Advice on next upgrade to a T390, looking to observe yeast and bacteria.

Do you have any microscopy questions, which you are afraid to ask? This is your place.
Post Reply
Message
Author
_plp_
Posts: 7
Joined: Fri Jan 10, 2020 11:23 am

Advice on next upgrade to a T390, looking to observe yeast and bacteria.

#1 Post by _plp_ » Tue Jan 14, 2020 12:12 pm

I am interested in observing yeast and bacteria and making up their shape.
I got an AmScope T390B with the cheaper phase contrast kit (no turret). The objectives in the PCS kit are described as planar, the 100x has a NA of 1.25.
When looking at samples, I find myself just using the 100x phase constrast objective which seems to work best for what I am trying to do.

What is the next logical upgrade I should plan for to increase resolution & experience, maximising gain vs. cost?

I have my eye on the eyepieces (no pun intended). To the touch these feel a bit on the cheap side and the eye relief is not long enough for someone with glasses.
The markings are WF10x/18 with 23mm diameter.

Another item I considered is a dark-field condenser that would work with the BF 100x objective.
Not sure if this setup would give me anything else above phase constrast other than potentially nice looking images.

Thanks

apochronaut
Posts: 2864
Joined: Fri May 15, 2015 12:15 am

Re: Advice on next upgrade to a T390, looking to observe yeast and bacteria.

#2 Post by apochronaut » Tue Jan 14, 2020 4:13 pm

Getting your microscope where you want it, will be a big d.i.y. project. and probably not worth the effort. Your limitations are based on the N.A. and degree of colour correction of the objectives, the precision and design of your sample preparations and the objective contrast plus the contrast techniques employed. I would also add the condenser colour correction and N.A. and illumination intensity. Yeast and bacteria are notoriously difficult to image with precision but obviously it can be done with a light microscope, otherwise photos and drawings from the pre electron microscope era would not exist.

Common phase contrast is only partially useful for small subjects, such as the ones you are targeting. It seems that the default phase system that is made by microscope companies is dark phase and most likely dark medium phase. While this is a good general purpose phase type, it has an undesirable degree of halo and shade off, which is highly detractive when viewing small subjects with important inclusions. Bright phase is a better system for yeast and bacteria, as is anoptral and other proprietary types such as B -Minus. B-Minus is particularly good for imaging small inclusions, such as those found in many bacteria and yeast pores. The phase halo, is not so objectionable with it as is the case with anoptral phase. Apodized phase controls the halo to a large degree. Read this link.https://www.microscopyu.com/tutorials/s ... -artifacts.
Since your microscope has 160mm fixed tube optics, the possibility to d.i.y. an improved phase system on your existing stand does exist, since there were multiple manufacturers of alternate phase systems using the 160mm system and the parts are often not that expensive. It takes time to find complete systems, though.

Dark field resolution is limited by the quality of the condenser employed, the quality of the high magnification objective(s) and the illumination capacity of the microscope stand. You will not be able to adapt your microscope to an acceptable level of dark field performance in order to image such small subjects, without again getting into more diy'ing, this time upping the illumination by a considerable level, buying into a fine condenser and at least one high resolution objective. You will need either a Paraboloid DF condenser or preferably a Cardioid DF condenser. Cardioids are mirror condensers and are free of ca, something necessary for high resolution DF. You will also need an iris diaphragm equipped 100X objective lens, preferably with an N.A. of 1.30 or higher. This usually implies either a fluorite or apochromat type.

It sounds like you prefer to keep your glasses on while at the microscope, so with that in mind, you need to find an acceptable pair of high eye relief eyepieces. These are designated by having a little icon of a pair of eye glasses on them. I believe that the Chinese make such eyepieces but they may be for the infinity corrected systems. I see them marketed as replacements for Bausch & Lomb , probably similar to the ones on a Balplan, which have very high eye relief.

For your purposes, if I were you, I would use my microscope as is and begin the process of assembling a more finely tuned system based on a second hand stand. Once you get enough of it put together, you could sell your existing microscope. Normally, such a microscope would bring very little but with phase, it should have some resale value. Both Nikon and AO had quite extensive phase systems; dark in several contrast grades, bright in the same. These show up on the used market. Something like an Optiphot or a Labophot, can be customized as to illumination output and parts for phase are fairly well represented on the used market. There were dark and bright in various contrasts and I think apodized too? Someone else might know.
With AO the picture is a little different. AO microscope stands were made with specific illumination capacity. AO series 20, 120 and 420 infinity corrected microscopes have rear mounted 100 watt illuminators , can use Cardioid oil DF condensers, as well as Toric Cardioid oil DF condensers, which have a wider illumination field, so can be used with lower power objectives. Both are very good for high resolution DF. The phase systems are inexpensive and the eyepieces are high eyepoint. The pre-infinity series 2/4 system had a broad array of phase optics, 26 objectives in total but by the time the infinity system came along they had reduced the number but still had bright ,dark and B-Minus, so enough to cover the basis for microbiology.

