Inverted microscopes cant do this....

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thomas.schwarz
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Inverted microscopes cant do this....

#1 Post by thomas.schwarz » Mon Dec 02, 2019 10:39 pm

Hello friends,

I would like an affordable DIC microscope and a lot of (all of?) the ones under 4000 euro on ebay are inverted. So i can imagine that for very deep fields, like cell cultures/pitri dishes/ bottles of fluid an inverted microscope is required.

So riddle me this, what can a non inverted microscope do that an inverted microscope can not do? In other words why is an inverted microscope bad (other than large size/heavy)? Why would I regret having purchased one and wish I had a regular one? Do inverted microscopes work with regular slides/coverglass? Does oblique lighting work on an inverted microscope? Dark field?

Thanks!
Tom

apochronaut
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Re: Inverted microscopes cant do this....

#2 Post by apochronaut » Tue Dec 03, 2019 12:21 am

The basic point of an inverted microscope , is that it removes the objective cluster from interfering with the specimen. This then allows larger specimens to be put on the stage as well as live specimens that have been grown in a culture or other aqueous medium. Specialized and sometimes quite large containers can be utilized fairly easily, and some of them are adapted to a broader use in the lab than just a slide.

Since close proximity of the condenser to the specimen is required in order to increase the N.A. of of the optical system being employed and in many cases oil or some other immersion medium is necessary for specific condensers to meet the system requirements, it then means that in order for an inverted microscope to achieve high resolution , the condenser then, becomes the impeding device to what is essentially the beauty of an inverted. For them to do what they do best, low N.A. long working distance condensers must be employed on them, so they essentially are limited to being a lower resolution instrument.

Of course they could/can be adapted for higher resolution viewing but then they become just an upside down microscope and why not just use a fine upright.
Last edited by apochronaut on Tue Dec 03, 2019 12:31 am, edited 1 time in total.

PeteM
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Re: Inverted microscopes cant do this....

#3 Post by PeteM » Tue Dec 03, 2019 12:30 am

Inverted microscopes for biological specimens are typically meant to look through a fairly thick (plus or minus 1mm or so) dish, rather that through a 0.17mm or so cover slip. To do this they will typically have either a fixed long working distance objective (say, 1.2mm) or one that can accommodate a variety of thicknesses of glass between the objective and specimen (say 0-2mm). Many inverted scopes are only equipped with up to about a 40x long working distance objective.

For the most part you can use an inverted scope to view slides up to about 400x with various tricks (correction collars, inverting the slide on the stage, etc. but it gets difficult at higher powers for mounted specimens. There the upright scope, thin cover slips, and oil immersion reigns (sometimes of both the objective and condenser) reigns. If you want to look at things in a Petri dish up to about 400x an inverted scope is great

Modern inverted scopes (tens of thousands) may offer all sorts of phase, DIC, standard fluorescence, confocal laser, etc. imaging and achieve higher resolution. But this won't be the sort of scope you find for a few thousand dollars.

On edit: much of this already covered by Apo while I was typing.

thomas.schwarz
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Re: Inverted microscopes cant do this....

#4 Post by thomas.schwarz » Tue Dec 03, 2019 7:00 pm

Ok. Wow Thank you!

...so... ...i am microbehunter, that is all, amd a kind one (just videos). So super information... what i do is go to my jars (several, large) collected from different points along a canal. I take a suction droper thing and take about 3 cubic cm of water (which is itself very much alive) slong with a JUNGLE of micro orgsnisms, put that in a pitri dish, go to my optica 290 tb, which i aöready returned, take the smallest droplet of the 3 cubic cm jungle, on a slide, cover glass, observe. Now i think you both indicated the the depth of the specimen i am describing is very, very small (thin). Yet.... i am diving, i am rising, they are diving, rising, left up, left up, no down, right, no other right, etc....

So... if i had my full 2 mm of petri dish fluid under micro observation, am i correct im assumimg that my microbes would more or less evade me without trying? I mean with an inverted microscope?

I think you both already answered that (?), for my money I can get to 40x with a wing and a prayer, and if u get there, stuff will be flying out if focus faster than I can say abakanoktwerstubikwyz. Yes?

PeteM
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Re: Inverted microscopes cant do this....

#5 Post by PeteM » Wed Dec 04, 2019 1:53 am

I think you've got it.

Trapping protists under a cover slip, maybe with some micro debris to get in their way for for them to stay put while feeding, gives you a better chance of having them in focus, at least in the Z direction. The higher the magnification, the less depth of field you can expect. There are also techniques to slow them down.

Perhaps Kurt and others who regularly use an inverted scope will have some ideas of how they track protists in their inverted scopes.

apochronaut
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Re: Inverted microscopes cant do this....

