MICAM Settings

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desertrat
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MICAM Settings

#1 Post by desertrat » Sun Feb 17, 2019 3:03 am

Thanks Bob and Doron for helpful advice on getting and processing images. Doron mentioned MICAM, a freeware microscope image capture program. And Bob has given advice on improving the images with image processing tools.

I decided to start a new thread so the existing threads where these were first discussed wouldn't be sidetracked from their original purposes. These threads were here:

viewtopic.php?p=63270#p63270

viewtopic.php?f=10&t=6799&p=63276#p63276

Experimenting with MICAM I was able to get rid of the banding problem that plagued my earlier use of the little 2MP eyepiece camera. One method is to turn down the voltage to the microscope lamp to the minimum setting. Even though the image is dim and yellow cast in the eyepieces, MICAM adjusts brightness and color balance to a mostly normal level. The second method is to use one of my telescope eyepiece filters, the 18% transmittance neutral density filter used for observing the moon. When the filter is put over the illuminator, it allows turning up the lamp voltage to get normal image brightness, and the banding likewise disappears.

MICAM has ended the difficulties of getting the best focus that was a problem with the Windows XP webcam image capture program. MICAM allows adjustments of image properties like contrast, color balance, etc before the image is captured, which does a lot of the work previously done with GIMP.

Bob suggested using more unsharp masking to further sharpen the image. To look for input, below are some images captured using the little 2MP eyepiece camera without the reducing lens. Using the reducing lens reduces sharpness too much when the most sharpness is needed.

These images are of a diatom from Klaus Kemp's 8 form test plate, Stauroneis phoenicenteron. The following webpage has data on its dimensions:

https://diatoms.org/species/stauroneis_phoenicenteron

The diatom was illuminated with a green 58 filter. The diatom is oriented vertically on the slide. The camera was rotated to make the diatom appear horizontal so more of it is visible in the rectangular image format. Instead of using the homemade condenser stop used previously, one of the darkfield/COL inserts I bought recently on Ebay was used. The smallest central stop, which produces darkfield with a 10X objective and COL with a 43X objective was used. The condenser focus, illuminator diaphragm, and condenser diaphragm were adjusted to get improved image contrast without closing the condenser diaphragm more than just starting to reduce the illumination circle on the back lens of the objective. This was to keep as much resolving power as possible. The objective used was an antique brass Spencer 44X 0.65 NA objective made in the 20s. I have been using these old brass objectives lately for routine work, and kind of enjoy exercising them. The first image is as captured with the eyepiece camera, and reduced to half its original size. The full size image doesn't show any greater detail, just more fuzziness.
S_Phoen_micam_0.jpg
S_Phoen_micam_0.jpg (38.93 KiB) Viewed 11122 times
The second image has had the unsharp mask tool in GIMP used. When the tool is opened, there is a dialog box with several parameters that can be adjusted. So far I haven't tried to learn exactly what they all do. I have accepted the default setting. If more sharpness is needed, I just run it again at the default settings. This image was with the mask used twice. Using the mask also increases contrast. The image contrast seen in the eyepieces looks more or less like this image.
S_Phoen_micam_a.jpg
S_Phoen_micam_a.jpg (64.03 KiB) Viewed 11122 times
The third image is with the unsharp mask run four times. Sharpness has increased, but contrast is now very high, and some artifacts are starting to appear. I could manually decrease contrast some to get it a little more normal looking. So, which one looks the best?
S_Phoen_micam_b.jpg
S_Phoen_micam_b.jpg (110.3 KiB) Viewed 11122 times
I first photographed this diatom in 2012 with my little polaroid digital camera using an AO 43X 0.95 NA apochromatic objective. I posted that image to the Yahoo microscope group, then posted it again here last year when I joined. Recently I tried to make the image again with the same equipment, but wasn't able to achieve the same sharpness. The little camera is a lot older and has seen some use. The autofocus sometimes doesn't work properly.

Tomorrow I will repeat what I did above using the apochromatic objective, and see how much difference in sharpness is visible.

Let me know how much unsharp mask produces the best looking image.
Rick

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75RR
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Re: MICAM Settings

#2 Post by 75RR » Sun Feb 17, 2019 7:26 am

Nice! I think one fine tuned unsharp mask pass and adjusting the levels will produce the better image.

desertrat wrote: The following webpage has data on its dimensions:

https://diatoms.org/species/stauroneis_phoenicenteron
Slightly off topic here ... is it me or does there appear to be 19 striae in 10µm?

