Oil Immersion

[Pat Thielen]

Right -- Here is a very newbie question. Don't laugh unless you absolutely have to. And if you do, have a nice beer to go along with it!

Awhile ago I got a new microscope and it came with an oil immersion lens... My problem is I have no real idea on how to use it as I've never used one before. A friend of mine was going to come over to show me some of this stuff but she just hasn't had the time. So, I've avoided using it as I've been rather frightened of my ignorance of such things. Anyway, now I need to use it -- I bought a really nice slide of Bacillus anthracis that I'd love to be able to see and photograph clearly.

So, my question is how do I properly use an oil immersion lens? I do have the oil, and I know I put a drop on the slide and swing the objective into it, but after that I have little knowledge. And, most importantly, how do I properly clean the objective and the slide when I'm done?

Thanks for any help any of you may be able to provide. And remember: When drinking your beer try not to blow any out through your nose. It can be most uncomfortable!

[lorez]

I'm too lazy to type, but I did find a pretty good (one of 1,000, probably) link.

flinnsci.com/teacher-resources/biology/frequently-aske...

Don't feel bad about asking a newbie question. When I was once working at a local university the prof asked one of the students to get the microscope ready to use with immersion oil. The student placed a generous drop of oil on the eyepiece.

lorez

[zzffnn]

1. Make sure your test slide is thin enough (some prepared slides are thick and uneven - an oil immersion objective may crash it without getting a focus).

2. Focus, as best as you as you can with your 40x NA 0.65 objective, onto test slide.

3. Put on oil, gently swing in 100x objective.

4. VERY SLOWLY turn the fine focus (never turn the coarse focus) one way or the other, while looking down the eyepiece(s) to see focus. Feel the fine focus, do not advance, if you feel resistance (resistance occurs when objective top lens touches on covet slip - any further advancement will produce a crash). Turn the fine focus the other way around, if you don't see focus and have felt resistance from one turning direction.

5. Remember which way you have to turn to focus with the 100x objective. Do the same next time. If it is off parfocality by over 30 microns, you can put on a parfocal shim ring to reduce the difference. If your fine focus has graduation, each small graduation is usually 1 or 2 microns (15 graduation marks equals 15 microns or 30 microns, depending on your scope).

You can clean off immersion oil with alcohol pads. Or simply lens tissue damped with 75% alcohol.

[zzffnn]

When I was once working at a local university the prof asked one of the students to get the microscope ready to use with immersion oil. The student placed a generous drop of oil on the eyepiece.

I am blowing water out of my nose (wish I had a better drink though, as that was very entertaining).

Did the student have too much ego to ask or was (s)he drunk?

That seems to be a novel way of forming a homogeneous optical system, the other way around. Did the student put some oil onto his/her eyeballs as well You know what I would tell that student to do, if I were the professor.

[Pat Thielen]

Thanks for the help -- I do appreciate it! And unlike the student, I know where the oil goes!

One further question: By using 75% alcohol (or other cleaning solution) to clean up the oil is there any chance the slide could be damaged? Such as the mounting medium being dissolved?

Thanks again! I hope to try photographing the wee beasties sometime this week. I also got slides of diatoms and a Dipylidium caninum (tapeworm) scolex that is very interesting and a bit scary. So, hopefully new photos will follow soon.

[zzffnn]

Not with the small amount and limited time of contact. Just don't wipe the slide with excessive force or amount.

You can wipe off alcohol with water, if that makes you feel better. Not really necessary though.

[75RR]

A link to a pdf on cleaning the microscope lenses (including oil) and one on why immersion "works"

http://www.olympusmicro.com/primer/anat ... rsion.html

http://microscopy.duke.edu/downloads/Th ... scsope.pdf

[lorez]

With regards to the student... I think the embarrassment was a sufficient lesson.

lorez

[JimT]

Pat, another alternative is naptha (lighter fluid) for cleaning the oil. Check this site:

http://www.microscopy-uk.org.uk/mag/ind ... clean.html

[NicoVB]

Great question!
And when you use oil on the condenser?
Is this only in combination with a oil objective, or is that independent?

Edit:
Found on Wikipedia:

Condensers and numerical aperture
Like objective lenses, condensers vary in their numerical aperture (NA). It is NA that determines optical resolution, in combination with the NA of the objective. Different condensers vary in their maximum and minimum numerical aperture, and the numerical aperture of a single condenser varies depending on the diameter setting of the condenser aperture. In order for the maximum numerical aperture (and therefore resolution) of an objective lens to be realized, the numerical aperture of the condenser must be matched to the numerical aperture of the used objective. The technique most commonly used in microscopy to optimize the light pathway between the condenser (and other illumination components of the microscope) and the objective lens is known as Köhler illumination.

The maximum NA is limited by the refractive index of the medium between the lens and the sample. As with objective lenses, a condenser lens with a maximum numerical aperture of greater than 0.95 is designed to be used under oil immersion (or, more rarely, under water immersion), with a layer of immersion oil placed in contact with both the slide/coverslip and the lens of the condenser. An oil immersion condenser may typically have NA of up to 1.25. Without this oil layer, not only is maximum numerical aperture not realized, but the condenser mat not be able to precisely focus light on the object. Condensers with a numerical aperture of 0.95 or less are designed to be used without oil or other fluid on the top lens and are termed dry condensers. Dual dry/immersion condensers are basically oil immersion condensers that can nonetheless focus light with the same degree of precision even without oil between the top lens and the slide.

I never paid any attention to this, learned on a normal dry brightfield condenser.
So on a darkfield (1.25) and NIC/PC DIC condenser (1.4), you need to apply oil?

[75RR]

So on a darkfield (1.25) and NIC/PC DIC condenser (1.4), you need to apply oil?

A condenser used without oil maxes at NA 0.95, whether it is a 1.2 or a 1.4, if you are using an oil objective i.e. one with an NA higher than 0.95 then you need to oil both the objective and the condenser in order to reach the potential NA of the objective.

[JimT]

One more from me as an alternative. I didn't want to mess with the mess of oil so I opted for a 60X dry obj. with an NA of .85. I am quite pleased with it and use it a lot more than I would a 100X.

Food for thought.

JimT