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Advice on gear for micro photography

 
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The Cove Online



Joined: 25 Aug 2016
Posts: 3

PostPosted: Tue Aug 30, 2016 5:43 am    Post subject: Advice on gear for micro photography Reply with quote

Hi Everyone,

First I'd like to credit Rik and Charlie amoung many others for your contribution to this field of both science and art.

My background is marine science and I've also had a passion for photography for some time.

I'm interested in photographing subjects such as algae, zooplankton and possibly some structural coral or polyps of similar size. So ideally I guess this would be a range of 5-10 microns (algae) and 200 microns (zooplankton).

I have access to an amscope microscope at home and some Olympus and Leica microscopes at work - I believe there is also some zeiss objectives lying around without a microscope. I am not interested in 'mediocre' photography and would seek to have a database of high resolution images, which can be printed of perhaps A3 scale. This is not for any work requirements, but more so because of a general interest in photography and the lack of good taxonomic reference images for algae, which may be used as an aid for species identification at work.

So gear wise, I suspect I will require focus stacking software - what are your recommendations here - zyrene? My current camera is a 5diii, which may pose a problem since it doesn't have the greatest megapixel count or density. I do have a 'perfect photo suite' which allows resizing images to significantly larger sizes for print with minimal quality losses.

I'm not quite sure on which pathway to take for the actual photography, nor what objectives I will benefit most from. In saying this, I'm referring to my confusion of the differences between photography through:
    *An objective to Canon lens mount via an adapter and either a tube lens (I'd use a 100mm macro) or bellows (not sure which are good for these).

    Connecting my camera directly to the microscope via a C-mount of some sort and any relevant adapters - not sure how to go about this route as I have tried looking into this before and always get confused or can't find the parts I'd need


So any advice on which methods, practices and equipment people would suggest to achieve this would be greatly appreciated.

For reference some of my greatest inspirations are those from Daniel Stoupin's freshwater gallery: http://www.microworldsphotography.com/Galleries
It was actually through his photos that I found out about this kind of photography, which otherwise seems quite illusive. So my goal is to try my best at resembling that quality of photography and subject matter.

A great member here who seems to specifically deal with work I'm trying to capture would be 'jacek':

http://www.photomacrography.net/forum/viewtopic.php?t=31507&highlight=

Thanks to everyone in advance.
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rjlittlefield
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PostPosted: Wed Aug 31, 2016 10:12 pm    Post subject: Reply with quote

First, thank you for the kind words. I hasten to say that you're interested in a regime where I am far from expert. Almost everything I know about microscopy is stuff that I have learned from other people. I'm hoping that other people will chime in with better advice.

That said, let me share some thoughts.

For the subjects that you're interested in, I expect you would be better off working with a conventional microscope rather than coupling an objective to your camera separately. The reason is illumination. Even for basic brightfield work, a simple condenser such as probably found on your Amscope will provide more light on subject than would be convenient to do separately. More advanced techniques such as phase contrast or DIC will definitely require the condenser (and probably a better microscope) because structured illumination is an intrinsic part of those techniques.

As for coupling a camera to your microscope, I suggest looking at the "afocal" technique in which you basically substitute a camera and its normal lens in place of your eye, looking through an ordinary eyepiece. See for example http://www.photomacrography.net/forum/viewtopic.php?p=99265 (using a trinoc head, but without projection optics) or even http://www.photomacrography.net/forum/viewtopic.php?p=56333#56333 (using a binoc head). That approach avoids a horde of difficulties that often pop up with direct connections through a trinocular head. With my own Amscope T490, going afocal through a normal eyepiece gives far higher quality than connecting a camera to the trinocular port. There is some loss of convenience, however, so if you have a scope that has a good trinoc head, using that would be a better approach for that scope.

For focus stacking there are a couple of good options. One of them is Zerene Stacker, which is software that I initially wrote for my own interests which mostly involve opaque subjects. Zerene Stacker does work well with transparent subjects also, and it has the advantage that its method of generating synthetic stereo works well with transparent subjects and arbitrarily complicated geometry. The other good option is Helicon Focus. Helicon Focus has the advantage of providing a "contrast weighted" rendering method (Method A), which with transparent subjects is less vulnerable to obvious artifacts than either of the methods provided by Zerene Stacker. The synthetic stereo method used by Helicon Focus does not work so well with transparent subjects, and in any case it is less faithful to the real subject than Zerene Stacker's method is. (See http://www.photomacrography.net/forum/viewtopic.php?p=55619#55619 for some discussion of that.) I believe that most or all of Daniel Stoupin's stereo images are made using Zerene Stacker. Jacek's postings have indicated both Zerene Stacker and Helicon Focus; I have no idea what motivates his choice about which one to use when.

