Images and comments

[Aenima]

Hi guys, have been trying to improve my technique - mostly by practice - / trial & error - and still have a long way to go.

[Apologies for the text between images, i wasn't sure where to post, but here seemed appropriate for the pics.]

A few things i've noticed, the achro objectives suffer a lot of CA when used in brightfield* and opening the condenser seemed to reduce this - leading me to believe that some kind of Oblique light would help me use the achros to better effect? [That was my thinking, anyway - and i could be quite wrong ]- allowing me to open the condenser wider, and also using more of the objectives aperture at the same time.

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cilliates brightfield

(*The worst of the CA seemed to be green tinge - i first assumed (right at the start) that these (mostly the aquatic) creatures were all green due to the high levels of chlorophyll/algae in the environment/ food chain and other similar reasons - but i soon realised that the green disappeared under the right circumstances and a lot of things were green when they shouldn't be. ) (also noticed green/pink CA even with open condenser, in addition to the usual blue around high contrast edges))

So with oblique lighting i think i managed to avoid some of the artifacts from the achro objectives and still capture some detail.

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bdelloid rotifer brightfield / Oblique






I found the detail a bit difficult to interpret, and it didn't look as much like the object as the bright-field shots did. So firstly i searched for APO objectives on the cheap, but these are not easy to find - and the ones i did find were old or damaged or both. So i went more in the direction of improving my grasp of illumination and tried to better my techniques for contrast and detail. It took me ages to realize that the objectives weren't wholly to blame for poor images, and my novice use of condenser and illumination was just as critical in the quality of the photos and views. Obviously phase contrast or DIC and whatever else cost a little too much in some cases, and i came across the interesting threads about the gradient filter and tried to make something similar to try out. Along with a blue filter and custom white balance to get a nice neutral background.
Not sure if i'm using the gradient filter properly, but i certainly came away quite impressed with the idea.

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rotifer 10x obj, gradient filter

Brineshrimp eye - focus stk of 5 shots

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copepo.jpg (287.09 KiB) Viewed 7384 times

I still have vibration as a possible issue - my dslr does jump quite a bit when taking a shot, even in live-view mode. I hope to extract some frames from a video to compare them with single images, as i don't have the means to separate the camera from the microscope, as yet, so video is the closest thing to a vibration-free image for comparison.

Anyway, yeah, this is about as far as i've got with my progress. Still a great deal to learn, and welcome critique at this stage.
For reference; My setup is the BHTU with D-plan achro objectives, a trinocular head with a NFK 2.5X proj. lens and solid connection to my DSLR. Shooting mostly in live view for convenience in framing/focusing as well as hopefully reducing shutter vibration.

ps. i have some leitz objectives, but noticing some CA even with a fluotar lens which i believe is a flourite lens? so it might be that the olympus projection lens doesn't quite supply the right correction to the leitz optics? (not really familiar with how that stuff works so i'm hoping someone can tell me whether there is a likely mismatch or i'm just expecting too much from the objectives)

P.p.s Another concern is the condenser itself (an ABBE type) - will there be a big difference between the ABBE and the achr/appl type? what effect does it have?

Thanks for any input. Thanks also to the members who gave previous advice - the more i learn, the more i realise how good it was!

Regards

Jay

[billbillt]

I see all of these as very good to superior images... Most folks here can't do half that good..

The Best,
BillT

[zzffnn]

I did not see big difference between Abbé vs Achr Apl condenser, but I did see much less CA from apo objectives (compared with Achr objectives).

Oblique does not always reduce CA; it depends, in some cases, it exacerbates CA.

[75RR]

Some very nice Oblique images + good comparison with Brightfield.

... or i'm just expecting too much from the objectives

I find that there is usually some CA that needs to be 'dealt with' in post processing.

Although the default illumination in microscopy is always said to be Brightfield (as in basic starter illumination),
I cannot recall at this moment any specific circumstance when observing or photographing protista and animalcules where Brightfield is in fact better than Oblique.

Oblique illumination, which creates a 3D effect, can be obtained in its simplest form by offsetting the brightfield port in a rotating condenser.

A similar effect can also be obtained by using a simple opaque mask close to the condenser's front aperture.
Opaque masks can be uncomplicated, such as the The Mathias arrow: http://www.microscopy-uk.org.uk/mag/ind ... lique.html
or quite sophisticated, such as the van Egmond Mask: viewtopic.php?f=15&t=4499
and the UGF (Universal Gradient Filter): viewtopic.php?f=28&t=2480&p=20658

Other types of Oblique lighting include COL (Circular Oblique Lighting): http://www.microscopy-uk.org.uk/mag/ind ... pjcol.html
and Hoffman Modulation Contrast: http://micro.magnet.fsu.edu/primer/tech ... intro.html

Another concern is the condenser itself (an ABBE type) - will there be a big difference between the ABBE and the achr/appl type? what effect does it have?

