100x Concerns

[linuxusr]

I am now spending much time using my 100x and it has opened a world not possible with my 40x as regards identifying internal morphology and appreciating geometry of various protista that now make up 1/3 to 1/2 of my FOV.

That being said, I'm still getting my feet wet. Two concerns:

I'm having a hard time being confident that I'm getting that oil drop on my front lens. Being that it's upside down, albeit at a ~ 45 degree angle, and that I'm low vision, it's not an easy task:

a. If I removed the objective, I could drop that drop dead-on. This, however, would be a bad idea as I would risk dropping the objective, it has to be threaded in at an exact angle (not easy), and I'd be allowing ambient dust to enter the open end,
b. Using immersion oil Cargille, Type A, I'd like to think that I could squeeze out a nice bead atop the nozzle, touch it to the front lens opening, and it would adhere. But this is not what happens. The drop drips down the side of the nozzle, and I have to apply more than one drop "to be sure."
c. Someone recommended placing the drop on a coverglass and bringing that to the lens. That doesn't work for me as the drop slides off because the lens is angled. **I could figure out a way to have my 100x at 90 degrees, then lift the slip perpendicularly. I'd have to be careful that in the rotation, a dry objective does not pass through my slide that already has oil on it. Perhaps I should reverse the process and get the oil on the objective first, then place the slide. What I like to do is get my specimen set up, before putting oil on the slide, with full koehler implemented and precise focus at 40x, so given parfocality, when I move in my 100x, I'm ready to go except for fine focus and light and condenser aperture.
d. I haven't used a glass rod, but I know I can use them for distributing debris on a slide from a hay infusion, so I was thinking that maybe I could get a nice bead of oil dropped onto the end of a glass rod, and use that to apply the oil. Has anyone tried that?

The second concern is that whereas Zeiss recommends coverslips no > 0.17 mm, my prepared slides use other items, for example, Carolina Biological Supply uses circles of glass that far exceed 0.17 mm. And my WD is limited. Yes, I'm quite fearful when I notice slippage in my specimen when using fine focus, knowing that the barrel of my objective is touching the glass! I'm writing to my Zeiss representative to confirm that my objective is spring loaded as I could not confirm this in the specs. If it is spring loaded, can I confirm this manually? I'm feeling that I'm OK because I observe that my front lens is recessed in the barrel by a few mm, meaning that what is touching the glass on my slide is not the lens but the metal barrel.

Thoughts, observations, opinions?

[Greg Howald]

Type B oil has higher viscosity than type A. Maybe the thicker oil would have a greater tendency to stay where you put it until the objective engages with the slide.
Sorry. Beyond that I don't have any other ideas.
Greg

[Hobbyst46]

...What I like to do is get my specimen set up, before putting oil on the slide, with full koehler implemented and precise focus at 40x, so given parfocality, when I move in my 100x, I'm ready to go except for fine focus and light and condenser aperture.

Yes, correct.

... I haven't used a glass rod, but I know I can use them for distributing debris on a slide from a hay infusion, so I was thinking that maybe I could get a nice bead of oil dropped onto the end of a glass rod, and use that to apply the oil. Has anyone tried that?

Yes, and it works fine.

The second concern is that whereas Zeiss recommends coverslips no > 0.17 mm, my prepared slides use other items, for example, Carolina Biological Supply uses circles of glass that far exceed 0.17 mm. And my WD is limited. Yes, I'm quite fearful when I notice slippage in my specimen when using fine focus, knowing that the barrel of my objective is touching the glass! I'm writing to my Zeiss representative to confirm that my objective is spring loaded as I could not confirm this in the specs. If it is spring loaded, can I confirm this manually? I'm feeling that I'm OK because I observe that my front lens is recessed in the barrel by a few mm, meaning that what is touching the glass on my slide is not the lens but the metal barrel.

The WD of a 100X objective is about 0.1mm. So very thick coverslips will blur the image.

Yet, having observed hundreds to thousands of slides with immersion, it is a straightforward operation that rarely results in significant damage.

First of all, press upwards, lightly, with the tip of your finger, on the end (bottom point when installed on the nosepiece) of the 100x objective. If it moves up easily, it is spring loaded (99.99999% it is). If not, might be it is stuck, which should never happen with any objective that is not really old.

Cargille Type A is fine, not overly viscous, is more fluid in warm climates than in cold climates but is always OK.
There is no need to limit the amount of oil between the objective lens and the coverslip. One drop, two drops, three drops, whatever. In principle oil drops are smaller than water drops but it is not important. After the 40X objective has shown the correct image (as you wrote), swing the nosepiece half-way, so no objective is above the coverslip. Place a drop (or more) of oil on the coverslip. Rotate the nosepiece further to bring the 100x into position. While doing it, do not look through the eyepieces! rather, look sideways at the coverslip, such that you see from the side how the lens approaches the coverslip. If in doubt, lower the stage with the fine focus some (NOT the coarse focus!). Then, observe through the eyepieces and adjust the fine focus.

Remember, that the 100x will yield an image, even in the absence of oil. It will be blurred, yes, but focusing will have an effect. The oil is mandatory for a sharp well defined image. If under immersion the image with the 100x is not focused, and focusing seems to have no effect, rotate the nosepiece half-way, put some more oil on the coverslip, and return the 100x objective to position.

One issue with immersion oil is air bubbles. You will know them when you see them, floating about and obscuring the view. Application of the oil with a glass rod rather than a pipette helps to avoid them.