#6 Post by apochronaut » Wed Dec 04, 2019 2:48 pm

There are a few ways that you can increase the depth of field and the depth of focus.
1) For some specimens, working at the maximum N.A. is not always necessary. The higher the N.A., the shallower is your depth of field. Lower N.A. objectives in most magnifications for many microscopes exist. Although there is a direct relationship between resolution and N.A., a lower N.A. does not necessarily mean that the image will be blurry, especially if ones goal is to track moving organisms in depth as well as laterally. It shouldn't matter that there is a small drop in resolution, if you can track something you wouldn't otherwise be able to with a higher N.A. objective. Some lower magnification objectives are also available with an iris diaphragm, allowing the N.A. to be selectively lowered and therefore the depth of field. Objectives with an iris diaphragm have been made down to as low as 48mm focal length( approx. 3X) by various mfg. Most of them are for 160mm tube lengths.

2) Dark Field. Dark Field , obtained through the use of a CA free mirror condenser, usually seen as a Cardioid type, has an odd characteristic of increasing the depth of field. I have not witnessed the same , with DF obtained through other types of condensers. Even at very high N.A.s, the depth of field can be quite stunning. In this case, high N.A. objectives can be used to great effect with the reduced aperture required for DF , increasing the depth of field.

3) Most of the objectives that are made for inverted microscopes can be used on standard upright microscopes too. For instance using a 20X .50 objective with a 1mm working distance, a live pond specimen can be prepared in a well slide with a thin slide, sub 1mm as a cover slip. In this way, much of the convenience that an inverted microscope offers can be had with a conventional microscope. Great depth of focus into the well of the slide is possible.

I'm sure there are other ways too.

Piter_
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Re: Inverted microscopes cant do this....

#7 Post by Piter_ » Sat Dec 07, 2019 9:33 pm

Hi guys.
Here is the main reason why inverted microscope is used in cell culture and biology.
Cells are grown in petri dishes:
Image
Or other similar thing, like ibidi slides:
Image
Cells are growing on the bottom and you cannot focus on them from the top, only from the bottom. Also you cannot turn the well up side down because all media will go all over your microscope, cells will die and so on...
From optical point of view there is no difference, apart from different orientation.
Thickness off the bottom varies between different plates, and you can find ones with 150 um thick bottom, suitable for high numerical aperture objectives. I would gladly get one of the older black inverted compound microscopes :). It is much easier to look on pond life using an inverted microscope :)

Hobbyst46
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Re: Inverted microscopes cant do this....

#8 Post by Hobbyst46 » Sat Dec 07, 2019 10:43 pm

Piter_ wrote:
Sat Dec 07, 2019 9:33 pm
Thickness off the bottom varies between different plates, and you can find ones with 150 um thick bottom, suitable for high numerical aperture objectives.
Thanks in advance for specific information: the ones with 150um thick bottom, with which high numerical aperture objective can they be used ?
Zeiss Standard GFL+Canon EOS-M10, Olympus VMZ stereo

PeteM
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Re: Inverted microscopes cant do this....

#9 Post by PeteM » Sat Dec 07, 2019 11:48 pm

Perhaps another question about Petri dishes and protists. I get how cells grown on the bottom will be in focus. But from my limited experience, give live protists any thickness of water to move about in (even under a cover slip) and they'll use pretty much all of it. How do we keep them fixed to the bottom in a Petri dish? My assumption has been that the Petri dish is great for sorting protists, running electro-physiology experiments, etc.; not so much for imaging protists at high (say, 250-400x) magnification

Piter_
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Re: Inverted microscopes cant do this....

#10 Post by Piter_ » Sun Dec 08, 2019 4:41 pm

Hobbyst46 wrote:
Sat Dec 07, 2019 10:43 pm
Piter_ wrote:
Sat Dec 07, 2019 9:33 pm
Thickness off the bottom varies between different plates, and you can find ones with 150 um thick bottom, suitable for high numerical aperture objectives.
Thanks in advance for specific information: the ones with 150um thick bottom, with which high numerical aperture objective can they be used ?
Sorry I misinformed you. The thickness is usually N1.5 ~180 um and refractive index ~1.5. May vary with producer some 10 um. Can be used with avery high numerical aperture objective. Bottom material can be plastic then it is cheaper. Can be glass then it is more expensive. Very expensive will be from quartz. They will also vary on thikness precision. More precise and "smoother" will be more expensive. Petri dishes usually too thick for use with high NA objectives. There are smaller size Petri dishes with a coverslip inlet, usually expensive. I have been making them from think plastic ones by drilling a hole in a dish and gluing a cover slip to it.
PeteM wrote:
Sat Dec 07, 2019 11:48 pm
Perhaps another question about Petri dishes and protists. I get how cells grown on the bottom will be in focus. But from my limited experience, give live protists any thickness of water to move about in (even under a cover slip) and they'll use pretty much all of it. How do we keep them fixed to the bottom in a Petri dish? My assumption has been that the Petri dish is great for sorting protists, running electro-physiology experiments, etc.; not so much for imaging protists at high (say, 250-400x) magnification
It is true that in case of paramecium or some other small speeder it will be very hard to catch them in focus. But for things which do not move it move slow it is very convenient. Things will sediment on the bottom where you can observe them. Also you can use a usual prep with cover a coverslip. Just put it with the cover slip down. :)

apochronaut
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Re: Inverted microscopes cant do this....

#11 Post by apochronaut » Sun Dec 08, 2019 6:55 pm

Yes. And the dishes are upwards of 5.00 each.

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