--Rik
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The Cove Online



Joined: 25 Aug 2016
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PostPosted: Fri Sep 02, 2016 6:04 am    Post subject: Reply with quote

Your very welcome and credit always due to those deserving for it.

Yes I didn't quite think of the illumination part for algae. I suppose you could set up a microscope slide and place a flash through the base but not ideal as you said. The amscope eyepieces are decent quality I've found, but the optical adapter I bought from them is a joke - I took this up with them and they continually delayed responses eventually to the point where they didn't respond at all after supposedly referring my request to a camera expert who refused to acknowledge the low quality of the item. I should have known that anything cheap with optical glass is going to cause issues, but I didn't expect the quality to be that bad (primarily a lack of sharpness).

We do have phase contrast microscopes at work, so that's one piece of the puzzle done. As I said, I am keen on high quality images even if it means a long time of saving for gear, but my aim of the post was to know what I should be purchasing or saving for. Techniques is the other side, which will come with experience and hopefully others opinions who have done the same sort of content.

Is the problems with trinocular heads because they require additional optics, which are of low grade glass or some other issue? Just keen to know how are you basing what determines a good trinoc microscope?

I didn't know you made zerene! That's some passion you've got right there Very Happy I imagine there is some heavy pre-editing before the stack is stiched to ensure no complications, but don't know what this would involve since I haven't really done much of this kind of work other than HDR stacks or HDR landscape panoramas.

What kind of objective's would these images be taken through? 40x - 100x? How would the focus be adjusted - cognisys stackshot? I'm not sure how I'd go about the increments given the flat nature and already small subject size.

Looking forward to hearing others techniques on the subject matter so please don't be shy if you have anything to add!

Thanks,
John
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rjlittlefield
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PostPosted: Sun Sep 04, 2016 7:48 pm    Post subject: Reply with quote

The Cove Online wrote:
Is the problems with trinocular heads because they require additional optics, which are of low grade glass or some other issue?

Beats me. When I think about it at all, mostly I just mutter something ungracious about the competence of the designer.

Quote:
Just keen to know how are you basing what determines a good trinoc microscope?

For me it's strictly functional -- if the image captured by the camera is close to as good as what I can see through the eyepieces, then it's a good adapter. In the ideal case the detail captured by the camera will actually exceed what I can see directly through 10X eyepieces, simply because at low mag the optical image will be better than what my eyes can resolve directly, but with the captured image I can pixel-peep.

Quote:
I didn't know you made zerene! That's some passion you've got right there Very Happy

I try to keep the relationship low key, so for me it's a mark of success that you didn't know. But when I offer comparisons, I figure it's only fair to mention that the software is specifically designed to do what I like. Yes, "passion" is a good word. If I were interested in making a lot of money, I should have kept my day job.

Quote:
I imagine there is some heavy pre-editing before the stack is stiched to ensure no complications

I wouldn't describe it that way. Typical workflow is either no pre-processing at all, for JPEGs direct from camera, or uniform and generic processing such as color balancing, exposure adjustment, noise reduction, and sharpening, for raws converted to TIFF or JPEG for stacking. If the frame includes any of the microscope's field stop, then sometimes it's a good idea to crop that off so that the software can realign images based on subject details. Again, that would be done for every frame the same way. The big manual steps come after stacking, when you start retouching to fix any artifacts that were introduced by the stacking.

Quote:
What kind of objective's would these images be taken through? 40x - 100x?

The field width on slide is typically around 10-20 mm divided by the objective magnification, so yes, for the size range that you've mentioned 40X or 100X would be reasonable.

Quote:
How would the focus be adjusted - cognisys stackshot?

Turn the fine focus knob. Lots of people do it by hand. If you need automation, then it's not difficult to do a DIY connection between the fine focus knob and a stepper motor controlled by StackShot or any of several other controllers that cost fewer $$. See for example http://www.photomacrography.net/forum/viewtopic.php?t=11008 .

--Rik
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NikonUser



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PostPosted: Mon Sep 05, 2016 8:41 am    Post subject: Reply with quote

I cannot see the problem.
Easy to get superb images with an Olympus BH2 and a NFK eyepiece; simply suspend a DSLR above the trinoc.

