects'

Hobbyst46
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Joined: Mon Aug 21, 2017 9:02 pm

Re: Experiments with afocal photomicrography: illuminations, spider threads...

#30 Post by Hobbyst46 » Tue Feb 13, 2018 10:52 am

MichaelG - thanks for the link. It is indeed illustrative. I was amused by the Hydrargium term. Even more vintage than my microscope...reminds me of the "Toxica" and "Separanda" pharmacy (apothecary) signs...
Zeiss Standard GFL+Canon EOS-M10, Olympus VMZ stereo

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