MicrobeHunter.com Microscopy Forum

[/size]Hello all! I recently arrived back home from a family trip, and my first thought was to play with my new stand! It is a trinocular AO Series 4 stand set up for phase contrast. With it I purchased a Minolta microscope adapter and a Canon 40D. The setup is pictured below.

I spent the better part of my day just figuring out the best way to set up the microscope adapter, deciding to opt out of the barrel to bring the Camera closer to the microscope. Here are some of my first photomicrographs!



While excited that i can take more professional images than through my phone, I am still unsatisfied with the quality of my images and would like the opinion of the community as to what I could do!

-Adnan

one additional image i forgot

Sorry ... I can’t see any of your images

MichaelG.

Your images are not visible.

Strange....they are visible to me. Let me fiddle around

edit: i think i fixed the issue, i hope it works!

Now I can see your pictures!

-Your camera is just fine, you should set it to silent mode and release from live view to have a shock free release.
- Is you camera parfocal to the eyepieces?
- Is your water layer as thin as possible?
- Is your condenser aperture and height set right?
- What eyposure times and ISO setting did you use?

Bob

Timemaster1212 wrote: ↑

Tue Jun 23, 2020 1:50 pm

Strange....they are visible to me. Let me fiddle around

edit: i think i fixed the issue, i hope it works!

It does indeed ... Thank You

MichaelG.

You really did a great job of isolating the organisms. Do you have a photo-processing program? You can increase sharpness, contrast and try to get a brighter background.

MicroBob wrote: ↑

Tue Jun 23, 2020 4:55 pm

Now I can see your pictures!

-Your camera is just fine, you should set it to silent mode and release from live view to have a shock free release.
- Is you camera parfocal to the eyepieces?
- Is your water layer as thin as possible?
- Is your condenser aperture and height set right?
- What eyposure times and ISO setting did you use?

Bob

So, let me tackle these in order
-My camera is somewhat parfocal, just small minor adjustments are needed and for that i use the live view on EOS
-My water layer was plenty thin, i suspected this at first and used tissue to take some water out
-I believe i set up my illumination correctly according to what i have read on kholer illumination
-now this was VERY experimental i tried to find a balance between ISO and shutter speed. i believe i was in the range of 1000 iso and 1/600th of a second for shutter speed. Today I focused more on this and the images do not seem to be too much better with a lower iso and a slower shutter speed.


These are all raw (not the format) photos straight from the camera. I have yet to search for editing software. I would like my pure images to be more or less well shot before i edit.

So these points are ok.
One important point is exposure compensation. The camera meters are tuned towards an 18% grey medium brightness. This doesn' t work when a faint object in water is photographed. Here you would have to dial in +1 or +2 stops. This would make better use of the cameras chips dynamic. More advanced cameras allow to adjust the JPEG-engines settings. In case your images fill only a small part of the histogram you could increas the contrast.
It also is a question of expectations. Most people show only their good shots. For zooplancton DIC and flash would be ideal plus the patience to use a 60:1 or 100:1 oil immersion objective.

So with this compensation what else should i do to increase image resolution? The live view shows a much sharper image than the actual photos being produced. So i am at a loss at this moment. I thought it may be the phase objectives but when replaced with a normal 43x, the image is nearly the same.

Your last images would alredy be a lot better with the exposure compensation. But you would run into a problem with the ISO vs. shutter speed. So ideally you would need more light.
Phase objectives somtimes work well for bright field, sometimes not. The tendency is that stronger phase objectives work worse. So you might do your tsting of the camera setup with a weaker objective to take this out of the equation.
Generally I think you are on a good way.

Forgive me. I copied your image and played a little with my processing program.

I would suggest that you fine tune your setup with a prepared diatom slide.

There are too many variables in a wet slide to do this properly.

DonSchaeffer wrote: ↑

Tue Jun 23, 2020 6:54 pm

Forgive me. I copied your image and played a little with my processing program. otherguysimage1.jpg

I do not mind at all! May i ask what you use? This already looks much better than the original imo!

75RR wrote: ↑

Tue Jun 23, 2020 6:56 pm

I would suggest that you fine tune your setup with a prepared diatom slide.

There are too many variables in a wet slide to do this properly.

This was what i was thinking. I just need to purchase one. Do you have a recommendation?

check out https://faststone.org/FSCaptureDetail.htm
This is a very convenient screen capture program which allows some very effective improvements in the image. I also use Corel Printshop Pro. I use this instead of Photoshop. It's much cheaper and allows you to own the software and update it as you wish.

Does the adapter have optics inside it or is it an empty hole? For some reason your camera is clearly giving out images that are way too dark.

no it does not, i actually suspected that it might be this and took out the only lens in the adapter that connects the minolta microscope adapter to the canon body. It actually made images sharper and a tad brighter, but that is it. This is why i am at a loss However it is fun to experiment! Thank you all for your help.

