Objective Testing

[MicroMan2]

Let's discuss various ways to assess objective clarity. Resolution and "planarity" are our main focuses for this discussion. I would use Cheek Cells to assess resolution, while not as convenient or as discerning ad diatom species, it sure is an economical alternative. What are some other ways to assess planarity and resolution.

[lorez]

A blood cell is a good test for both resolution and 'planarity'. The unstained epithelial cell is also an excellent choice. Since it is almost transparent it is a good specimen to demonstrate the effect of the aperture on contrast. The diatom is fun. The presence of bacteria in a specimen is also a good test, especially if you have a bench mark specimen.

lorez

[75RR]

Microscopists have been using Diatoms since Victorian times to test Objectives. If it works ....
By the way, what do you mean by "Planarity" ?

[zzffnn]

Microscopists have been using Diatoms since Victorian times to test Objectives. If it works ....
By the way, what do you mean by "Planarity" ?

It means that the objective center and periphery focus at the same time (when center is focused sharply, periphery should be in sharp focus too, if your subject is flat and wide enough).

Most diatoms and protists are not good test subjects for planarity (with thick and small diatoms, you will never know if the out of focus area is caused by the diatom itself having variable thickness or the objective lacking planarity).

If your primary interest is protists, you don't really need plan objectives above the 20x objective, since your subjects are rarely flat/wide enough anyway.

Victorian microscopists have been using blood smear as test subject too. It depends on what your primary focus is. For planarity, blood smear (or an unstained microtomed plant thin section) is better than diatoms. For resolution / contrast / effective aperture without considering planarity diatoms work very well.

[75RR]

To test for Field Flatness I would agree that a plant sample sectioned by a microtome is probably best.
I wonder if there is an ideal plant and/or type of section that would help to show up the width of the flat field.

[zzffnn]

^ carrot would work, though it may be boring?

[75RR]

^ carrot would work, though it may be boring?

I am sure mrsonchus can come up with something better.

[Peter]

Hi All,
To test for plan (flat field) I would recommend a fixed and stained smear from a culture of small bacteria, these will readily show up any out of focus and are much thinner than most other microscopic objects.
Just my thoughts.
Peter.

[apochronaut]

^ carrot would work, though it may be boring?

You have flat carrots in Texas? Just what did BP put in the Gulf of Mexico, anyway?

[zzffnn]

It seems that you are not used to Texas or US. Big corporations can fund research to make lots of weird organisms. A pig with human heart, a mouse with human ear, pink fluorensent fish or a square watermelon, you name it. Some, such as the square watermelon, don't even need lots of funding or science/research.

[apochronaut]

Well, my carrots all have legs and sometimes sex parts too. I think they might be an interspecific hybrid, with the bunnies who eat them.

[JimT]

OK, getting funny. Plant cells (even one cell thick) are three dimensional and the wall of one cell may be (Is probably) thicker or thinner than another. Diatoms are also thick. And a carrot, forget it. Even a Texas carrot

One thing I have is a slide with a I mm scale divided into 5 UM segments. Perfectly flat and also a good reference for the size of critters.

[gekko]

I like JimT's idea of using a stage micrometer, and remember to use a reasonably large aperture to limit depth of field. Another thought occurs to me: what about focusing on a tiny "dot" in the center of the field of view, then, without changing the focus, move the stage in each of the four directions until the "dot" goes "sufficiently" out of focus? Would that work?

[apochronaut]

Hi All,
To test for plan (flat field) I would recommend a fixed and stained smear from a culture of small bacteria, these will readily show up any out of focus and are much thinner than most other microscopic objects.
Just my thoughts.
Peter.

Peter points out a really critical point both for field flatness and possibly resolution. The thickness of the sample has a major affect on an objective's performance. Printed resolution test slides are useful too. Diatoms can be tricky because of the sample thickness and as well the effect of the mounting medium, which if not balsam or some other high n medium will lower the objective's apparent performance. Older slides, balsam mounted are nice.
It would be possible to have a bacteria data base to compare to. I'm sure there are many other classes of subjects that could serve the purpose; mold spores, pollens. Thin is good. I have found the 8 form diatom slides, that are widely used for resolution, a little lacking because of the relatively thick low n medium used for mounting. Thicker species, usually used for lower resolution objectives, mounted along with smaller more detailed ones, can slide over or under each other, if subjected to the slightest accidental pressure. It can't be good.

For flatness and coma, a cheap micrometer reticle can be used, as well. Dark lines against a bright back ground, will show curvature of field and colour fringing readily.

[zzffnn]

I read somewhere that KK 8 form diatoms are mounted in RI 1.4 or 1.7 mountant onto cover slip (I cannot remember which RI was it, though 1.7 seems to be more likely)?

[zzffnn]

OK, getting funny. Plant cells (even one cell thick) are three dimensional and the wall of one cell may be (Is probably) thicker or thinner than another. Diatoms are also thick. And a carrot, forget it. Even a Texas carrot

One thing I have is a slide with a I mm scale divided into 5 UM segments. Perfectly flat and also a good reference for the size of critters.

I meant a carrot microtomed to 1 micron section. Would n't that be flat, thin and wide enough? I don't know for sure. Printed resolution targets may be better, but a dedicated one can be expensive? I remember seeing one retailed for $300 and considered myself not serious enough to use it.