Image
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Re: MICAM Settings

#3 Post by MichaelG. » Sun Feb 17, 2019 8:37 am

desertrat wrote:Experimenting with MICAM I was able to get rid of the banding problem that plagued my earlier use of the little 2MP eyepiece camera. One method is to turn down the voltage to the microscope lamp to the minimum setting. Even though the image is dim and yellow cast in the eyepieces, MICAM adjusts brightness and color balance to a mostly normal level. The second method is to use one of my telescope eyepiece filters, the 18% transmittance neutral density filter used for observing the moon. When the filter is put over the illuminator, it allows turning up the lamp voltage to get normal image brightness, and the banding likewise disappears.
I don't mean to divert your new thread off its MICAM topic, Rick, but may I ask what type of lamp and lamp controller you are using?

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Re: MICAM Settings

#4 Post by Hobbyst46 » Sun Feb 17, 2019 9:25 am

75RR wrote:Slightly off topic here ... is it me or does there appear to be 19 striae in 10µm?
I also count 19 stria in 10 microns. The original text there says >14 in 10 microns. Yes, 19>14, so it is OK.

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Re: MICAM Settings

#5 Post by Hobbyst46 » Sun Feb 17, 2019 9:40 am

desertrat wrote:...
Of the above posted sharpened photos of the diatom, I personally like the third (sharpest).

Intuitively, I think that repated application of the same unsharp mask is like a power series, inceases the sharpness (and artifacts) too steeply.

For excersize, I copied the initial image of "your" diatom and fed it to ImageJ - a free software, very powerful, very friendly when you understand the logic. ImageJ includes an unsharp mask with two parameters: "Radius" (in pixels) and "Mask weight" (a fraction, 0.1-0.9). In my hands, tweaking BOTH parameters sharpened the diatom better than repeated use of the same mask.

With your permission, I can post some results of those calculations.

Probably, the parameter values vary from image to image, should be found by trial and error. I would like to find an "objective" neutral math tool which tells that the image is sharp. Otherwise, the quality of the sharpened image is within the eyes of the beholder.
Last edited by Hobbyst46 on Sun Feb 17, 2019 10:06 am, edited 1 time in total.

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Re: MICAM Settings

#6 Post by MicroBob » Sun Feb 17, 2019 10:01 am

Hi Rick,
I'm no image editing expert myself but as far as I know usharp masking should be carried out once as the very last step of image editing and setting the final image size. From your three images I find the second on just fine - good aparent sharpness and little artifacts. When this would be too little you should better play with the settings of the tool than running unsharp masking more than once.

With a small chip camera I would always prefer a lot of light as they tend to break down in low light conditions. So I think that your new solution against the banding creates a new bottle neck in you imaging process. Also the yellow light doesn't make the best use of the sensors capabilities. Would it be possible to build up a DC lamp for you microscope?

I edited your original file, just one short try: I opened it with gimp, split it into colour channels, used the green channel, adjusted the histogram and applied unsharp masking. The result could be a bit less contrasty and grainy.

Bob
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Rick Diatom 1.png
Rick Diatom 1.png (215.79 KiB) Viewed 11073 times

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Re: MICAM Settings

#7 Post by 75RR » Sun Feb 17, 2019 10:25 am

I am going to follow MicrobBob's cue on this ...

Using the first image: Black and White with Green filter, adjusted Levels + sharpen
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x.jpg
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Re: MICAM Settings

#8 Post by Hobbyst46 » Sun Feb 17, 2019 10:52 am

Yet another attempt:
Original diatom opened in ImageJ, Split channels into red, blue and green, (naturally) red and blue are very weak so are dumped, the "green" (now grey) was sharpened once by an unsharp mask with following parameters: Pixels=2.0, Mask weight = 0.85.
Also, the same unsharp procedure applied to the original photo, without splitting channels.
Attachments
1- diatom.jpg (green) 2.0 pixels 0.85 mask weight.jpg
1- diatom.jpg (green) 2.0 pixels 0.85 mask weight.jpg (110.5 KiB) Viewed 11066 times
4- diatom unsharp mask ImageJ 2.0 pixel 0.8 mask weight.jpg
4- diatom unsharp mask ImageJ 2.0 pixel 0.8 mask weight.jpg (87.53 KiB) Viewed 11066 times

desertrat
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Re: MICAM Settings

