Oil immersion tolerances

[coominya]

I experimented with the 100x obj tonight (with oil) and managed to crack a cover slip. There was a lot of plant material under it and possibly some grit. Is it necessary to exclude all this from the sample and have just organisms in water?

[75RR]

I experimented with the 100x obj tonight (with oil) and managed to crack a cover slip.

Been there - done that :)


Fundamentals of light microscopy and electronic imaging / Douglas B. Murphy.

[zzffnn]

Yes, that will help a lot.

Filter your sample water with pore size of around 0.4mm will remove many plants and sand particles, while keeping most protists. If you know the exact size of subjects you want (say 0.05mm), then you filter out other things with the next closest pore size (say 0.06mm).

Also sand particles precipitate faster than protists. So use a pipette to draw the precipitated layer immediately above the sand particles layer will also help.

[Hobbyst46]

Filtration through a 25 micrometers mesh size net can be extremely slow, since small debris particles clog the filter.
Filtration with a 0.3-0.6mm net is fast. Such nets are often included in supermarket infusion packages (herbal mixtures, "herbal tea") or air freshening products, scented soap bars etc. I usually avoid filtration by careful withdrawal of liquid from the top layer above the sediments in the tube, using a pipette. Protists are there, they do not sink to the bottom.

To bring an immersion objective into observation position, I look directly at the the space between objective and the coverslip, NOT through the eyepieces, so that the objective NEARLY touches the coverslip. Only then do I look through the eyepieces and with the fine focus raise the objective until the specimen is in focus.

[Charles]

I think the pipet procedure would be a good solution.

The way I do it so as not to crash the coverslip (as long as the objectives are somewhat parfocal) is to first focus with the 10X, then switch in the 40X and fine focus, swing the objective out of the way, put your drop of immersion oil on the slip and swing in the 100X. Only use the fine focus to refine your focus. If the objective actually contacts the coverslip, focus the other way.

[MicroBob]

There are oil immersion objectives for cover slip use an one for use without coverslip. It is difficult to keep them apart as the immersion oil has the same refractive index as glass and teh cover slip doesn't matter as long as there is enough room for it.
I habe a Leitz NPL Fluotar 50:1 n.a.1,0 that is meant for use without cover slip and has a work distance of 0,22mm. It can be used with coverslip but there is no tolerance at all for overly thick cover slips or thick preparations.

I use some immersion objectives without spring loaded front too. When in doubt where I am I release the clamp that holds the slide and start to move it slightly back and forth while I lower the objective with the fine focus.

[einman]

Hobbyist46-"To bring an immersion objective into observation position, I look directly at the the space between objective and the coverslip, NOT through the eyepieces, so that the objective NEARLY touches the coverslip. Only then do I look through the eyepieces and with the fine focus raise the objective until the specimen is in focus."

That is exactly what I do as well.

Alternatively you can use protist flaggers. They are unionized so you have to obtain them by the dozen. They position themselves within the field of view and wave you in..LOL

[MicroBob]

My problem with "nearly touches" is allways that the drop of oil connects long before it really becomes critical. With a spring loaded objective no problem ...

[charlie g]

Great questions you raise on use of oil-immersion 100X objectives...thanks for posting this thread.

Everything starts with the specific 'specs' of your 100X oil-immersion objective. What coverslip...vrs: 'no cover slip' is the spec of your 100X oil-objective? What is the 'working distance spec' of you oil-100x objective?

The specs of this very 'tight' tolerance range of function for your objective...well this tolerance dictates just how 'thick a sandwich' you can plop under this setup and focus/ and observe a specimen...without a coverslip shattering due to pressure from the microscope objective.

Grit in a slide, type of slide...whatever you plop onto your microscope stage must be 'thin enough overall sandwich thickness'...for your setup optics to perform.


So you need learn and respect the total thickness of the sandwich ( glass slide+ specimen in media+ coverslip thickness+ immersion oil bridge) you plop on your microscope stage to observe with your particular oil-immersion 100X objective.

