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Using a new microscope with my son, we cannot see images beyond a certain magnification:

Eyepiece: 25x - Objective lens: 4x   --> OK  
Eyepiece: 25x - Objective lens: 10x  --> OK  
Eyepiece: 25x - Objective lens: 40x  --> not focused  
Eyepiece: 25x - Objective lens: 100x --> not focused  

In the last two cases, we try moving the stage along all its range, without getting anything.

Same issue using a 10x eyepiece.

On the 100x objective lens "OIL" is written; does it mean the observation needs to be done through oil instead of air? Or just that oil is used inside the objective?

We did not prepare the leaf; we just put a small piece of it between two slides.

Are we doing anything wrong?

Observing a leaf at 250x, we can see tiny round structures (I think barely bigger than what can be possibly seen by our eye): are those the cells?

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    $\begingroup$ Can you tell us the other writings on the objective lenses ? Do you also have a 10x ocular in your kit ? How are you preparing the leaf for observation ? $\endgroup$ – Eliane B. Jan 1 '17 at 19:48
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    $\begingroup$ Generally speaking, strong objective will focus very close to the sample. (Be careful of lens-sample collision though) $\endgroup$ – Eliane B. Jan 1 '17 at 19:48
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    $\begingroup$ It's been thousands of years since I've used a microscope, but shouldn't 100x be an oil-immersion magnification? Also, looking at a pre-fixed set of slides first should help you to get the hang of things. Looking at cells takes practice. If you have none, buy some. The very thin skin between two layers of an onion needs little preparation, and is a great home-made intro to plant cells. A scraping of the inside of your cheek mixed with saline (easy to make) is also a good home-made intro to animal cells. $\endgroup$ – anongoodnurse Jan 1 '17 at 21:13
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    $\begingroup$ Oh, btw, it's possible that the things you're seeing at 250x are chloroplasts. Round and green and very small. $\endgroup$ – anongoodnurse Jan 1 '17 at 21:16
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    $\begingroup$ Just a guess, but putting the piece of leaf between two slides may not let the objective get close enough to focus at higher magnification. There are cover slips made of much thinner glass that are used for this purpose. $\endgroup$ – jamesqf Jan 2 '17 at 5:53
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This is probably the problem: "We did not prepare the leaf; we just put a small piece of it between two slides.".

I suppose you mean you put a leaf fragment between two regular microscope slides, those measuring 26mm * 76mm (1in. * 3in.)?

That won't work. Long story short: microscope optics/objectives are designed with a specific working distance in mind, which is not an entiraly free choice, but dictated by the laws of physics/optics. An average achromat 40/0.65 has a working distance of about 0.6 mm. An average achromat 100/1.25 has a working distance of about 0.2 mm. An average microscope slide has a thickness of about 1 - 1.2 mm...

In the... well... regular microtechnique, a specimen is put on a slide and covered with a very thin coverslip, having a thickness of around 0.15 - 0.17 mm.

Also, as others mentioned, using a 25x eyepiece in combination with a 40/0.65 or a 100/1.25 is kind of overkill. With that combination, you enter the realm of "empty magnification". In short: structures appear larger, but no new structures are revealed and the image becomes more and more fuzzy.

And yes: you need to use immersion oil with the 100x objective, but keep in mind this is a very demanding objective to be used, on the part of the microscopist as well as on the part of the slide preparer.

Using a microscope is not all that simple for novices, but there's an easy way to find out -if no image can be produced-, if it's a microscope problem or an inexperienced user problem: take as a specimen an as thin as possible large object, such as a leaf of cigarette paper, and try to focus on it, using low power and gradually use stronger objectives up to 40/0.65. If an image can be produced, even if it's rather blurred at 40/0.65, the microscope is okay.