#9 Post by desertrat » Sun Feb 17, 2019 4:30 pm

Thanks for all the replies. This will give me plenty to work on for awhile. The unsharp mask in GIMP has several parameters that can be adjusted. I guess I need to spend some time to learn how to get the best adjustments with a single application of the mask.
MichaelG wrote:I don't mean to divert your new thread off its MICAM topic, Rick, but may I ask what type of lamp and lamp controller you are using?
The incandescent lamp is in the base of my AO 4 Series microscope, and is similar to an old fashioned automotive tail light lamp bulb. It illuminates an optical train in the base that does modified Kohler illumination. It's powered by a separate step down transformer with several taps in the secondary winding. There is no control circuitry involved. Several output voltages can be selected from 2.5V to 7.5V. The lamp is rated for 6.5V or thereabouts.
Rick

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Re: MICAM Settings

#10 Post by MichaelG. » Sun Feb 17, 2019 4:40 pm

desertrat wrote:
MichaelG wrote:I don't mean to divert your new thread off its MICAM topic, Rick, but may I ask what type of lamp and lamp controller you are using?
The incandescent lamp is in the base of my AO 4 Series microscope, and is similar to an old fashioned automotive tail light lamp. It illuminates an optical train in the base that does modified Kohler illumination. It's powered by a separate step down transformer with several taps in the secondary winding. There is no control circuitry involved. Several output voltages can be selected from 2.5V to 7.5V. The lamp is rated for 6.5V or thereabouts.
Thanks for that, Rick
Strange that the camera should apparently show less banding at both high and low settings than it does in the mid-range.
... I need to have a think about that one.

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Re: MICAM Settings

#11 Post by desertrat » Sun Feb 17, 2019 4:46 pm

MichaelG. wrote:
desertrat wrote:
MichaelG wrote:I don't mean to divert your new thread off its MICAM topic, Rick, but may I ask what type of lamp and lamp controller you are using?
The incandescent lamp is in the base of my AO 4 Series microscope, and is similar to an old fashioned automotive tail light lamp. It illuminates an optical train in the base that does modified Kohler illumination. It's powered by a separate step down transformer with several taps in the secondary winding. There is no control circuitry involved. Several output voltages can be selected from 2.5V to 7.5V. The lamp is rated for 6.5V or thereabouts.
Thanks for that, Rick
Strange that the camera should apparently show less banding at both high and low settings than it does in the mid-range.
... I need to have a think about that one.

MichaelG.
I'll throw in a little more odd complication. The banding is still there at higher voltage selections, unless the 18% ND filter is used. Why that is, I have no idea. I'm not planning on trying to analyse this. I'm happy I was able to get rid of the banding without having to set up a smooth DC power supply for the lamp in the base. I'm interested to see any theories posted here...
Rick

A/O 10 Series Microstar
A/O 4 Series Microstar
A/O 4 Series Phasestar
A/O 4 Series Apostar
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Several old monocular scopes in more or less decrepit but usable condition

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Re: MICAM Settings

#12 Post by Hobbyst46 » Sun Feb 17, 2019 4:57 pm

desertrat wrote:
MichaelG. wrote:
desertrat wrote: The incandescent lamp is in the base of my AO 4 Series microscope, and is similar to an old fashioned automotive tail light lamp. It illuminates an optical train in the base that does modified Kohler illumination. It's powered by a separate step down transformer with several taps in the secondary winding. There is no control circuitry involved. Several output voltages can be selected from 2.5V to 7.5V. The lamp is rated for 6.5V or thereabouts.
Thanks for that, Rick
Strange that the camera should apparently show less banding at both high and low settings than it does in the mid-range.
... I need to have a think about that one.

MichaelG.
I'll throw in a little more odd complication. The banding is still there at higher voltage selections, unless the 18% ND filter is used. Why that is, I have no idea. I'm not planning on trying to analyse this. I'm happy I was able to get rid of the banding without having to set up a smooth DC power supply for the lamp in the base. I'm interested to see any theories posted here...
A DC lamp, or a LED driven with a constant current, is the most stable light source. Hopefully this mode will become possible for you. The ND filter sounds like a brute force solution, even if it apparently works.

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Re: MICAM Settings

#13 Post by desertrat » Sun Feb 17, 2019 5:05 pm

MicroBob wrote:...With a small chip camera I would always prefer a lot of light as they tend to break down in low light conditions. So I think that your new solution against the banding creates a new bottle neck in you imaging process. Also the yellow light doesn't make the best use of the sensors capabilities. Would it be possible to build up a DC lamp for you microscope?...Bob
Hi Bob, I forgot to mention that the image brightness ratios in my trinocular head are fixed with no adjustments. Most of the light goes up the vertical tube, with a fairly small fraction going into the binocular head. I don't know what the fractions are, but when the image in the binocular tubes is normal brightness or even a little dim, the image in the vertical tube is very bright, even a little uncomfortable for me to view with an eyepiece. So I think the eyepiece camera is getting plenty of light.
Rick

