MicrobeHunter.com Microscopy Forum

While trying to figure out what to get for dinner I noticed they sell yeast cubes for practically peanuts in the store. I figured yeast alone are probably a bit boring but how about feeding them to one of my cultures and see who or what likes to eat them. Apparently anything with a mouth big enough was stuffed with yeast a couple of days later. Here I show you an amoeba I thought looked pretty, notice in the second shot a corn starch granule in a large vacuole and on top of it a single yeast cell in its own vacuole (this particular culture was fed with a grain of boiled corn several weeks ago). You can see that during the process of digestion the yeast contents get emptied and the cell wall remnants get digested later. There is quite a difference in their brightness as seen by phase contrast.







DIC images taken with a 40/0.95 apochromat, phase contrast image taken with a Ph2 Neofluar 40/0.75

great images. i guess yeast are the potato of the protistatariat.

Very nice images Wes and a cool description !

Nicely imaged!

You can see that during the process of digestion the yeast contents get emptied and the cell wall remnants get digested later. There is quite a difference in their brightness as seen by phase contrast.

An interesting comparison, I find that phase tends to get ignored when one gets DIC - this clearly shows that it shouldn't be.

Many thanks apochronaut, mintakax and 75RR!

75RR wrote: ↑

Thu Oct 10, 2019 2:57 am

An interesting comparison, I find that phase tends to get ignored when one gets DIC - this clearly shows that it shouldn't be.

I completely agree, when I got all the components for setting up DIC I ignored other illumination techniques. Its interesting to re-discover phase contrast and darkfield. Certain objects like tiny grains and bacteria are somewhat better visualized with phase contrast in my experience.

Very attractive and educational photos.

Wes wrote: ↑

Thu Oct 10, 2019 6:17 am

Many thanks apochronaut, mintakax and 75RR!

75RR wrote: ↑

Thu Oct 10, 2019 2:57 am

An interesting comparison, I find that phase tends to get ignored when one gets DIC - this clearly shows that it shouldn't be.

I completely agree, when I got all the components for setting up DIC I ignored other illumination techniques. Its interesting to re-discover phase contrast and darkfield. Certain objects like tiny grains and bacteria are somewhat better visualized with phase contrast in my experience.

One of the problems with phase, is that it's development became ignored by most companies, once it had matured enough, and a dark phase medium contrast version became more or less established as a norm. That's what you get, if you buy a " phase contrast system" , unless it is otherwise designated.
The reason for that is probably due to that version's valuable application in life sciences but for many other applications, it is not as usefull as such types as bright, anoptral, or B Minus.
The undesirable halo and shade off ,that are the targets of a lot of complaints, probably seemed difficult to insurmountable at one time and in the face of rising interest in D.I.C. and then the emergence of Hoffman modulation and confocal, r & d money that might have gone to phase at one time, wasn't there.
Only a couple of companies, actively continued the evolution of phase in a limited way and offered more options than a 4 or 5 objective spread in dark phase but 75RR's point wasn't lost on some of them and I think there has been a resurgence in development. The halo problem seems to be getting tackled, with apodized phase and by hitching phase to more colour corrected objectives as well as
It would be nice to see the AO Polanret concept picked up again and tweaked, where both the amplitude and frequency are adjustable on a continuum, rather than the system relying on the static matching of plates.
Phase does offer a rather flat image but it can be fiddled with to squeeze some relief out of it. It does offer a more real representation of the image than D.I.C., which in addition to putting up fake relief, always looks like something is struggling under a freshly applied layer of latex paint. Please, at least sweep the wall free of critters before you paint it.
Another contrast enhancement technique, that of slit condensing( somewhat akin to Hoffman or rather, Hoffman is a modified form of slit condensing) got short shrift as well. I only know of one company that offered a comprehensive slit condensing system. Then there is COL, oblique and Rheinberg. A lot can be done , fiddling with condensers, stops and polarization.

apochronaut wrote: ↑

Thu Oct 10, 2019 12:49 pm

...Phase does offer a rather flat image but it can be fiddled with to squeeze some relief out of it. It does offer a more real representation of the image than D.I.C., which in addition to putting up fake relief, always looks like something is struggling under a freshly applied layer of latex paint. Please, at least sweep the wall free of critters before you paint it.
Another contrast enhancement technique, that of slit condensing( somewhat akin to Hoffman or rather, Hoffman is a modified form of slit condensing) got short shrift as well. I only know of one company that offered a comprehensive slit condensing system. Then there is COL, oblique and Rheinberg...

For quantitative analysis of living cell images, regardless of the relief "satisfaction", phase contrast can be better than DIC, notwithstanding the halos, due to the high contrast between cells and surroundings as well as to the flatness of the image.

Stunning!

I believe these are Nuclearia (from a sister group to the fungi). Apart from the overall shape, which they share w/ vampyrellids, the adhering bacteria are a clue: Nuclearia typically has a mucus sheath studded with embedded bacteria, small flagellates and other debris.