Yes, yes, I have shattered microscope slide coverslips in a quest to enjoy a 'fixed permanent slide/specimen/coverslip preparation...ouch! Charlie guevara

[coominya]

Fundamentals of light microscopy and electronic imaging / Douglas B. Murphy.

Thanks for that. Found a copy online

Great idea about the supermarket filtering devices Hobbyst46
The pipettes are a good idea too for those who suggested that. I have a pack and have been using them to include some sludge off the bottom in my sample jar but will go 5mm or so above that level now.

To bring an immersion objective into observation position, I look directly at the the space between objective and the coverslip, NOT through the eyepieces

Thanks.

Thanks for all the great ideas guys. So glad I joined here.

[coominya]

I'm using old gear at the moment, no spring loaded objective it seems.

[einman]

Hmm..I did not see what type of stand you have. The objective looks like an Olympus objective.

[zzffnn]

I'm using old gear at the moment, no spring loaded objective it seems.

I could be totally wrong. But it actually looks like a retractable (spring loaded inside) objective to me. Please push down on objective shell tip (not on lens) to see if it retracts.

[Hobbyst46]

Having looked at the manual of your Olympus ECE-Bi, I think that it is from the 1960-1970 era. I am 99% sure that the 100x objective is an Olympus spring-loaded of a short parfocality length - 36mm rather than the 45mm DIN. I think I saw a similar one somewhere around - I will check within a day or two and report.

[coominya]

Please push down on objective shell tip (not on lens) to see if it retracts.

Yes, you are right! It was jammed (old age perhaps) but a really good push got it moving again. It's free but does it now require any servicing? Like a drop of isopropyl alcohol around the edge? It takes about 15 seconds to fully emerge after being depressed and the effort to get it moving initially would certainly be enough to crack a cover slip.

[MicroBob]

In the current state it is not very usable because it changes forus on its own. The optical parts are in the inner barrel. Asprint surrounds this inner barrel or is behind it and then there is somthing like a screwed in ring that hols it together against the sprint pressure. You might have a look if you can unscrew this ring without getting deeper into to optics. Then clean everything and put it together without oil or with just a tiny little sewing machin oil.
I wouldn't recommend to use fluids on the objective before dismantling.

[coominya]

You might have a look if you can unscrew this ring without getting deeper into to optics. Then clean everything and put it together...

That makes sense. The view through that objective isn't that flash anyway, plus it is just an old practice scope that I purpose to learn on. If I screw it up I wouldn't cry (much)

[Hobbyst46]

Just checked. Your HI 100x1.30 objective appears to be an Olympus 36mm parfocality, oil immersion, not a phase, the metal collar IMO can be screwed out so the spring-loaded inside is visible. There should be a longitudinal slot in the fixed part. A single tiny drop of a light machine oil into that slot should start smooth recession of the lenses into the barrel.

[75RR]

You might want to try warming it up in an oven (to get the old grease flowing again) before you open up the insides and start playing Doctor!

[Hobbyst46]

You might want to try warming it up in an oven (to get the old grease flowing again) before you open up the insides and start playing Doctor!

75RR, I Googled the m.p. or s.p. of Canada Balsam (that Olympus objective is probably 40+ years old). And in the "Basic Optics and Optical Instruments: Revised Edition By Naval Education" found that Canada Balsam softens at about 125F, so 52 Celsius. In my opinion, the oven must be fairly accurate to avoid damage to the objective.

[75RR]

This was the recommendation I received at mikroskopie-forum.de a few years ago when I asked about a stuck Zeiss-Winkel 40x and 100x from my first microscope, a Standard Junior KF

Hi Glen

one possibility is to warm up the stuck objectives in a oven to about 50 °C for some hours. May be then the problem is solved. Next step could be to bring in 1 or 2 droplets of Xylen in the cleavage of the stuck parts. And if this doesn't help warm it again.

Good luck!

For the record ... unfortunately it did not work for me. I did not however have access to Xylen. That might have made the difference I suppose.