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    $\begingroup$ "We did not prepare the leaf; we just put a small piece of it between two slides." Nice point... a slide is too thick for a 40x objective. $\endgroup$ – Always Confused Apr 22 '17 at 10:14
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    $\begingroup$ Sorry. I'm not a native English speaker, but in Dutch and, as far as I know, in French and German as well, any object put on a slide for microscopic examination is considered "prepared" for -, being it as a temporary mount. Ah well, I'm not a linguist, unfortunatly. $\endgroup$ – guest Apr 22 '17 at 10:55
  • $\begingroup$ Logically it is true...irrespective of language... whether you are preparing 'chemically' or preparing just with a blade and forceps, or just preparing by hand; temporary or semi-permanent or permanent; coverslip or another slide or naked... you are 'preparing it for microscopy'. $\endgroup$ – Always Confused Apr 22 '17 at 11:05
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Eyepiece: 25x - Objective lens: 40x ---> not focused

Are you sure it can't be focussed? Objective lens like 40x or 45x normally have very near-focus (can be less than 1 mm) and also a shallow depth-of-field.

To focus the slide at 40X objective; at first focus it with 10X objective. Then rotate the turnstile at nose-piece and bring 10X objective. (In some lower quality microscope with the adjustment screws you may need to bring the main-portion of microscope upwards, distant from the slide. Otherwise the long 40x lens may hit the slide or drag the coverslip. However good-quality microscopes doesn't have this problem). Now it is time to focus the 40X.

To easily do that; at first bring the 40X lens tip just 1 or 2 mm distance from cover-slip. (Just like sometimes while a known-titration we blow the titrant from the burette quite high-rate upto certain value; and subsequently slow-down). Now take only the fine-adjustment screw and bring the objective backwards (and very carefully forwards... so that the lens doesn't hit the cover slip).

  • Note-1. I didn't worked with 25X eyepiece; but I worked up to 15X eyepiece; that normally gives wonderful clear picture. But in some microscopes having expandable draw tube, higher magnification with similar 15X eyepiece I've seen, but gradually high expansion of drawtube (and refocus of course) makes image gradually more blurry. But focus was not impossible.

You could also try eye-pieces of 10X and 15X values.

  • Note-2: Use thin and uniform cover-slip (such as Blue-star (trade-name)) and try to make your sections more thin. See if that cause any improvement.

  • Note-3: Do not use bare specimen (no-coverslip)... it may damage the lens which is very close to specimen.

Eyepiece: 25x - Objective lens: 100x --> not focused.

The 100X objective lense is usually a Oil-immersion lens. I.e. between the coverslip and 100X lens tip; the light pass through a specialized oil instead of air.

What I've seen if I do not use the oil; I do not get the focus even when the lens tip touches the coverslip.

  • Note-1: Cleaning of oil immersion lens: Do not use xylene (though some protocol says that). It damages the eyepiece, cause blur, and may make it permanently non-functional. Most of our university-teachers forbidden strictly to use xylene for lens cleaning. Use a soft cotton cloth, at first moist, then dry (or alternatively wet and dry for several times). However cotton and xylene to be used to clean the coverslip quickly with minimal damage/friction of the microscopic specimen.

  • Note-2: If use oil; make its use neat and clean way. Do not contaminate oils to other lense.

  • Note-3: For common purpose we do not need the oil-immersion lens. It is used only to view very tiny things like chromosomes, bacteria, cell-wall ornamentation, pollen walls, fungal spores and such type of things. Average plant and animal cells' contours (and tissues) could be seen well under the 10X objective and 10X to 15X eyepiece. Sometimes students' microscope come without the 100X objective; the place for it on the turnstile kept closed with a screw-cap.

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25X is a mighty strong ocular (eyepiece) lens, certainly for home use. This means with the 40X objective you are already at 1000X overall. It would take a seriously thin, well mounted and well lit specimen for you to see a good image. This assumes the optics are of sufficient quality.

I recommend using a 10X objective. This will give you 400X with the 40X objective. To use the 100X objective, you will need to insert a drop of oil between the objective and the slide. "Immersion oil" is made especially for this purpose.

At the 400X, or higher, magnification you will need to slice tissue very thin. But there are a couple ways to view cells without the knowledge or tools for preparing sections. To view plant cells, do an internet search for "onion cell wet mount". To view some animal cells (your son's), search "cheek cell slide".

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