See Charles Krebs' website for his earlier equipment; and his images, they are incredible

Here are some images of Volvox using a BH2
http://www.photomacrography.net/forum/viewtopic.php?t=10640&postdays=0&postorder=asc&highlight=volvox&start=0
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Nikon camera, lenses and objectives
Olympus microscope and objectives
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Charles Krebs



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PostPosted: Mon Sep 05, 2016 2:59 pm    Post subject: Reply with quote

Welcome aboard!

I would say a compound microscope with a properly set-up trinocular tube is your best bet.
Quote:

My current camera is a 5diii, which may pose a problem since it doesn't have the greatest megapixel count or density.
Shouldn't be a problem at all. In most possible "scenarios" you will likely be using a 2.5X projection type eyepiece in the trinocular tube. One thing you will quickly realize is that much (most?) compound microscope imaging is already well into "diffraction territory" so the pixel density demands are lower than might be expected.

I can't speak to Amscope, never used their equipment. Seems their scopes are OK, but I have never heard anything about components made to adapt a SLR to their microscopes. Fortunately, it is actually easier to get a decent 2.5X projection type photoeyepiece appropriate for a 24x36mm sensor than is is to get a a low power one appropriate for APS-C sensors. Nikon and Meiji made/make such 2.5X eyepieces, as does Olympus. (Olympus NFK series is "corrective" and should really be used only with their LB Series objectives.) If you are after high quality results you must realize that while Zeiss, Olympus, Nikon, and Leica (and some others) are all capable of superb images, the various optics may not be "interchangeably" compatible. It might be a good idea to take in inventory on specifically what is available to you ( "I believe there is also some zeiss objectives lying around without a microscope")

One thing that need to be considered at the outset is the lighting methods needed or desired. Brightfield, oblique brightfield, and darkfield are the basics and possible with just about any scope. Phase contrast, DIC, and fluorescence require additional components that may not be available for some brands. DIC sounds like something you would really find useful, but it is elusive in that older versions are tough to find (and still very pricey), and current (newer) versions are hugely expensive to set up. Nonetheless, looking ahead, it would probably be a good idea to select a system where at least this option does exist as a possibility even if it seems out of reach initially.

The optics are very important. If you are after high quality photographic images you will certainly want the option of using "higher end" objectives such as Plan Fluorites or Plan Apos. Most of the "big 4" manufacturers have extensive line-ups of objectives ranging from basic inexpensive achromats through Plan Fluorites and Plan Apos (and even highly specialized ones beyond that). Many of the Asian brands will offer one or two levels of achromats, some of which are apparently quite good, but for imaging purposes fall short of the ones mentioned above.

Selecting this equipment is, in concept, not really that difficult. But there is a huge amount to choose from and many of the details that might not be initially obvious can be important to know. It is certainly easy if there is an unlimited budget, but 99.999% of us don't have that luxury so some research on existing options is important.
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The Cove Online



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PostPosted: Thu Feb 23, 2017 4:17 am    Post subject: Reply with quote

Hello again everyone.

Just an update, I have not abandoned the micro scene. I've been coming back and forth reading what I can about the different setups people have and general information about photomicroscopy. I think I get deterred at times seeing as I find most of the formulas on magnification, aperture and similar concepts to be quite daunting. I'm certainly not a numbers man so I think this is worsened by my lack of knowledge of optics in general. I don't have too much free time to really search the forums so it's sometimes hard to find targeted information.

Perhaps I should have consulted the pros first, but I went and threw myself in the deep end and bought a 100X Mitutoyo M PLAN APO SL 0.55 objective. For some reason the idea of connecting the objective to the camera always seemed a lot easier for me then to hook it up to a microscope. It'll also allow me to do work from home and not have to fiddle with the microscopes at work (and I don't want to rely on my Amscope one at home). Maybe later down the track I'll take advantage of the phase contrast Leica's we have at work. For those reasons, I'm planning on using my camera despite the problems I may face with lighting. I did look into the working distance of the 100x objective before purchasing, which seems to rival some 10x objectives at 13mm, but whether this is really a positive I'm not entirely sure given that the resolution issues that may come about at this magnification? I remember there were two properties - resolution and something else I cannot recall that affect the ability to discern detail in the image but again couldn't grasp the concept fully. I chose the objective because of the price I got it at and the high WD so I can still light the subjects. The rated resolving power was 0.5 compared to 0.3 to the other 100x Mitutoyo (HR version), though the WD of the HR was 2mm. Not sure how the aperture difference of 0.9 compared to 0.55 impacted the quality (resolution) of the image obtained here. I figured the higher working distance was a better trade off in allowing more images to be taken for stacking though I could be missing the mark there.