[coominya]

Just checked. Your HI 100x1.30 objective appears to be an Olympus 36mm parfocality, oil immersion, not a phase, the metal collar IMO can be screwed out so the spring-loaded inside is visible. There should be a longitudinal slot in the fixed part. A single tiny drop of a light machine oil into that slot should start smooth recession of the lenses into the barrel.

That sounds like simple enough, I have some small syringes with needles attached I use for delicate oiling. I haven't touched it as yet, never wise to rush into maintenance of unknown mechanical things, they never seem to be as simple as the manual or logic suggests.

One thing I have noticed, and I'll mention it here, is that using the 40x or 100x objectives produces masses of wriggly lines visible inside my eyeball, floaters of a sort. I have tried adjusting the condenser to avoid them but to no avail. They do diminish with increased light intensity but that's no real solution.

[coominya]

For the record ... unfortunately it did not work for me. I did not however have access to Xylen. That might have made the difference I suppose.

What do you use to clean the oil off the objective 75RR?

[Hobbyst46]

One thing I have noticed, and I'll mention it here, is that using the 40x or 100x objectives produces masses of wriggly lines visible inside my eyeball, floaters of a sort. I have tried adjusting the condenser to avoid them but to no avail. They do diminish with increased light intensity but that's no real solution.

I suspect that you might modify the way you look through the eyepieces. The problem is not related to the condenser IMO.

If you already had experimented with immersion, I would first of all carefully and gently remove any oil that remained on the objective front lens. This is done very gently with lens tissue or (some think it is better) Kimwipe or Q-tip. Replace the tissue or tip after each swipe. Better still, absorb excess oil by touching a tissue to the objective, then swipe. Once or twice should suffice. Check if the 40x objective is not smeared with oil - it happens. Use a clean specimen slide with no oil on it.

Then switch to the 10x objective, open the iris such there is plenty of light on the specimen, take a piece of frosted glass or translucent "drawing" paper or even a translucent packing paper, hold it against the eyepiece. You see a spot of light on it. Increase the distance between the paper and the eyepiece, the spot becomes smaller in diameter. Notice the distance at which the spot is at a minimum. That is the eyepoint of your eyepieces. AT THIS DISTANCE YOUR EYES should be during an observation session.
If you wear eyeglasses, and the eyepoint is about 20mm away from the eyepiece, you need not take off your glasses while observing, because your eyepieces are designed for eyeglass wearers.

One more tip if I may. Always look through the eyepieces with both eyes, in a relaxed manner, do not try to strain your eyes or "wink" like taking aim at something. This holds whether the eyepoint is near the eyepiece or not.

Hope these help.

[75RR]

What do you use to clean the oil off the objective 75RR?

I have been using 70% isopropanol, though here is Spain it is mostly available at 99%.
I will have to test it when I obtain some and see which I prefer. One can apparently change the % by adding distilled water.
I apply it with cotton swabs which I wrap on a bamboo stick. See page 9 of the Clean Microscope: viewtopic.php?f=15&t=1428

... using the 40x or 100x objectives produces masses of wriggly lines visible inside my eyeball, floaters of a sort.

I find that the viewing angle has a lot to do with it. Try adjusting your chair, your posture or the height of the microscope.

[Hobbyst46]

I have some small syringes with needles attached I use for delicate oiling.

In the case that you do take off the collar to expose the spring-loaded part - and I am not recommending this, just saying it might be possible - I suggest that you do not inject oil into any crevice that you see. Just put one tiny drop in contact with the exposed inner part.

[Hobbyst46]

To remove very persistent oil I start with octane (light petroleum). Yet isopropanol is also very good, I take it straight - 95-100% (technical grade, available from an educational science equipment store). When pure, it is a better solvent for oil, I believe.

[billbillt]

Hi,

"One thing I have noticed, and I'll mention it here, is that using the 40x or 100x objectives produces masses of wriggly lines visible inside my eyeball, floaters of a sort. I have tried adjusting the condenser to avoid them but to no avail. They do diminish with increased light intensity but that's no real solution."

I think we all have this....

BillT