Microscope 101: Oil Immersion Lens Technique

Oil immersion is a necessary technique for high powered microscopy, however few modern microscope companies seem to provide information what exactly that means! How is a novice microscope user supposed to know these things without at least a little insert or blurb in the manual about it? It honestly confuses me every time I see a list of specifications on a manufacturer or  brand name website, with no explanation as to what they are or how they benefit me–why not capitalize on that educational opportunity?

But, I digress. Oil immersion is pretty much exactly what it sounds like.

The 100x objective lens has a very narrow aperture on it, so it is difficult to get light into it because of how scattered light gets through air as a medium.

By using a relatively thick oil, usually cedarwood oil, light is kept from scattering as much, allowing more of it to maintain the correct angle to enter the lens, and thus your sample becomes viewable and easier to focus on. Pretty nifty!

Most companies provide small sample bottles with your microscope, however you will eventually need to get more, as a drop a time from a 7 mL bottle won’t last too long, depending on your frequency of usage of the microscope.

 

Here’s what to expect in your box:

So, how do you perform oil immersion? Really quite simple.

1. Place your sample on the microscope stage, and center it using the 4x objective.

Be sure that your sample is properly setup (slide, water drop, sample, cover slip) before starting. An improperly mounted sample will cause problems before you even get started. If you need a refresher on how to do so, you can check out my guide here: How To Properly Mount a Slide

You want to easily focus on your sample and center on the part you want to view with the 100x. But, this is difficult to do on the first try with the higher powered lenses. So, it’s best to start with your lowest power objective available–on compound microscopes, usually that’s a 4x objective. I use mostly AmScope microscopes and Omax microscopes, and both come with 4x out of the box.

2. Once centered, gradually work your way up through the objectives.

Gradually moving up in magnification allows you to fine tune the positioning of the sample, ensuring that the desired element of your sample will be centered in the image when you make it to the 100x objective. Pretty simple, nothing fancy at this step.

3. When you reach the 100x objective, raise the objective up, and place a drop of immersion oil on top of the cover slip.

Be sure you have a cover slip on your sample. You don’t want your oil mixing with the sample or water, and you certainly don’t want to get your sample or water on (or in!) the objective lens.

The drop should be thick enough so that it beads up, so not so thin that the lens cannot be immersed in it. The lens, in this situation, is referring to just the glass tip of the 100x objective.

4. Slowly adjust the focusing knobs to bring the lens to be just immersed in the oil drop.

You don’t want to break the slide, so be careful with your adjustments. The focal point is very, very close for a 100x objective, AND you have to have it immersed in the oil. So there’s a lot of things to pay attention to at the same time.

It may take you a few tries if it’s your first time. If you need to try again (because the oil smeared, for example), simply wipe off the oil from both the objective lens and the cover slip (carefully to avoid damaging your mounted sample). Don’t let the oil dry on either of them, but especially the objective lens–that will take a little more finesse to clean off, but all is not lost unless it gets inside your objective somehow.

5. View your sample, using only the fine focus to make minor adjustments.

If you adjust it too much when it’s oil immersed, you could break out of the oil, or smear it. Worse case, you may break your slide and have to start over from the beginning of the whole process.

Bonus Tip: What do I do if my immersion oil dries up on my objective lens?

A quick tip I’ve learned from a few manufacturers is that you can actually spray a tiny bit of butane lighter fluid onto your objective, and wipe it off thoroughly with a kimwipe, or any non abrasive lens cloth. Alternatively, you can use turpentine (paint thinner), but be extremely careful and use a small amount.

If you aren’t familiar with Kimwipes, you can pick up a pack (probably one of the most useful things to have around the house, especially if you’re getting into microscopes) online. I haven’t seen them in stores, but here’s what they look like (or if you need a pack, you can click below and order as many as you’d like for cheap online):

Alcohol and ammonia should NOT be used with objectives because the glues and concretes used when placing the lens can be broken down by such chemicals.

Bonus Tip: I’ve run out of immersion oil. What type do I buy? And from where?

These days, it’s getting increasingly difficult to find immersion oil in bulk. I’m not 100% sure why, but generally you’ll want at least 1 small bottle so that you have an easy to use dripper or pipette to drop it onto the slide with. Imaging trying to pour just a drop from a large gallon sized container! What a mess that would make!

I did spend a bit searching, and was able to find a decent sized supply bottle, as well as a quality small bottle as well. Here are my suggestions for immersion oil when you’ve run out:

Small Bottle:

Large Bottle:

That’s pretty much it in a nutshell! Just remember to wipe off any immersion oil before it dries with a kimwipe–your life will be much easier than if you let it dry.

If this helped you, be sure to leave me a comment below and let me know! I’m happy to help answer any questions!