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Re: MICAM Settings

#14 Post by desertrat » Tue Feb 19, 2019 1:18 am

Since everything was set up from the previous session, decided to photograph the diatom with two different objectives. Below, on the left is an AO 43X apochromat NA 0.95, in the middle is an antique Leitz 6L 45X NA 0.65, and on the right is the antique lacquered Spencer 44X NA 0.65 objective used to make the images at the beginning of this thread.
43_45_44.JPG
43_45_44.JPG (45.4 KiB) Viewed 10977 times
Having greater difficulties with the autofocus on my aging little Polaroid digicam. It looks it may not be in service much longer. :( These were taken outside under an overcast sky against a cardboard background without flash to avoid all the inscriptions being washed out.

First up is the image from the apochromat. The image in the 15X compens eyepieces was definitely a bit sharper than any of the similar magnification achromats produced. My image capture equipment just isn't capable of getting the image in its full sharpness. Am now using the unsharp mask tool only once after adjusting its three parameters. These are radius, amount, and threshold. After using the unsharp mask, adjusting the contrast using brightness curves instead of the basic brightness and contrast tool. I tried using the unsharp mask before and after downsizing the image, and didn't see any obvious visible difference. If anything, it seemed to work a little better for me by using it before downsizing. Below is the apochromat image.
Stau_phoen_43apo.jpg
Stau_phoen_43apo.jpg (72.36 KiB) Viewed 10977 times
When the image is reduced in size enough to post here, the punctae with the smallest separation cannot be easily seen. Below is an inset of the right hand side of the above image before it was reduced in size. The punctae can be seen more clearly.
Stau_phoen_43apo_inset.jpg
Stau_phoen_43apo_inset.jpg (32.46 KiB) Viewed 10977 times
The images from the diatom database linked in the first post were focused differently, so the diatom's punctae were visible as bright dots instead dark dots. I'm wondering if this was done because it gave the punctae improved visibililty and greater accuracy? Tried this with the above objective during the same session, but with limited success. Was able to visualize the punctae as bright dots only over a limited section of the diatom. With stacking of multiple images it might have worked over more of the diatom. It's obvious those images were made with much better equipment than what I'm using. Below is my "bright punctae" attempt. The green filter was removed from the illuminator and the image color fringes were eliminated with complete desaturation and turned into monochrome gray scale.
Stau_phoen_43apo_brtfld.jpg
Stau_phoen_43apo_brtfld.jpg (62.72 KiB) Viewed 10977 times
The last image was made with the antique Leitz 6L 45X NA 0.65 objective.
Stau_phoen_45X_6L.jpg
Stau_phoen_45X_6L.jpg (67.31 KiB) Viewed 10977 times
It obviously saw a lot of use from students in a college that ceased to exist long ago. The plating is worn off the end and the brass underneath has developed an age patina. The lens element there shows no sign of damage however, no pits or scratches. Getting images with good contrast has always been a bit difficult with this objective. There doesn't seem to be any visible haze on the interior elements, but a small amount might be possible. I have cleaned the two surfaces accessible, not wanting to try to disassemble the objective to reach the inner elements. When using it on stained bacteria slides, it gives very good images with only a tiny bit less contrast than the other uncoated achromats. Was unable to get good "bright punctae" images with this or any of my other achromats. I'm wondering if that focus adjustment only works well with more highly corrected objectives?
Rick

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Re: MICAM Settings

#15 Post by Hobbyst46 » Tue Feb 19, 2019 8:43 am

Your AO 43X is a very beautiful piece of equipment.
And the diatom photos are fine!
I prefer the dark punctae photo over bright ones because their physical status is holes, but, on the other hand, they are illuminated from below, so should be tiny light sources...yet, the final resolution of the bright punctae is about the same as that of the dark ones.

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Re: MICAM Settings

#16 Post by 75RR » Tue Feb 19, 2019 8:57 am

I find the punctae are nicely resolved.
After using the unsharp mask, adjusting the contrast using brightness curves instead of the basic brightness and contrast tool.
You might want to try adjusting the Levels before you adjust the contrast.
Having greater difficulties with the autofocus on my aging little Polaroid digicam.
Can you set the camera to infinity?
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Re: MICAM Settings

#17 Post by MicroBob » Tue Feb 19, 2019 9:17 am

[quote="desertrat"] even a little uncomfortable for me to view with an eyepiece. So I think the eyepiece camera is getting plenty of light./quote]

Hi Rick,
I'm sure that your camera gets enough light then to make a good image. I would say when it starts to get a bit dim for the eye the camera might also start to find it a bit dim when connected to the same port.

