My First Images with Canon DSC4000

[Roldorf]

Hi two lightly edited (LightZone) images of a mosquito my wife killed in the fridge. SLR sharpen only.

IMG_0004_lzn.jpg (141.34 KiB) Viewed 8242 times

IMG_0001_lzn.jpg (159.04 KiB) Viewed 8242 times

Are these eggs?

[mrsonchus]

Congrats on the camera - these look a good start to me.
A nice even illumination is a very promising start.
They look a lot like eggs to me also.

Keep it up.

[Roldorf]

@mrsonchus.

Thanks for the encouragement (much needed).
I have tried a self made darkfield patch from Olivers video, which seems to work quite well even on this very opportunistic slide (squashed under a coverplate with water).

IMG_0001_1_lzn.jpg (154.6 KiB) Viewed 8230 times

Still needs some work I think.

[mrsonchus]

Excellent, the detail in the row/s of hairs along the legs is showing nicely and well-focused. Great to see you progress with your imaging. Everyone here started at the beginning and gradually learned the craft, with the considerable and very valuable help and feedback from this multi-talented forum.
Coming along nicely I'd say - practice the brightfield and then follow-on to the other techniques. Once the basics of brightfield are mastered, which of course includes the correct use of a microscope and camera combination, the rest of your adventures will be based upon a good solid foundation.
An idea would be to get some prepared slides which will help with this, any price or quality really at this stage - enough to learn microscope use and configuration.

Keep up the good work!

[Roldorf]

Trying out ToupView for image stacking. A 7 image stack of pollen from a Lilly in our garden.

Stack Lilly Pollen resize.jpg (221.72 KiB) Viewed 8147 times

Seems like I have a long way to go.

But at least I can now see the whole structure of it.

Edit:- Just looked at the original images used to make the stack and there are hardly any artifacts on them. Is this a problem with ToupView? or could it be Gimp2 that I used to scale the image for submission.

Added this stack done in Picolay.

Picolay Lilly.jpg (116.73 KiB) Viewed 8136 times

[mrsonchus]

Hi again, another very good early-image! I wouldn't worry too much re the stacking performance ins-and-outs.... The main point here I think, stacking notwithstanding, is that you have clearlyu got illumination to a good standard, nice and even. The essential details of the lattice on the Lily pollen's surface are visible - the network of truly tiny (not that easy to resolve well anyway) 'lumps' or chain-like arms that form the net-like covering.
A long whaile back I too did the very same thing as I was learning the craft, took the ubiquitous and very feature-rich Lily and examined this pollen. You've definitely captured a lot of detail here old chap.

Your images here are better than you think - these are not images taken of a perfectly flat, stained-for-contrast, permanently-mounted microscope slide with cover-slip, they're raw 'pollen on a plate' type images, a very difficult subject for any brightfield 'scope.

Here's a link to my Lily pollen adventure back in 2015. I had the advantage of some chemicals and stains to help clarify the pollen structure, and only really achieved a little more than your first images here! At the time I used 'Combine ZP' free version (hence the watermark on some images) stacking program (I now use 'Helicon Focus' std version paid-for).

Stacking is hard enough to master, this type of specimen is also difficult, your results are very encouraging. I'd get som prepared slides and learn with those - thus enabling you to concentrate on the technique rather than fighting also with a difficult specimen...

Nice work, great start, interesting posts - please keep them coming as I like to see how you're getting on, as I certain many others here do...

[Roldorf]

Hey mrsonchus,
Just looked at your pollen adventure from 2015 very nice images. I wonder why the pollen I had was 'round' whereas yours were elliptical.

I edited my last post adding a stack of the same images with Picolay and cropped the image with windows `Photos`. Took a bit longer to process but they seem a little better, or is it just me.

[Roldorf]

Prepared slides would be OK I guess but I really like the adventure of going out and seeing what I can find to look at. Much more exciting.

[Roldorf]

Just went back to make a second stack and the pollen has 'DEVELOPED'. Maybe I should take it outside before it takes over the house. Anyone know what is happening (I am not a biologist) just a curious person.

Crop Pico Stack Pollen2.jpg (57.72 KiB) Viewed 8123 times

[mrsonchus]

Hey mrsonchus,
Just looked at your pollen adventure from 2015 very nice images. I wonder why the pollen I had was 'round' whereas yours were elliptical.

