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Oil Immersion Objective Question

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#1 Otto Piechowski

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Posted 11 December 2013 - 09:25 PM

I have a 100X objective with a 10X ocular for 1000X.

I have the oil to place on a specimen for use with the 1000X.

The images I get, say of bacteria, or cell detail, is clear.

But, there is no more detail seen with the 1000X oil immersion objective than with the 400X non oil objective. Further, the images seen with the 100X objective at 1000X are of equal clarity and detail transmission whether I view with oil or without oil.

Am I doing something wrong? If so, what do I need to change? Or is this the expected behavior of the proper use of an oil immersion objective.

Thank you.

Otto

#2 EJN

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Posted 12 December 2013 - 02:33 PM

To really get the most out of an oil immersion objective, you must
have an Abbe or achromatic condenser with an N.A. (numerical aperture)
of 1.25 or greater, with an iris diaphragm. The diaphragm is set wide
open, and slowly closed until the best image is obtained. For extreme
resolution, the top element of the condenser is also oiled to the
bottom of the slide. Illumination is important too, Koehler illumination
(you can Google it) is the preferred method; a built-in or external
illuminator with a condenser lens and a frosted glass will work if the
light is bright enough.

Also, the immersion oil should have a refractive index equal to glass, 1.52

#3 Otto Piechowski

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Posted 12 December 2013 - 03:08 PM

EJN,

That was very helpful.

A few more questions, please.

The 100X objective has the notation stamped on it: 1.25
The oil I am using is labeled 1.5150.
Are these compatible (right for each other)?

Thank you.

Otto

#4 EJN

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Posted 12 December 2013 - 04:02 PM

1.25 on the objective is the N.A. and that is the normal value for non-ED glass
oil immersion objectives. The immersion oil has the right value too, so they are
exactly right for each other.

#5 Hikari

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Posted 12 December 2013 - 05:34 PM

100X objectives are right at the limit of optical microscopy. With the loss of DoF, they tend not to look a lot better than a 40X, just larger.

Your oil objective should look really bad without oil. Is there some oil dried on it? Perhaps you need to clean it. A dirty objective performs poorly too.

#6 Otto Piechowski

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Posted 12 December 2013 - 07:07 PM

I have been cleaning the objective regularly with 70% isopropyl alcohol.

Is it possible that the condenser needs to be of a certain type? I vaguely remember reading something about a # of .95 not being useful and of 1.25 being useful. I don't know which mine is.

But, the resolution of the 100X is identical to the 400X even when with no oil at all.

Otto

#7 Otto Piechowski

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Posted 12 December 2013 - 07:25 PM

I just tried it again. Oil on condenser touching bottom of slide. Oil on slide which is in contact with the bottom of the oil immersion objective. Nice clear view. When all the oil is cleaned off the condenser, and objective and slide; with multiple swipes of isopropyl alcohol and a commercial microscope cleaner and tissue paper, and all are dry, the views through the oil immersion, dry, are just as sharp as with the oil.

And the dry and wet oil immersion images have equal resolution with the 400X air objective which provides nice sharp contrasty images.

The scope is a binocular AO Spencer from the 1930s or 1940s I would guess. The microscope is a 40X, 100X, 400X. It did not come with an oil immersion objective and probably was designed only for the 40/100/400. The 100X objective, I picked up from surplus shed.

Otto

#8 EJN

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Posted 13 December 2013 - 12:10 PM

...the views through the oil immersion, dry, are just as sharp as with the oil.

That's just odd. I have 2 oil immersion objectives, without oil the image is
blurry and washed out. With oil it becomes sharp and contrasty. The difference
is not subtle. Without being able to see and look through your setup, I really
have no idea what else to suggest.

I also see better resolution - I have a slide of mixed diatoms, and on a particular
crescent-shaped diatom, what appear as fine lines with the 40x objective resolves
into closely spaced dots with the 100x oil immersion objective.

#9 Otto Piechowski

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Posted 13 December 2013 - 02:46 PM

EJNā€¦could it be a problem with having the wrong condenser? And, (you a pretty good at these things) is there a test you can think of I can do to determine if the condenser is the problem?

Otto

#10 EJN

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Posted 13 December 2013 - 06:32 PM

The condenser is not going to make that big a difference unless
it is one of those those that are built into the stage, those
are usually N.A. 0.65 and somewhat inadequate for an oil immersion
lens. No condenser at all is doubleplusungood.