[Hobbyst46]

Hi guys, have been trying to improve my technique...

1. First of all, congratulations on your approach and progress, most of the photos you posted as very good and beyond. I hope to achieve your level of photomicrography one day.

2. IMHO, the "realistic" image of an object is less important in microscopy than it used to be. The image depends on the illumination. The advantage of phase contrast and DIC over their "alternatives" like UGF is that they are universal and well defined. When you exactly align the optical (expensive and brand-specific) components, you get predictable and reproducible phase/DIC images. Oblique or UGF are more empirical and the results are variable and depend on many variables. However, for esthetic purposes, Oblique is not inferior to other expensive illumination modes.

3. CA can be caused by several factors (which do not nullify one another...). I would suspect a slight mismatch between the camera (whether focally or afocally installed) and the microscope optics. I am saying this because I am using all-Zeiss color corrected optics on the microscope, and a Canon Camera, and there is still CA. Neofluar objectives ( = fluotar, fluorite etc) and Planapos reduce CA, but do not eliminate it completely. For your p.s. question, I doubt that the Olympus projection lens will supply the right correction to any leitz optics - it would be an (expensive) trial and error matching.

The BHTU is an excellent microscope, I would stay with Olympus objectives and not try other brands.

Another concern is the condenser itself (an ABBE type) - will there be a big difference between the ABBE and the achr/appl type? what effect does it have?

From my experience with Zeiss, and based on eye vision through the eyepieces only (no photography), the Abbe is definitely inferior to the Achromat Aplanat from the aspect of CA.

[Aenima]

I see all of these as very good to superior images... Most folks here can't do half that good..

The Best,
BillT

Thanks BillT - i am pretty happy with some of the latest results. Although for every useable shot there are a hundred in the recycle bin. hehe. ;P i have the forum here to thank for the advice - but also have been very lucky finding decent equipment for good prices, and i only really knew what to look out for as a result of that advice.

zzffnn - thank you for the comments. Yes, i do have an APO objective and it 'seems' almost completely free of CA - was happily around £50 ebay - the Zeiss 25x 0.65 4080953 slim silver one with black ring and sprung tip - but it does tend to run out of working distance with thick water samples and hits the coverslip every now and then. I have wondered whether anything can be done to gain a bit more room - adjusting the projection lens or something? Bit of a long shot though. Maybe a zeiss projection lens will work?

75RR - wow, some great links. Thank you.
I should clarify, the 'gradient filter' i mentioned above is the same as the UGF that litonotus member wrote about, and the last 3 images i posted were taken with it. (using zzffnn's blue-tack approach) Very interesting creation, and surprisingly easy to make and use. Big thanks to the original poster.

The CA issue i think i did try using adobe's CameraRAW software because it had a 'lens correction' option that gives you a feature supposed to remove CA, but it doesn't seem to recognise the green/pink CA in my microscope images. I think it just selectively desaturates areas of high edge contrast - i'm looking around hoping for some plug-ins/programs/tutorials/techniques that might work for reducing CA in micro images. Does anything spring to mind for post-processing CA?

Hobbyst46 - just caught your reply before posting - thank you. Great info, makes a lot of sense. Yeah my only reason for the leitz objectives is that i started on a SM-LUX from ebay, (and had to wait a while for olympus stand to pop up cheaply there too), and i was very impressed with it's quality - but i can fully imagine the mismatch prevents that same leitz image quality when used on the olympus. Was worth a try though especially as i already had the objectives.

Anyhoo - thanks guys for the input and replies. Unfortunately i have an endless supply of questions that pile up as i continue to use the microscope - i try to avoid pointless ones via websearch and forum reading - but some things are hard to find answers for, and i do miss a lot of info. So i'm grateful for the feedback.

[75RR]

The CA issue i think i did try using adobe's CameraRAW software because it had a 'lens correction' option that gives you a feature supposed to remove CA, but it doesn't seem to recognise the green/pink CA in my microscope images. I think it just selectively desaturates areas of high edge contrast - i'm looking around hoping for some plug-ins/programs/tutorials/techniques that might work for reducing CA in micro images. Does anything spring to mind for post-processing CA?

One method to remove specific CA. colours:

In Photoshop (and I believe in other Post Processing photographic software) one can select the specific chromatic aberration colour that is to be eliminated by using the eyedropper in the Hue/Saturation window.
In Photoshop this is under Image → Adjustments → Hue/Saturation
First select a general colour from the dropdown menu, one that is closest to the CA you wish to eliminate, lets say ‘cyan’ for example.
You then activate the eyedropper, place it over the colour that you wish to eliminate, and sample a more specific hue.
The next step is to move the Saturation slider almost all the way over to the left, at least to the minus 75-90 range.
Care must be taken that this specific colour is not present in other areas of the image, if it is, those areas will have to be isolated in order to avoid desaturating them.