Bob

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Re: MICAM Settings

#18 Post by mrsonchus » Tue Feb 19, 2019 11:10 am

Hi all, a really interesting thread this one.
I had a quick look at W-pedia and found the following info which seems relevant, namely because the relationship between the camera's pixel-size (i.e. the size of each physical element of it's sensor, in µ) and the resolving capability of the sensor - interesting and a good pointer maybe to an important criterion when selecting a CMOS camera for microscopy?

Here's a snippet from W-pedia, lots of technical bits I don't really understand much, but some that I do.
++++TEXT COPIED FROM W-PEDIA++++++++++++++++++++++++++++++++++++++++++++++++++++++++
Sensor resolution (spatial)
Some optical sensors are designed to detect spatial differences in electromagnetic energy. These include photographic film, solid-state devices (CCD, CMOS detectors, and infrared detectors like PtSi and InSb), tube detectors (vidicon, plumbicon, and photomultiplier tubes used in night-vision devices), scanning detectors (mainly used for IR), pyroelectric detectors, and microbolometer detectors. The ability of such a detector to resolve those differences depends mostly on the size of the detecting elements.

Spatial resolution is typically expressed in line pairs per millimeter (lppmm), lines (of resolution, mostly for analog video), contrast vs. cycles/mm, or MTF (the modulus of OTF). The MTF may be found by taking the two-dimensional Fourier transform of the spatial sampling function. Smaller pixels result in wider MTF curves and thus better detection of higher frequency energy.

This is analogous to taking the Fourier transform of a signal sampling function; as in that case, the dominant factor is the sampling period, which is analogous to the size of the picture element (pixel).

Other factors include pixel noise, pixel cross-talk, substrate penetration, and fill factor.

A common problem among non-technicians is the use of the number of pixels on the detector to describe the resolution. If all sensors were the same size, this would be acceptable. Since they are not, the use of the number of pixels can be misleading. For example, a 2-megapixel camera of 20-micrometre-square pixels will have worse resolution than a 1-megapixel camera with 8-micrometre pixels, all else being equal.
++++++++++END OF COPIED TEXT+++++++++++++++++++++++++++++++++++++++++++++++++++++++++


The bright/dark phenomenon of focus Z-planes is the same that I encounter with projections on the surface of pollen grains. Focusing down onto the tip of such a protrusion the first focus is a bright one, the lower focus then becomes a dark one. I always use the bright and 'first encountered from above' one. This is also seen when examining for example sieve plates in plant phloem elements, where the bright 'version' always gives me more information and a sense of the actual 3-dimensionality (albeit an extremely shallow one!) of the perforated sieve-plate.
Seems to me that the choice of which to use may be a matter of preference if empirical and critical methods aren't involved, when perhaps a deeper look into the subject becomes necessary.

I'm really enjoying this interesting thread, and couldn't resist chiming-in with the teeny bit I've experienced in this area.

Keep the info coming chaps, very useful thread indeed.

John B. :)
John B

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Re: MICAM Settings

#19 Post by 75RR » Tue Feb 19, 2019 11:38 am

I had a quick look at W-pedia and found the following info which seems relevant, namely because the relationship between the camera's pixel-size (i.e. the size of each physical element of it's sensor, in µ) and the resolving capability of the sensor - interesting and a good pointer maybe to an important criterion when selecting a CMOS camera for microscopy?
This excellent article by Charles Krebs explains this simply and clearly; it also adds the objective's resolution to the equation.

http://krebsmicro.com/relayDSLR/relayoptics1.html
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Re: MICAM Settings

#20 Post by mrsonchus » Tue Feb 19, 2019 4:03 pm

That's a great link 75' - a really good explanation and indeed a way to consider the choice of sensor-size as matched to a 'scope's objective-set, or perhaps the most used/critical objectives therein.
Looking forward to reading the link-article and it's subsidiaries this evening.

Thanks 75'.

John B. :)
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Re: MICAM Settings

#21 Post by MicroBob » Fri Feb 22, 2019 8:34 pm

Hi together,
there is a thread in the german Mikroskopie-forum.de that is about a similar camera as Rick's:
https://www.mikroskopie-forum.de/index. ... ic=33588.0

The problem is not yet solved, but the gathered information is quite interesting.
The banding at high illumination is experienced too and Lutz has in addition a problem with the sharpness in brightfield while it is fine with DIC.
This really seems to be a much more interesting problem than it looks at first sight.

Bob

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