Hobbyst46 wrote: ↑

Thu Oct 10, 2019 1:27 pm

apochronaut wrote: ↑

Thu Oct 10, 2019 12:49 pm

...Phase does offer a rather flat image but it can be fiddled with to squeeze some relief out of it. It does offer a more real representation of the image than D.I.C., which in addition to putting up fake relief, always looks like something is struggling under a freshly applied layer of latex paint. Please, at least sweep the wall free of critters before you paint it.
Another contrast enhancement technique, that of slit condensing( somewhat akin to Hoffman or rather, Hoffman is a modified form of slit condensing) got short shrift as well. I only know of one company that offered a comprehensive slit condensing system. Then there is COL, oblique and Rheinberg...

For quantitative analysis of living cell images, regardless of the relief "satisfaction", phase contrast can be better than DIC, notwithstanding the halos, due to the high contrast between cells and surroundings as well as to the flatness of the image.

There has been a lot of preliminary work to standardize some practices. An old post here, with a bad photo of the rather crude older document but it serves to support the point.
viewtopic.php?f=15&t=5032

Hobbyst46 wrote: ↑

Thu Oct 10, 2019 8:41 am

Very attractive and educational photos.

Thank you Hobbyst46.

Bruce Taylor wrote: ↑

Thu Oct 10, 2019 1:54 pm

Stunning!

I believe these are Nuclearia (from a sister group to the fungi). Apart from the overall shape, which they share w/ vampyrellids, the adhering bacteria are a clue: Nuclearia typically has a mucus sheath studded with embedded bacteria, small flagellates and other debris.

Many thanks for the ID Bruce! Its great that you frequent this forum as I get to learn a quite a bit about the protists I encounter thanks to your killer ID skills.


Speaking of illumination techniques I find relief phase contrast interesting

http://zeiss-campus.magnet.fsu.edu/refe ... 9-2007.pdf

Haven't yet had the patience to properly explore, maybe someone who has can chime in.

Wes wrote: ↑

Thu Oct 10, 2019 10:42 pm

Speaking of illumination techniques I find relief phase contrast interesting

http://zeiss-campus.magnet.fsu.edu/refe ... 9-2007.pdf

Haven't yet had the patience to properly explore, maybe someone who has can chime in.

Had a quick look. I think calling it Relief Phase is a misnomer.

Looks like plain old Oblique using Phase objectives to me.

Wes wrote: ↑

Thu Oct 10, 2019 10:42 pm

Speaking of illumination techniques I find relief phase contrast interesting

http://zeiss-campus.magnet.fsu.edu/refe ... 9-2007.pdf

Haven't yet had the patience to properly explore, maybe someone who has can chime in.

Thanks for posting the link! It is pleasuring reading material, displaying nice tweaks of phase contrast, but somewhat distant from the present post, so maybe worth a separate post in Illumination Techniques.

Beautiful! I don't have DIC and have only recently started investing in phase contrast additions to my vintage Leitz microscopes. These phase contrast photos have definitely given me something to aspire to.

75RR wrote: ↑

Fri Oct 11, 2019 6:23 am

Looks like plain old Oblique using Phase objectives to me.

You are basically right, its a combination of the two.

Hobbyst46 wrote: ↑

Fri Oct 11, 2019 7:51 am

Wes wrote: ↑

Thu Oct 10, 2019 10:42 pm

Speaking of illumination techniques I find relief phase contrast interesting

http://zeiss-campus.magnet.fsu.edu/refe ... 9-2007.pdf

Haven't yet had the patience to properly explore, maybe someone who has can chime in.

Thanks for posting the link! It is pleasuring reading material, displaying nice tweaks of phase contrast, but somewhat distant from the present post, so maybe worth a separate post in Illumination Techniques.

You are welcome. You can find a lot of interesting historic articles on the Zeiss resource website.
http://zeiss-campus.magnet.fsu.edu/refe ... /index.php

Aslan wrote: ↑

Fri Oct 11, 2019 11:42 am

Beautiful! I don't have DIC and have only recently started investing in phase contrast additions to my vintage Leitz microscopes. These phase contrast photos have definitely given me something to aspire to.

Thanks Aslan, phase works surprisingly well with amoeba. Here are a few more from the same sample. The third one shows the bacterial halo that Bruce mentioned.

apochronaut wrote: ↑

Thu Oct 10, 2019 12:49 pm

D.I.C., which in addition to putting up fake relief, always looks like something is struggling under a freshly applied layer of latex paint. Please, at least sweep the wall free of critters before you paint it.

Hi Wes,
this is an interesting observation and a nice DIC/phase comparison.
I think DIC is so popular among amateurs today because it gives a look that fits better to todays ways of imaging things. The gritty look of phase contrast images is more in line with coarsely structured, high contrast b/w newspaper photos. Today every picture is digitally edited and smoothed, videos and sound too. They even change the whole look of cinema films to some strange faded colours. But the scientific use of phase contrast is clearly there. It is nice that the Zeiss West DIC revolver condensers have Ph2 and Ph3 phase annuli included.
I just bought two Zeiss objectives on ebay, one og them a Neofluar 16 phase. Both completely right through delaminated! Fortunately the seller was an honest person and reimbursed without any discussion when I showed the images taken through the phase telescope.

Bob