... but it does tend to run out of working distance with thick water samples and hits the cover slip every now and then. I have wondered whether anything can be done to gain a bit more room ...

Do not think much can be done to gain more room, by which I think you wish to focus deeper. It is my understanding that for optimal results not only does the cover slip have to be the stated thickness (it is a design parameter) but the subject has to be as close to the underside of the cover slip as possible. You should perhaps be looking at ways to having less water under the cover slip. Placing absorbent paper to the edges of the cover slip is one way to achieve this.

[billbillt]

"The BHTU is an excellent microscope, I would stay with Olympus objectives and not try other brands."

That is part of the fun with all of this stuff.. Trying different things.. It does no harm to try other brands of objectives and oculars... who knows, you may find a combination of different brands that works well FOR YOU...

BillT

[Hobbyst46]

A brute force way to have critters swim or stay close to the cover slip: decrease coverslip area. The water depth is more or less fixed since you do not want to press on it and crush the swimmers. But, a 15x15 coverslip decreases the volume water by 2-fold relative to a 22x22 coverslip (say) and since the depth is fixed, the probability that the critter is near the coverslip is doubled. Just smear petroleum jelly (not nail polish!) around the slip to seal and prevent evaporation and drying.

[zzffnn]

Jay, I would second what 75RR said (on removing excess water under cover slip). With the same apo objective, there is not much else you can do to increase its working distance. Unless you change to another apo objective of same magnification with less NA and specified longer WD.

I don't have experience with projection lens.

[wporter]

Aenima,

Make sure that you are not, in post-processing, removing colors other than those caused by CA. For instance, if you observe and image a prismatic specimen, the colors may be be from dispersion by the specimen, rather than any CA resulting from optical components. I suspect that in the great last image of a copepod, that many of those colors are from the specimen and not the optics.

A good way to detect real CA colors is to look at the edges of an opaque black-and-white specimen at various positions across the field of view. You can then tune your post-processing settings so that when those same settings are applied to a biological specimen, they are not overdoing the color removal.

[Hobbyst46]

@Aenima

I have checked mfgs stated working distances for similar magnification and NA objectives.

The WD of a Zeiss Planapo 25x0.65, mfg (a batch apparently later than yours - different catalogue no.) is 0.14mm.

The WD of a Zeiss Neofluar 25x0.60 is 0.54mm.

The WD of an Olympus S Plan Apo 20x0.70 is 0.55mm.

I could not find that an Olympus fluorite objective with a ~20 mag and NA>0.45 exists.

[MicroBob]

Hi Aenima,

plancton objects are difficult to photograph in high quality. When you see pictures that are considerably better than yours then you can be sure that quite a few tricks have been applied.
One trick ist to remove the object from the sample with a pipette and place it individually under a separate cover slip with just the necessary amout of water. Not easy to do .
The objective is calculated for the object to be just below a 0,17mm cover slip. For higher power dry objectives the cover slip should be 0,17mm or slightly below and the water gap very thin. With an oil objective the cover slip thickness is less critical as long as it is not much too thick.

When you photograph objects with little colour information you can just use one colour channel to get rid of chromatic errors. When you make a b/w image from this single channel image it will be sharper than the original colour image.

Keep on with your work, you are on a very good way.

Bob

[Aenima]

Thanks guys - really good info and helpful replies.

The idea regarding smaller coverslips sounds good - i am trying to wrap my head around the mathy bit - haha, i'm thinking it meant if there is less horizontal space, the creatures will be more likely to use vertical space leaving them near the top more often?

One thing regarding the smear that keeps water in, very useful to know, but what i often tend to rely on, for easier critter photography, is the steady drying out that (i think) forces the coverslip down onto the contents - it seems to pin the cilliates in place for a while, ultimately squishing them - but for a while just before then, they seem to sit still. (Although they're still very difficult to photograph even then, and i usually have more luck with larger subjects/lower magnification.)

I have some 'delta lab' cover slips 24mmx24mm that are labeled Nr.1 - which seem like decent quality. Also some amscope 22x22mm, which actually state .12-.16mm thickness - so will try those, being smaller in size and close to the correct thickness.

Thanks again for the suggestions and advice. Much appreciated.

Regards

[Hobbyst46]

if there is less horizontal space, the creatures will be more likely to use vertical space leaving them near the top more often?

Yes, exactly.

