MicrobeHunter.com Microscopy Forum

Hi folks,
A couple of images from Travis Wetland, Christchurch, New Zealand
Not sure of where they fit in the great taxonomy of things but guess the 2nd image is Paramecium.
Images with Chinese 20x and 10x phase objectives, first with one of my home made oblique stops.

Looks like Paramecium for sure! Great shots! The first one seems a bit stress as it is about to burst. Maybe because of pressure from the cover glass.

Thanks hkv,
Yes I suspect you are right about the stress on the first one, it wasn't moving.
The holes were constantly changing size and moving.
All the others on the sample were swimming freely.
It makes me wonder how thin they must be to be able to swim freely between a slide and coverslip.

Hi Grahame,
nice images!
When you apply a drop of water next to the cover slip it is sucked between cover slip and slide and your water layer becomes thicker again to keep the objects alive. It you remove some water with the tip of a paper tissue you can stop them from moving up and down in a too thick layer of water.

Bob

Thanks Bob,
What you say has just made me think.
So does a larger drop of water create a larger space between the slide and the coverslip?
Within reason.

Grahame wrote:Thanks Bob,
What you say has just made me think.
So does a larger drop of water create a larger space between the slide and the coverslip?
Within reason.

Yes, for sure. Large drop, large gap. The surface tension of the water lifts the glass sort of. I use Microbobs method as well. Add water with a pipette close to one of the sides of the cover slip and the water is sucked in under the glass and lift it. Use a piece of paper close to the side and water is sucked out lowering the glass. I use this all the time when I fail to get the resolution up during shooting. In many cases, poor resolution is due to the water film is too thick so the subject comes too far away from the surface of the cover glass. A slight push on top and then suck out the water with a piece of paper will do the trick. Especially oil immersion objectives are very sensitive to water film thickness. That is why I prefer my water immersion objectives which are much less sensitive. I often get better resolution with a 60X water immersion at NA/1.20 than with the 100X Oil at NA/1.4

Grahame wrote:It makes me wonder how thin they must be to be able to swim freely between a slide and coverslip.

An approximate answer: assume a single drop of water on the slide, fills the gap under a 22x22mm coverslip up to the rim, without overflow. The volume of a regular drop is 0.05 cm^3, or 50 mm^3. The area is 484 mm^2. That makes the liquid layer thickness 0.103mm, or 103um. Any slimmer creature will move freely.

Thanks hkv,
You have just helped make something go click in my old brain.
Basic principles but new way of thunkin.

Hobbyst46,
Thanks for the maths
I can understand that.
The "aquariums" I use are .2 mm high
Now with a big drop of water I can get .1
I'm now wondering if I have created problems for myself by using aquariums.
I do like free swimming critters.
Cheers for more things to think about

This water gap thing is among the tricks to acheive images like Håkan does.
When you just take some water with mud and sand you are far away from this resolution because the water gap will nevert be perfect for the single object you wan't to take a photo of. It's always good to have an idea about the work that is necessary for a level of image quality. Just to have the right expectations.

Hi Bob,
Yes I should probably re thunk the way I work.
But it was definitely planned.
Those 2 images took around 300 shots and 2 plus hrs to get them.
Doesn't mean I had it planned properly, possibly never do
Probably I should start sucking water out, but that will bring critters into my tissue.
I attempt to get all critters back into my aquarium jars after a foto shoot.
Lots of things to balance.
All good fun

Most of us here in the forum do microscopy as an enjoyment. Some people enjoy it to acheive the best photographs, some take a photo only for documentary purpose and just enjoy what they see it their eyepieces. It also is an ethic question : I myself will only rarely contribute to the development of science so it is a question whether I should severely damage plants or animals for my recreational purpose. Great images of one small critter, dying in the process will help the others to survive by gathering attention for them and their needs on the other hand side. So no need to get dogmatic here.

For myself I have found out that I do better when staying away from perfectionism. I got to the point that I'm already very satisfied with my work, when it is good enough for my recreational purpose. At the same time I enjoy it very much to watch the process and results of people who are determined to get better and better results. I'm thankful for being allowed to take part in that journey, and I'm always interested in the details of their methods. But only occasionally I try to follow them and rarely try to compete with them. I'm just interested in many many things but in a non-competetive way.

Seeing images from the slides of Robert Koch has helped me here - they often look as if slapped togehter by small children.

One thing should always stay in view: It is great to do something as a hobby - because you can do it just the way you want!

Bob

Hello Gramhme, and thanks for the specific mention that these ciliates ( Paramecium genus) came from a 'wetland local'. I think your half of our globe is going into: spring/summer?

Rather than thinking math formulas...simply understand that microscope objectives often are made to function with a coverslip...or are made to function with no coverslip.

We can 'cheat/ ignore the spec's of an objective...scan a slide with: 4X, 10X, 20X objectives and observe a water droplet with no coverslip, to seek targets of interest...but then to image-capture a target object/organism...it's best to use the objective specs: cover slip required.

When a coverslip is part of your wetmount slide 'sandwitch'...this is when you pay attention o depth of water column under the slides coverslip. It becomes easy to observe the 'sweet spot water film/ water column thickness'...your the observer peering with your microscope ..you see when your higher magnification objectives can focus and track a target easily...and with crisp focus.

We simply learn how much/ or how little sample fluid to have on a slide , before we plop a cover slip on this droplet..your goal is that 'sweet spot' water film to permit use of your: 40X, 60X, or 100X objectives.

Please consider use of the large rectangular coverslops...rather than the 'tiny' square cover slips. There is so much more area under a large rectangular cover slip wetmount slide...vrs a simple square coverslip.

My question to You is: did you 'photo shop' the images you posted, and which we enjoy? There is no hints of debris, only an organism crisply in focus in your posted images here.

thanks for your forum sharing! Charlie Guevara, finger lakes/US

Hi Charlie,
Thanks for the tips.
Yes we are well into spring and the variable weather that brings.
More like winter out there this morning.
Yes I use photoshop.
In my world it's been as much a part of the imaging process as the camera since I discovered it way back at version 5.0 in 1998.
Generally on these images it's only noise / scratch / dust removal, sharpening and clean up the background.