Does the microscope have a light adjuster for how much light goes to the trinocular port vs the eyepieces?

The series 4 trinocular, at least the one i have, has a push/pull lever that lets me direct the light either for my binoculars or for my photo tube.

Congratulations on your first attempts.
One thing is certain that your photos are too dark. I suggest you change the light in the microscope for a stronger one. Try to play with different white balance settings. I also suggest using the smallest ISO 100-400. I also allowed myself to copy your photo and play a bit in PS.I hope you don't mine.

No i do not at all. This too looks better than the original, however it is still lacking crispness. Maybe at this point the objectives do matter? That image was shot at 43x, so resolution should already be low correct? If so would, for example, an APO objective perform remarkably better?

Timemaster1212 wrote: ↑

Wed Jun 24, 2020 2:38 am

No i do not at all. This too looks better than the original, however it is still lacking crispness. Maybe at this point the objectives do matter? That image was shot at 43x, so resolution should already be low correct? If so would, for example, an APO objective perform remarkably better?

“Remarkably” is probably a bit too much to expect. Member Apochronaut has done some very nice comparisons if you do a little searching. This is one that shows some real world differences, but I’m sure I’ve seen others, as well.

viewtopic.php?f=5&t=4420&hilit=40x+objective+comparison

Ah that’s interesting! I’ll keep practicing, and in the mean time look for a diatom test slide, i think this can help me fine tune a set up.
Edit: the only issue is sourcing one

Good images are all about getting your ducks in a row.

Good and plentiful light is a must. If the source is halogen or tungsten then a blue filter is required.

If your 43x objective has a Numerical aperture (NA) much over 0.65 then the thickness of the cover glass can be critical.

Köhler must be setup correctly http://zeiss-campus.magnet.fsu.edu/tuto ... flash.html

If your microscope is not Köhler capable then make sure the top of the condenser is just 1 or 2mm below the slide. (Medium and high power objectives)

and the camera must be parfocal with the eyepieces. viewtopic.php?f=15&t=882

I source my diatom slides from Klaus Kemp (in the UK) Unfortunately he has a backlog at the moment and is not taking any orders for now.

http://www.diatoms.co.uk/

https://www.diatoms.nl/

http://www.diatomshop.com/

Ideally (long term), you would want a Test Slide, something like the one below.

But in the meantime and at a pinch, you can use just about any second hand diatom slide bought on Ebay.

What you want is a consistent repeatable subject with fine detail.

One that will let you compare results over time.
.

I've been experimenting with a relatively cheap microscope and camera setup myself. I found that having some sort of lens in between the camera sensor and objective can be very beneficial, but also found that my phone camera ended up with far better results. An afocal setup with my mirrorless worked best, but I really could not find a way to mount it reliably. I've seen excellent results with microscopes using photo-projection eyepieces, but that's out of my current price range. Right now I'm using this adapter which produces decent results and offers an approximate 10x magnification. There is some heavy field curvature on the edges though (even with a plan objective), so cropping is necessary for me.

That being said, post-processing can really take an image to the next level. I also tried my hand in Photoshop with one of your images with some success. I prefer Seb28's result though. He did a great job with the noise reduction and removal of chromatic aberration. Keep us posted on how your troubleshooting goes, would love to see how it turns out.

Timemaster1212 wrote: ↑

Tue Jun 23, 2020 5:58 pm

have yet to search for editing software. I would like my pure images to be more or less well shot before i edit.

Hi,
it looks like we have all been 'playing' with you images !
So I thought I would have a go with GIMP https://www.gimp.org, freeware for both Windows and Linux. (leaving more funds for the microscope etc ) You have maybe found it in your searchings by now ? Something to try while you work on it all being "well shot".

I am no expert here are my humble efforts ,
brightness, contrast, curves and some sharpening (unsharp mask and wavelets) Also a Gaussian blur high pass filter in greyscale layers to remove some of the background gradients which became rather intense after all the furtling !

Wow this is great! It seems i have a lot to do! I appreciate all the helpful tips and i love seeing people mess with the images haha. I believe a better light source and possibly figuring out the adapter set up with diatoms will be my next step. In the mean time I’ll try my hand at photo editing. I have indeed heard of gimp and even dabbled with it for past school robotics projects.

Timemaster1212 wrote: ↑

Tue Jun 23, 2020 7:05 am

one additional image i forgot

Whilst you are dabbling, and folks are clearly interested in messing with your images ... Could you perhaps provide a tiff file, or a RAW file from the camera, so that we can look at the channels.
This image in particular shows some obvious colour fringing, which I would like to investigate further.

MichaelG.