I rarely oil my condenser to the slide, that is really only useful
for getting the last few percent of possible resolution, and generally
makes too much of a mess to be worth it. An un-oiled Abbe condenser
is operating at an N.A. of ~ 0.9 to 0.95, and is good enough 99%
of the time.

The only thing I can suggest is after focusing with the oil immersion
objective, remove the eyepiece and look down the drawtube. The iris
should be adjusted so that it just barely protrudes into the edge of the
illuminated circle seen on the back of the objective lens. Reinsert the
eyepiece, if the image seems too low in contrast, close the iris slightly
more. Closing it too much will *seem* to increase contrast, but the
resolution goes down quickly.

If the condenser is incapable of illuminating the full apparent aperture
of the objective, then it is either too low or inadequate.

The only subjects where I see considerably more detail using the 100x objective
over the 40x objective are diatoms, bacteria, yeast, & blood cells.

What type of illuminator are you using? With my old B & L microscope with
a substage mirror, I use a 9-LED light source with a diffusing filter.
If the mirror is plano-concave, use the concave side. However, if you
use Koehler illumination you should use the flat side of the mirror.

One other thing, if you use an illuminator with a clear bulb without a
diffuser, while the image may *look* bright and sharp, it is severely
limiting resolution because it makes the microscope essentially operate
like a pinhole camera.

#11 Otto Piechowski

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Posted 13 December 2013 - 07:05 PM

You are being very helpful EJN. Thank you.

I need a little instruction about setting up the specimen for viewing. Let's take blood. I assume I want to smear the blood. Then do I want to cover it with a cover slip or not? If not, do I want to "fix" the smear to the slide somehow so that the oil doesn't loosen and wash it around; say by heating the underside of the slide?

Otto

#12 Hikari

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Posted 13 December 2013 - 08:12 PM

Otto, you always use a cover slip. The cover slip glass plus the oil is what the objective is corrected for.

The fact you view with and without oil is the same is just odd. I can't get an oil objective to work without the oil. The view is just blurry. Your condenser NA will not affect that. Ideally, your condenser NA should be equal or greater than you objective NA, but you are sill going to use a condenser with a smaller NA, it is just not an an efficient illuminator.

#13 Otto Piechowski

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Posted 13 December 2013 - 09:17 PM

Will (Hikari) and EJN,

Thank you for walking me through this.

I did get a drop of blood and put a cover slip on it. Then I put the oil drop on top of the coverslip and used the 100X objective. The cells seen were larger and the details were more easily seen, then with the 40X objective at 400X. I now understand and see what is meant and what you mean by saying the 100X oil immersion objective provides a better view.

Now, having said that, I want to suggest for your consideration a saying from amateur astronomy. It is said, and it is true, that once something is seen in a finer and larger (aperture) instrument, once the detail is known to exist (say on Mars or the moon), it will then be able to be seen with a somewhat less fine and smaller aperture.

Applying this to my views of the blood. It seems that what I saw at 1000X was bigger, just as contrasty and good resolution, and the detail was clear. But once I knew the detail to look for, I could always find the same at 400X, though it took some active peering. Would you agree, or not agree with this assessment?

And now a question about the blood I was looking at (mine). There were all of the nice round cells. I believe those are red blood cells. There were also cells of the same size as the red blood cells but with all sorts of small bumps and projections on them. There were quite a few of these as well; almost as many as the perfectly round red blood cells. Are these bumpy-cells also red blood cells but somehow deteriorated or degenerated from some gases or lack of gases (oxygen?) in the solution?

Otto

#14 Hikari

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Posted 14 December 2013 - 12:33 PM

Otto, not seeing an image, it is hard to know what you are exactly seeing. You could have damaged your cells in prep, but without an image, it is hard to know.

Also, I run a microscope lab for researchers and my background is in imaging, not biology. So, if there the cells are diseased or they are something else, I probably could not give you an answer.

#15 EdC

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Posted 29 December 2013 - 07:54 PM

Bumpy red cells = called crenated blood cells. Could be an abnormality or it could be an artifact caused by something getting into the sample, maybe salt, loss of water by osmosis. Google "crenated blood cells" and you will be taken to many articles with pics.

Have fun!

I've done some slides where they show up myself.

#16 aatt

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Posted 11 January 2014 - 01:38 PM

Other types of cells?Lacking in color? Probably white blood cells






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