By the way: I have once read that paramecium is attracted to light. If this is true, could that inspire something? like shine a light beam near the top side of the coverslip?...hopefully will check literature if it works and how to accomplish...

[apochronaut]

The advantage of using a condenser with a higher degree of colour correction is mostly seen off axis. If your goal is to capture the largest field as is possible, then the chromatism inherent with abbe condensers will cause background chromatism and some resolution loss, increasing with distance from the axis. This will be perceived more readily with highly corrected objectives, than with achromats. Aplanatic or aspheric condensers go a long way towards rectifying off axis chromatism and coma and the addition of achromatism to the condenser on top of that, especially when using highly corrected objectives, clears the way for better wide field imaging, especially with sensitive cameras.
If one is photographing smaller areas of the field or cropping to a smaller center frame, then an abbe condenser might suffice, better yet an achromat but it needn't be an aplanat necessarily, although it seems that most achromats are anyway.
There are some inexpensive high N.A. abbe type condensers out there, 1.3 or even 1.4 N.A. These are unlikely to be any better for most applications, than a common 1.25 abbe condenser, only with very high N.A. non plan objectives.

Your images are extremely good, given that I gather there hasn't been so much fussing with them. That is pretty hard to accomplish and with fairly simple tools. Too much of what goes up on this forum or any other for that matter is overly messed with.

[Radazz]

I think these are beautiful images.

I try not to compare my images to some of the DIC images here, I find I'm happier that way.


Radazz

[Aenima]

The advantage of using a condenser with a higher degree of colour correction is mostly seen off axis. If your goal is to capture the largest field as is possible, then the chromatism inherent with abbe condensers will cause background chromatism and some resolution loss, increasing with distance from the axis. This will be perceived more readily with highly corrected objectives, than with achromats. Aplanatic or aspheric condensers go a long way towards rectifying off axis chromatism and coma and the addition of achromatism to the condenser on top of that, especially when using highly corrected objectives, clears the way for better wide field imaging, especially with sensitive cameras.
If one is photographing smaller areas of the field or cropping to a smaller center frame, then an abbe condenser might suffice, better yet an achromat but it needn't be an aplanat necessarily, although it seems that most achromats are anyway.
There are some inexpensive high N.A. abbe type condensers out there, 1.3 or even 1.4 N.A. These are unlikely to be any better for most applications, than a common 1.25 abbe condenser, only with very high N.A. non plan objectives.

Your images are extremely good, given that I gather there hasn't been so much fussing with them. That is pretty hard to accomplish and with fairly simple tools. Too much of what goes up on this forum or any other for that matter is overly messed with.

apochronaut - apologies for the delayed reply, - thank you for the detailed info regarding condensers - funnily enough, i have actually got a achr/appl 1.35 condenser that i picked up for a silly (in a good way) price a few months ago - a nikon model that fits the olympus scopes - i had intended to follow the tutorial by the member carl who turned his nikon condenser into a darkfield one by inserting a stop up inside it. However when i got it i realised i'd messed up with the condenser type having extra glass, and the darkfield stop wouldn't fit inside.
The good news though was i can still use it on the BH2 as it is, and perhaps still benefit from additional correction. I stored it away due to the non-starter DF project, as well as the fact that it doesn't cover the 4x and lower field, and i use 4x a lot.

Anyway, I do like to try for a wide field, but i think the olympus 2.5x projection lens is stopping me from including the full image from the objectives - the ideal lens apparently for the APS-C sensor was the 1.67x lens which is rarer and costs a fortune. I think the result is that only the center portion of the field is recorded. I'm guessing that means some of the edge artifacts are outside the FOV, and the full resolution is lost, giving me a 'zoomed in' type effect. So to get a wider image i need to use a much lower mag. objective than if i had the right projection lens.


As for the amount of processing i do - the usual workflow would be first to use photoshop's excellent noise filter to reduce high iso blotches and a certain amount of CA along with it, i might up the saturation afterward. (maybe use Match colour to neutralise white balance). Then a gentle high-pass sharpen - if applicable i also try to blur out the debris around the object. I am still figuring this step out as it often leaves a blatant outline from the lasso selection, especially when there are gradients in background brightness.

But otherwise that's all.

I started a new flickr account for microscope pics, and there are a few rotifer images that are straight from camera (also with the use of the gradient filter) - not sure if it will display here but this is a link to one of them. https://flic.kr/p/272H6pS

Thank you Radazz, and thank you to everyone else for your kind comments and advice.

[Hobbyst46]

@Aenima
I entered the Flickr and observed your photos, and enjoyed them!

[Aenima]

@Aenima
I entered the Flickr and observed your photos, and enjoyed them!

thank you very much Hobbyst46

Hopefully will add more photos soon.