Ultimately, to image such small subjects accurately, you will need to refine your sample preparation as well. That means no shortcuts as to the quality of slides, coverslips and strict adherence to your sample thickness and adjustment of your illumination beam. Higher grade microscopes also offer better options for modest filtering of the illumination, creating better contrast conditions and the optics usually have superior contrast over what average grade Chinese optics provide.

_plp_
Posts: 7
Joined: Fri Jan 10, 2020 11:23 am

Re: Advice on next upgrade to a T390, looking to observe yeast and bacteria.

#3 Post by _plp_ » Tue Jan 14, 2020 5:07 pm

Apochronaut, thank you for taking the time to write such a thorough reply.

As suggested, I will probably source eypieces with a higher eye relief and will stick to my current setup without changes until I learn enough to start thinking on the next step.
I will start by researching the terminology you've used in your post!

Do you have a link to a well imaged bacteria slide (done with affordable amateur equipment) I can use for comparison to what I am able to see on my setup?

Thanks again.

MichaelG.
Posts: 1611
Joined: Mon Apr 10, 2017 8:24 am
Location: NorthWest England

Re: Advice on next upgrade to a T390, looking to observe yeast and bacteria.

#4 Post by MichaelG. » Tue Jan 14, 2020 8:26 pm

_plp_ wrote:
Tue Jan 14, 2020 5:07 pm

Do you have a link to a well imaged bacteria slide (done with affordable amateur equipment) I can use for comparison to what I am able to see on my setup?
This video by our host will probably disappoint you ... but it’s a ‘reality check’
https://youtu.be/aZzRygip5XY

You need a Scanning Electron Microscope if you want to reveal any significant structural detail :(
https://pixels.com/featured/e-coli-bact ... llips.html

MichaelG.
Too many 'projects'

apochronaut
Posts: 2864
Joined: Fri May 15, 2015 12:15 am

Re: Advice on next upgrade to a T390, looking to observe yeast and bacteria.

#5 Post by apochronaut » Wed Jan 15, 2020 4:04 pm

_plp_ wrote:
Tue Jan 14, 2020 5:07 pm
Apochronaut, thank you for taking the time to write such a thorough reply.

As suggested, I will probably source eypieces with a higher eye relief and will stick to my current setup without changes until I learn enough to start thinking on the next step.
I will start by researching the terminology you've used in your post!

Do you have a link to a well imaged bacteria slide (done with affordable amateur equipment) I can use for comparison to what I am able to see on my setup?

