MicrobeHunter.com Microscopy Forum

If you look at DIC images of yeast they are maybe the best you could dream for:
https://www.youtube.com/watch?v=PTGYLYru2Sw

My question: what are the small, round and fast moving objects between the yeast cells?

Leitzcycler wrote: ↑

Tue Dec 26, 2023 7:51 pm

If you look at DIC images of yeast they are maybe the best you could dream for:
https://www.youtube.com/watch?v=PTGYLYru2Sw

My question: what are the small, round and fast moving objects between the yeast cells?

Yeast use multiple methods of reproduction. Budding is well known where they undergo mitosis, producing a stand alone daughter cell, a clone that quickly separates.
The daughter cell doesn't always separate though, and under certain conditions will stay attached, subsequently budding itself and so forth creating a colony with many cells still attached. It is kind of like mycelium but comprised of globular sections rather than longitudinal hyphae. Possibly what hypha evolved from. Yeast are fungi but considered fungi imperfectii.
The third mode is sexual. In this mode the cell undergoes mitosis and then each division, meiosis , producing 4 individual haploid cells ; 2 a haploids and 2 alpha haploids. These are called spores and are much smaller than a yeast cell, free ranging and any a and any alpha can sense mating factors from the other type, grow a protrusion called a shmoo towards it and mate to produce a diploid with possibly a different genotype than their parent. If they didn't mate with their sibling.
Most of those smaller bodies that are undergoing more rapid brownian movement than the yeast cells are probably haploid yeast cells although a few are so small that they might be cocci of some sort.

Theoretically a yeast strain, shouldn't be that consistently different from another strain of the same species if there is sexual reproduction going on. Is it possible that one of the hallmarks of cherished yeasts in brewing and baking are that they have had the genes that control sexual reproduction extirpated, allowing their keepers to maintain valued consistency?

Thanks, another question:

bodies that are undergoing more rapid brownian movement

What about the movement of the organelles inside the yeast cells? Is that brownian movement as well or rather a result of cellular function and metabolism? Or both?

I wouldn't think much of it, due to the chemical and electrical organisation of the cell but thermally driven random motion might be there some. The cytoplasmic viscosity would be pretty controlled and maybe more resistant to it.
I certainly seldom see what I would think was brownian motion in a cell but it might be hard to determine if it is or just part of the cellular workings, since much of that and for many cells is only being slowly revealed.

What about the movement of the organelles inside the yeast cells? Is that brownian movement as well or rather a result of cellular function and metabolism? Or both?

Here is a timelapse video, taken in light-grey darkfield; a dry darkfield condenser under a 1.25 oil Lomo objective (it covers about 15 minutes of real time):

https://streamable.com/mgelak

[Used sticky tape to hold both ends of the coverslip ! ]

There are some cells that show very little internal movement ..so maybe it is mostly metabolic.

..failed darkfield is not such a failure. I wonder if a ~90x phase annulus with an oil objective might work similarly ..

I wouldnt have tried it where it not for zzfn's COL suggestion and osterport's green filter made things look better

I think more likely, use a 70 to .75 objective. with your dry DF condenser. Maybe even less.
For phase diaphragms. They can provide good DF with objectives of considerably lower N.A. than they are designed to provide phase wiith. In the systems I use, I get good 10X DF with the 40X diaphragm and passable 20X DF with the 97 or 100X diaphragms. The 20X is a little on the dusk side but all of those 20X objectives are .50 N.A. I suspect if they were the more common .40 or even .45 it would be better.

I think more likely, use a 70 to .75 objective. with your dry DF condenser. Maybe even less

The dry darkfield condenser made a good annular illuminator for the 1.25 oil objective.

The yeast cells dont seem to be the right target for darkfield.

Quite a nice set of three photographs in the middle of this Miscape article:
http://www.microscopy-uk.org.uk/mag/art ... .html#ref2

Some interesting links at the bottom of the article too.

Chas wrote: ↑

Fri Dec 29, 2023 12:49 pm

I think more likely, use a 70 to .75 objective. with your dry DF condenser. Maybe even less

The yeast cells dont seem to be the right target for darkfield.

Yes. Yesst samples can look like a serving of tapioca, when not thin enough or when lit with too much scattered incoherent light. Outside of BF, which can be all over the place, each lighting technique is highly engineered to provide reasonably predictable wavefronts that are used to define the details of the sample, and yeast cells emerge more resolved and defined when those techniques are directed towards it with the precision built into their design.
Occasionally, some benefit can be found in hybridizing techniques but one risks the possibility that one will cancel the other, rather than be additive. Rarely does the latter happen. An assessment should include whether any details are available that were unnavailable from either of the contributing techniques or are there enough cummulative details that the hybrid technique seems in itself an improvement over either of the contributors. Yeast is actually a pretty good tool to assist in illumination tinkering because it reveals failures quite easily.

apochronaut wrote: ↑

Tue Dec 26, 2023 11:03 am

An iris on a 40X is not uncommon but usually it is on objectives with higher N.A.s. Reducing from .65 will quickly result in a loss of resolution. This brand that you reference , Leichert. I have not seen it and a 40X with an iris is unusual. I know Reichert had a 40X with iris.
It is very difficult to add an iris. Stand alone small irises of various sizes do exist but you would have to find a location in the optical tube to fit it. Putting it in an objective would be tricky,
Prior to the advent of plan optics, when the objective lens stack was all down in the lower part of the barrel, some companies had a two piece barrel. A lens section and a distance piece that threaded into it to position the lens section the correct distance from the nosepiece in order to establish the correct parfocal length. Leitz had an optional iris section that just threaded in to replace that distance piece, turning any so fitted objective into an iris objective. Irises were sometimes used in Pol microscopy as well as in DF.
You can get a similar effect by reducing the N.A. of the condenser with the condenser iris. I often use the condenser iris when I am viewing through PC but usually as a method of increasing contrast with certain subjects. It would follow that depth of field would increase some too.