I edited my last post adding a stack of the same images with Picolay and cropped the image with windows `Photos`. Took a bit longer to process but they seem a little better, or is it just me.

Ah, stacking, it's a bit of a struggle to know quite how they work, even after several years I only really understand as much as I need to re stacking methods. I'd just make sure you make a note of the settings you use so you have a record when it goes well.

Your pollen may be round as many pollen types will swell, become rather more spherical than when on the plant, disguising their more distinctive shape, when they are placed in liquid, usually just water, for observation. The effect you see in the later images is a bursting of the grain and consequent spillage of internals - sounds awful!

My stained images are I think of 'fixed' pollen grains caught-up in my preparation of tissue for sectioning, remaining quite close to their natural form as fixed plant tissues ideally will. I think the methylene-blue stained pollen was stained in water, not sure now.
Basically the best approach at first is to try a few methods, as each type of pollen may react differently to the next....

Try dry, onto slide and observe, in water with coverslip on top, with and without stain of with different types etc.... There really is no formulaic method that works with all specimens - the fun and satisfaction is in the exploration, and of course the sharing here of your exploits!

If I get a chance I'll make a short video or post of a few basic techniques that are able to give good imaging quickly from fresh plant material.

Keep it up - very enjoyable to follow your adventures.

[Wes]

I really like the darkfield image! Also the second pollen shot is nice.

What microscope do you use and how do you couple the camera to it?

[mrsonchus]

Hey mrsonchus,
Just looked at your pollen adventure from 2015 very nice images. I wonder why the pollen I had was 'round' whereas yours were elliptical.

I edited my last post adding a stack of the same images with Picolay and cropped the image with windows `Photos`. Took a bit longer to process but they seem a little better, or is it just me.

If you're running Windows 10 I'd recommend Windows-Photos too, [edit & create]->[filters]->[enhance] as a really fast method of sharpening and enhancing an image - I very often use this as it saves loading Photoshop and gives very good results indeed. I used to regard such 'built-in' facilities as second-best, but this version with Windows 10 is very good indeed and only takes seconds.

More please!

[Roldorf]

Hi Wes,

Thanks for the compliment.

To answer your questions:-

1. The microscope is a Bresser Science Infinity with a trinocular head, so the camera goes into the photo tube (trinocular head part).

2. The camera is a longer story.
I first bought a 5 mp USB microcam from Bresser shortly after I bought the microscope as I had wanted to try to fix an old Canon camera which had a live view function on the photo port first. Unfortunately that didn't work and I made do with a Sony DSC RX100 by holding it on one of the eyepieces for pictures for reference. My reason for wanting a camera on the port was to view images on a screen rather than having to look through the objectives all the time. Also there are two of us (my wife) who have the same hobby and can only afford one microscope so instead of constantly swapping chairs I thought it would be good to have a separate screen.
The microcam was O.K and did its job 'but' I thought it would be nice to have a connection by wifi and to be also able to display the image on a much larger screen like my TV which has 4 HDMI inputs.
I came across an advert for the Canon DSLR 4000 camera (18 Mp) and it had everything I needed for the scope including live view shooting. Lots of microscopy websites recommended it as a good camera for microscopes and as it is to be a permanent fixture on the scope the fact that it had a reinforced plastic lens mounting didn't really matter.
The deciding factor was the price. I bought it new as a special offer including lens and bag for 249 Euro.
I had thought it would be an easy matter to connect the camera to the trinocular tube with a T2 ring and bought one for a canon, and that is where the story really begins.

To follow on and hear more about this you need to start reading here:
viewtopic.php?f=9&t=7628

Hope you find it interesting and not as frustrating as we have found it.

[75RR]

Your last image: Crop Pico Stack Pollen2.jpg came out nicely. What are the differences between that one and the previous ones?

[Roldorf]

Hi mrsonchus,
I will try a dry mount later on today and see how it turns out.

[Roldorf]

Hi 75RR,

The second one was taken with the same settings but only has 4 images in the stack as opposed to 7.

I also used the 'Olympus Background Subtraction Toolkit' to try to get an even background without so much noise.
That was the only post processing I did other that the stacking with Picolay.

I was a bit disappointed with it though. It has only a very small window to work in and trying to pick points for the resulting background is a pig in a poke. You have no idea which colour you are picking until the resulting background comes up on the second image window, as you are not able to zoom in.

I think I will have to install Photoshop on this laptop. It's a very old version from 2006 so I will have to see if it has the stitch option on it.