Thanks again.
I went into my image files and picked out a few pictures of bacteria, yeasts and mould spores( about the same size as yeasts) that should illustrate the possibilities for you. There were 3 microscopes involved in taking the pictures and all were done with the same camera. The camera body is available for not much over 200.00 now, if you look around. The most expensive of the microscopes; a 100 watt halogen Reichert Diastar 420 set up for high resolution dark field was around 700.00 as configured at the time it's photos were taken. Another, a first generation 18 watt tungsten AO 10 with a complete set of phase objectives ( 9 planachro and achro) cost about 450.00 as it is currently configured and the third; an AO-Spencer series 4 , about 400.00 with it's 14 achromat phase objectives. So, while none of these systems were dirt cheap, none are outrageously expensive, either, especially when compared to what is available as new instruments. Building up systems such as these takes some time; that's why I suggested keeping what you have while focusing on building up a more precise second hand system. New doesn't necessarily mean good. Some of the objectives in my series 4 were made in the 1940's....used for one of the pictures here in fact.
Attachments
Yeast and bacteria infecting a bad sauerkraut batch. 100X planachro dark phase.
Yeast and bacteria infecting a bad sauerkraut batch. 100X planachro dark phase.
DSC02997 (1024x542).jpg (193.97 KiB) Viewed 454 times
This is a demonstration picture utilizing offset filter wheels in the illumination pathway. The thin stick like structures are gliding bacteria that are essentially invisible in normal bright field. 40X planfluor objective.
This is a demonstration picture utilizing offset filter wheels in the illumination pathway. The thin stick like structures are gliding bacteria that are essentially invisible in normal bright field. 40X planfluor objective.
DSC02617 (1024x575).jpg (192.79 KiB) Viewed 454 times
100x dark phase. Contaminant of milk. Possibly B. Cereus.
100x dark phase. Contaminant of milk. Possibly B. Cereus.
DSC02610 (1024x625).jpg (298.95 KiB) Viewed 454 times
Spores of the unusual mould Wallemia Sebi. DF 100X 1.32 planapo.
Spores of the unusual mould Wallemia Sebi. DF 100X 1.32 planapo.
DSC02405 (1024x629).jpg (256.73 KiB) Viewed 454 times
This image has been excessively digitally contrast enhanced in order to reveal details of what are very transparent organisms. They are presumably human blood parasites. The 8 or so x .3 micron bodies appear to have spores attached or possibly joints where longitudinal division is taking place.Dark field with a planapo 100X 1.32 objective. Cropped.
This image has been excessively digitally contrast enhanced in order to reveal details of what are very transparent organisms. They are presumably human blood parasites. The 8 or so x .3 micron bodies appear to have spores attached or possibly joints where longitudinal division is taking place.Dark field with a planapo 100X 1.32 objective. Cropped.
DSC02294 (1024x576).jpg (194.54 KiB) Viewed 454 times

apochronaut
Posts: 2864
Joined: Fri May 15, 2015 12:15 am

Re: Advice on next upgrade to a T390, looking to observe yeast and bacteria.

#6 Post by apochronaut » Wed Jan 15, 2020 4:38 pm

The following images demonstrate 3 different phase types used to view the same sample. This is a yeast and bacteria culture, grown from a center plug removed from a baked loaf of sourdough bread.
Attachments
97X 1.25 Dark M phase achromat
97X 1.25 Dark M phase achromat
DSC02935 (1024x575).jpg (147.55 KiB) Viewed 451 times
97X 1.25 Bright M phase achromat
97X 1.25 Bright M phase achromat
DSC02936 (1280x719).jpg (132.39 KiB) Viewed 451 times
97X 1.25 B-Minus L phase achromat
97X 1.25 B-Minus L phase achromat
DSC02939 (1280x719).jpg (109.04 KiB) Viewed 451 times

User avatar
wporter
Posts: 283
Joined: Wed Oct 21, 2015 10:18 pm
Location: USA

Re: Advice on next upgrade to a T390, looking to observe yeast and bacteria.

#7 Post by wporter » Wed Jan 15, 2020 5:07 pm

Really well-done exposition and images, Phil. Very nice comparisons.

I wish we had more of this kind of answer on this forum; good images of this or that to support a point are generally worth much more than mere verbal descriptions.

MichaelG.
Posts: 1611
Joined: Mon Apr 10, 2017 8:24 am
Location: NorthWest England

Re: Advice on next upgrade to a T390, looking to observe yeast and bacteria.

#8 Post by MichaelG. » Wed Jan 15, 2020 5:16 pm

Seconded !!

MichaelG.
Too many 'projects'

apochronaut
Posts: 2864
Joined: Fri May 15, 2015 12:15 am

Re: Advice on next upgrade to a T390, looking to observe yeast and bacteria.

#9 Post by apochronaut » Fri Jan 17, 2020 1:04 am

A picture IS worth a thousand words............sometimes. Sometimes only 2 , or 1 multisyllable word will do.

A problem arises when a picture requires one or many verbal keys, especially when it is being presented to someone quite unfamiliar with what they are looking at.

Hobbyst46
Posts: 2307
Joined: Mon Aug 21, 2017 9:02 pm

Re: Advice on next upgrade to a T390, looking to observe yeast and bacteria.

#10 Post by Hobbyst46 » Fri Jan 17, 2020 11:17 am

Apochronaut, thanks for posting the comparison. Excellent display of methods, tailored to the audience.
Zeiss Standard GFL+Canon EOS-M10, Olympus VMZ stereo

_plp_
Posts: 7
Joined: Fri Jan 10, 2020 11:23 am

Re: Advice on next upgrade to a T390, looking to observe yeast and bacteria.

#11 Post by _plp_ » Mon Jan 20, 2020 4:52 pm

These pictures make very clear what is possible with differen configurations.
Thanks for posting, very educative.

Post Reply