1st day of 2024, I got some improvement with an immersion condenser, the image quality got much better:
1. Following suggestion from apochronaut, I narrowed down the Keole illumination, the DoF is much improved, where you see not just the dark mess in the middle, but some details from completely dark to light dark, see the yeasts in green box. For big yeast, it's still quite dark inside ( too thick? )

Then I dip a drop of oil onto the condenser, the image is much improved. looks like NA 0.75 should pair with higher NA condenser:
1. You can see a lot more details in the yeast cell
2. every yeast cell somes with darker core inside and some light dark area surrounded.

Today is good day, let's enjoy it! I wish everyone here has got a great 1st day in 2024!

Enjoy the video ( better play in 1080p)

Leitzcycler wrote: ↑

Wed Dec 27, 2023 9:04 am

What about the movement of the organelles inside the yeast cells? Is that brownian movement as well or rather a result of cellular function and metabolism? Or both?

That might actually be measurable if you can resolve the organelles finely enough: if there's a mean displacement proportional to the square root of time, then the motion could be Brownian. But you'd have to do the measurement over many cells, and you'd have to account for the relative motions of the cells themselves. This also assumes no interaction between organelles.

Not being a biologist, is there a spatial relationship between organelles, or can they be distributed uniformly/randomly?

Looking at some recent papers online, it sounds like there's a very clear transport process inside cells, and that the inside of a cell is nowhere close to being homogeneous, which is a fairly key assumption of Brownian motion. Actin filaments, microtubules, etc. - there's a whole microstructure to the cytoplasm that they didn't teach us about in freshman biology

So it's _very_ unlikely that you'd see Brownian motion of the organelles, but you could do the experiments if you so chose.

We really live in a golden age: 20 years ago, I'd have had to go to a university library to read about this, digging through papers from a bunch of different journals. I can do it on my cell phone now

Edit: here's a link to Nature's site - https://www.nature.com/scitable/topicpa ... -14052932/

which gives the size of microtubules at ~20 nm, so you'd need an electron microscope to resolve that.

To go back to the original question , in analyzing phase contrast performance based on many yesrs of utilizing various versions if it, the biggest sdvantage of p.c. is that it maximizes the N.A. of the objective. It can't create something out of nothing but it does unmask the vision of the objective, assisting it to see through occluding factors that stop the objective from achieving it's full potential.
The first is illumination coherence. Phase is designed to do that.
The second of these is contrast enhancement.. Contrast is seriously degraded by numerous optical defects. Phase contrast is blind to many of those defects.
The third is maximization of resolution.Objectives have a peak resolution capacity determined by the N.A. but in order for that to be realized, a number of debilitating optical problems must be neutralized, principally scatter and interference. Phase removes much of that.

Objectives in general are manufactured in categories. Roughly, these are and have been since the 19th century : student, general purpose, laboratory, advanced laboratory and research. Although this is very vague and the actual grades are much more refined and specific, this rough classification serves to make a point and that is that appllying phase contrast to an objective, will accelerate it's capability up to the next tier of classification.
For instance a 40X general purpose objective is .65 N.A. and an achromat.The next level of objective would be .85 N.A. and also an achromat. Usually this incresde in N.A. yields better contrast, resolution and colour correction at the expense of depth of field and working distance. Adding phase contrast capability to a 40X .65 achromat objective increases both it's contrast and resolution up to the point that it peforms about as well as a good perfoming 40X .85 objective, with two advantages. It will have slightly better depth of field and slightly greater working distance. Phase also seems to provide slightly improved colour correction, so the normal colour correction benefit usually found with higher N.A. objectives is matched by the better phase performance .
Generalky, phase will highlight details better than the higher grade objective too, although in actuality it has no better resolution.

I have an iris that is inserted between the objective and turret. It was made for Leitz objectives. I have never actually used it but such devices do exist, although quite rare.

einman wrote: ↑

Mon Jan 15, 2024 3:51 am

I have an iris that is inserted between the objective and turret. It was made for Leitz objectives. I have never actually used it but such devices do exist, although quite rare.

In the days before plan was an expected feature of an objective, most objectives of under 8mm focal length or roughly 20X and up magnification had their optical stack in a very short barrel.only about 10 to 20mm long. That optical section when joined to an empty parfocalizing section provided the needed parfocal length. Leitz threaded the optical section to the parfocalizing section. Their DF condenser kits had an extra device with it's own fitted port in the case as well as the oil immersion condenser. You could buy a kit including either a parfocalizing section with funnel stop for your intended Leitz objective or a parfocalizing universal iris section. These would have been used likely with any objective over about .90 N.A.for DF

Presumably you could buy the part separately too.

It seems like the price of entry for phase contrast these days is about $150, with a full set of objectives, annuli, and phase telescope coming in at $250 or so. Does this seem right? Any better deals to be had?

That depends on the microscope involved and whether you are looking to acquire new equipment or used equipment but in general, I would be surprised if someone could find a condenser in good shape with 4 diaphragms, 4 good matching objectives, and a pc telescope for much less than 250.00 . Maybe , if already on a used microscope, adding in the value of the stand. Even 4 decent BF objectives and a quality centering condenser should be that much and certainly if you wanted resolution and contrast that could nudge phase contrast's capability.