I was reading up on some setups that incorporate a Raynox as a Tube Lens, though I'm not entirely sure on what the principles behind the inclusion is for compared to say just using empty tubes or bellows. Speaking of which, I don't know if it would be appropriate for use with a 100x objective as I've only seen it with 50x or less setups. This seems to be another key concept I don't quite understand yet - focal lengths and magnification. The setup I'm referring to can be seen in the link below from 'mawyatt:'
http://www.photomacrography.net/forum/viewtopic.php?t=29586

In the mean time I've went ahead and gotten some 52mm adapters since the 42mm has the potential for vignetting on a FF camera. So far I've purchased or intend to purchase:

BD M26 36tpi microscope objective to M52 Item#201643906071 http://www.photomacrography.net/forum/viewtopic.php?t=12404
55-52mm adapter (Step down Male-Female) Item # 281809543929
55-49mm adapter (Step up F-M) Item # 151826423625
Raynox 250 lens 49-43mm (F-M) Item #
43-52mm adapter (step down F-M) Item # 121591808435
17-31mm length Helicoid for fine focus with 52-42mm threads (F-M) Item # 272559616595
42-52mm adapter (Step up F-M) Item # 291834238381
NEED A 52MM FEMALE - FEMALE adapter HERE
52mm (M) - Canon EF Mount Item #220946213277
4 sets of 9, 16 and 30mm extension tubes with EF Mount Item # 320841459055

Would the first item listed from Jinfinance be ok for the Mitutoyo objective? Not sure on threads being 0.706 (Mitu) or 0.75 (Nikon) as discussed:
http://www.photomacrography.net/forum/viewtopic.php?t=12404

I can't seem to find a 52mm female-female adapter to connect the setup to the extension tubes. I'm also not sure of how many tubes I should be attaching - again comes back to my lack of understanding in calculating magnification and focal distances/aperture properties.

Still yet to be purchased is the black flocking to prevent light reflecting from the extension tubes as I'm not sure where to get this in AUS or if I'll just have to ship it over.
So what remains include:

A stage for either the camera and/or subject - ideas here? I've seen people using Newport stages, is this an alternative to a Stackshot system or are they used to move both the camera/subject? An alternative to the stackshot system I found was the Wemacro rails, which come at a much cheaper cost; not sure on flexibility or useability of their system though, but it's a substantial difference in price.

Lighting: I plan on using a pair of off camera Yongnuo/Canon 600EX-RT flashes.

Adjustable Iris: Not sure of what benefit this plays.
If there's anything else useful I should be aware of, or other general reading somebody recommends feel free to send me some links please.


Thanks,
John
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ChrisR
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PostPosted: Thu Feb 23, 2017 4:35 am    Post subject: Reply with quote

A quick small answer - 52mm females : http://www.photomacrography.net/forum/viewtopic.php?p=163172#163172 is one way. Ebay seller Rafcamera often has new designs.
Flocking - Protostar from the US - shipping is significant. I have some sticky backed black fuzzy shelf lining/pot base material (like "Fablon") which is fairly good, and cheap.
Iris - softens the transition from in-focus to fug, Try ebay Jinfinance, many options. Others do them too now, in M42 etc.

That Yongnuo flash: Be VERY certain it'll actually do what you want with a paticular Canon. Canon move the goalposts . I have Y one of that model which isn't 100% compatible despite the vendors claims. Yongnuo know all about it, but the vendor doesn't want to know because I didn't test all features for a couple of months, and it's not worth the hassle. "Upgrade" would cost too much.
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dolmadis



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PostPosted: Thu Feb 23, 2017 5:54 am    Post subject: Reply with quote

Quote:
4 sets of 9, 16 and 30mm extension tubes with EF Mount Item # 320841459055


I can't find this item by its number but are they 52mm extension tubes?

Thanks


John
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ChrisR
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PostPosted: Thu Feb 23, 2017 6:19 am    Post subject: Reply with quote

No:
http://www.ebay.com/itm/DSLR-Macro-Extension-Tube-Ring-For-CANON-EOS-50D-5D-7D-/320841459055
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dolmadis



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PostPosted: Thu Feb 23, 2017 6:37 am    Post subject: Reply with quote

Thanks. Hope the OP notes that because I think that 52's are needed.

The only source I have found is on Amazon UK, and I think .com, but personally I think that they are quite expensive for 4 sets.

Anyone have a cheaper source?

Thanks

John
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ChrisR
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PostPosted: Thu Feb 23, 2017 8:17 am    Post subject: Reply with quote

Nikon K intermediate rings are 52mm. They are very useful, especially if you have a Nikon. Often too expensive though.
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