[mrsonchus]

Hi Roldorf, why not give the free 'trial version' of either Combine-ZP or Helicon Focus a try - that's what I did and eventually decided to treat myself to the basic paid-for version of Helicon Focus.
A tip, where choice of stacking method is available (usually three types - 'Depth Map', 'Weighted Average' or 'Pyramid') choose the 'Pyramid' option.
The other two also have settings for say, width of feature to be selected, level of smoothing - these are OK but counter-intuitive.
I find for example that a 'finer' detail width of perhaps 5 will actually not stack finer details (e.g. pollen-grain exine spines) as well as setting this to 35 - a 'less-fine' setting?.... I thought (intuitively at least) that 'finer' meant finer details in the result - but it appears to mean finer 'blur' will be selected in the source images and so the higher 35 setting will stack select and stack less-than razor-sharp edges in the source images - far more appropriate for photomicroscopy I find.

The 'Pyramid' option doesn't have these width and smoothing settings - it's just selected and set-off. The Pyramid setting (as the sofware's built-in advice pop-up says) is very good for images with overlapping features such as the semi-transparent plant tissue, especially in brightfield images....
I find the Pyramid stack in Helicon Focus to be the best of all.

The above said, Toupview's 'maximum contrast' setting is also nearly always very good indeed.
Image selection for stacking is important, and not always benefitting from a high number of images for stacking - this can introduce so much overlapping detail that a stacked image becomes confused and not representative of the actual specimen - this is definitely the case for pollen. I as you, started at the very beginning with pollen, as it's ubiquitous, attractive and just plain convenient I suppose.

Good luck, you're going well.

Explanation re pollen stacks of semi-transparent/translucent pollen -
it's important to 'start at the top' with pollen-grain focus - i.e. the point highest - nearest to the objective - and stop when you get halfway-down with your stack images - as otherwise you'll begin to add images that will overlap and make the stack poor or at least innacurate....

[Roldorf]

Thanks for that I never thought about only going halfway down but that makes perfect sense after all we don't have x-ray vision.

I tried your suggestion of not using water and this is what I got.

A single image no stack Lilly pollen grain Latest version of Gimp (2.10.12 just downloaded and installed) to scale image. Will have a look to see if it has stacking.

40x lilly pollen scaled.JPG (107.22 KiB) Viewed 8045 times

And lo and behold they are not round.

[mrsonchus]

Haha! Now that's a beauty! You're off & running here!

Try only about 3-5 images layers, only to halway of course, and see how you get on....

Nicely done my friend, looking good.

p.s. Some of the harder pollen-grains (or at least less inclined to 'suck-up' water) will not explode or distort in water - for example the Dandelion, Sonchus, Milk-weed species have a pollen-grain with a very hard 'fenestrated' grain with lovely prominent spiny ridges, and unlikely to distort in water.
Keep the pollen on the anther while staining - just dip the whole thing in stain or leave the whole thing (anther or stigma with pollen on - not whole plant) in stain, rinse by immersing in clean water, then the pollen-grains may be prodded off of the anther/stigma onto a slide - this technique enable pollen to be handled without losing it all....

Great work, keep it up, watching with interest and enjoyment.

[Roldorf]

I shall forget stacking with Gimp I haven't been able to get it to work. Maybe someone else on the forum will have a go. It is very nice at editing probably as good as Photoshop with the latest version 2.10.12. This is a 7 image stack of the last pollen grains, dressed up in MS picture and sharpened and background cleaned in Gimp. This is the last effort on the Lilly. Want to try something else tomorrow.

Darkfield Lilly 1-7 crop edit.jpg (379.51 KiB) Viewed 8033 times

[Roldorf]

The outer skin of a garden pea pod showing stomata in pod.

[mrsonchus]

Another nice image.
The two valves of the pod are thought to be modified leaves, the outer surface would be the lower surface of that leaf.
Interesting to see the arrangement of the cells surrounding the stomate guards-cells also, as they're of many types and are often taxonomically significant...

Great image, interesting subject too!

[Roldorf]

Yes I had never thought to look and had a visitor today who wanted to look at something under the microscope only thing I had to hand were some peas out of the garden (which I had been eating raw Mmmmm) so managed to peel off a piece of the outer skin.

[mrsonchus]

That my friend is the spirit of inquisitive-investigation that has led us all here!

Well done!