Another issue is that pressure of the lens, through the oil, on the coverslip can shift or stress the specimen under the coverslip. Yet neither the coverslip or lens will be damaged. It has happened to me, so there is a slight risk to the slide. Yet nothing serious to worry about.

Arrange the objectives in the nosepiece such that you always turn the nosepiece in one direction, like 4x>10x>40x>100x, to minimize the risk of bringing a dry lens with oil. This way, when you are done with immersion, the next lens is 4X which has a huge WD so is unlikely to dip in oil.

Good luck. It is really fairly easy.

[dtsh]

d. I haven't used a glass rod, but I know I can use them for distributing debris on a slide from a hay infusion, so I was thinking that maybe I could get a nice bead of oil dropped onto the end of a glass rod, and use that to apply the oil. Has anyone tried that?

Some bottles of oil come with a rod attached to the cap. I have a bottle of Cargille (30cc/1fl.oz) that includes such an applicator.

This looks to be the same as the one I have, the description states it has the applicator and all else looks identical.
https://www.amazon.com/AmScope-Microsco ... B0099360HG

and a link to type B that also states it has an applicator
https://www.amazon.com/AmScope-ML-B-1-M ... B0186GTK5U

[Rossf]

Hi linuxusr -I’ve used the technique where you lower objective till it just touches the oil drop on the coverslip then rotate the nose back and forth just slightly to “bump” out air bubbles. I’ve also heard of people using a fine brush to apply oil to front of objective as well as place a drop in normal fashion on coverslip -some 100x objectives have a small half sphere recess in the front element which can make oil immersion a bit more tricky.Actually some of my inverted non oil objectives have this type of recess-just an optical design quirk I guess-Try to get some decent coverslips-I got some Zeiss #1.5 .17 coverslips on eBay quite cheap with very fine tolerance of .05mm either way and they have improved immersion work quite a bit.Actually they improve performance of all objectives 40x up.
Regards ross

[PeteM]

My preferences:

1) Immersion oil with the tube shaped long nose plastic dropper built in. Far easier than a glass rod or conventional dropper to put a drop of oil where you want.

2) If your objectives aren't already parfocal, use parfocal shims to get the 4x through 40x pretty much on focus. It's OK and maybe even desirable if you need to focus a tiny bit closer for the 100x or other oil immersion objectives.

3) Go through the normal progression of scanning at low magnification and find some detail you want to see better at 40x dry.

4) Rotate the turret so you have a clear area of the slide between to 40x and 100x oil immersion. Put a drop of immersion oil right on the tiny spot of the cover slip that's now illuminated by the condenser.

5) Gently rotate the 100x into place. If your objectives are near parfocal you'll have an image. By gently rotating /swiping the objective into place you're less likely to have air bubbles. Tweak the fine focus for perfection.

[MicroBob]

By gently rotating /swiping the objective into place you're less likely to have air bubbles.

I have the habit to swing once into and through the oil puddle and backwards in again. This reduces the risk of trapped air under the front lens, especially with objectives that are not already oily at the tip. Usually not necessary but it is just a brief additional movement.

[lorez2]

"I'm having a hard time being confident that I'm getting that oil drop on my front lens. Being that it's upside down, albeit at a ~ 45 degree angle"

I don't understand this.

Thanks,

lorez

[BramHuntingNematodes]

Maybe the whole stage is tilted up 45 degrees is all I can figure mayb a picture might help I never heard of this problem before

[PeteM]

My reading was that the OP was trying to apply the oil directly to the objective, rather than the slide, and had the objective rotated out of position to make the attempt. Hence the desire for a sticky immersion oil and repeated mentions of applying the oil directly to the objective lens. Either that or he was getting a jump on April 1st?

We do call them oil immersion objectives; not oil immersion cover slips . . . someone starting from scratch could make this mistake.

And maybe that explains why so many imagine they want a 60x dry objective instead of a 100x oil objective???

Perhaps someone has made a 30 second YouTube video showing how easy oil immersion really can be -- and will link it here?

[linuxusr]

@lorez
@BramHuntingNematodes

Per PeteM:

My reading was that the OP was trying to apply the oil directly to the objective, rather than the slide, and had the objective rotated out of position to make the attempt. Hence the desire for a sticky immersion oil and repeated mentions of applying the oil directly to the objective lens.

Yes, this is correct. Actually, I apply both to the slide and the objective but since applying to the slide is not an issue, I had only mentioned the objective. I believe I have a solution but must get to my scope first to try it out. I will report later.

@ ET AL:

Thanks for your thoughtful posts. I'll get back to you in the next few days while I try some stuff out on my scope and dig more into this topic. E.G. Viscosity choice is subjective and/or dependent on objective factors such as ambient temperature and does not affect n refractive index.

[linuxusr]

@Hobbyst46
@dtsh
@Rossf
@PeteM
@MicroBob

With the help of your posts and playing with my scope last night, I solved the problem of applying immersion oil to my 100x. The problem is that I was preparing my slide first: With my specimen focused on my 40x with Koehler illum. implemented, this forced me to apply oil to my 100x while it was angled. Now I reversed the process. First I oil the 100x: Drop the stage and rotate in the 100x at 90 degrees in the path of the optical axis perpendicular to the stage. I then squeeze out 2 or 3 drops, making a single large globule on a microscope slide. A slide gives me better control than a coverglass. Holding the slide with both hands I raise it perpendicularly to the objective, touching it to the barrel, where it adheres at the center and, likely penetrates to the front lens, since there is a space of several mm. I then rotate the 100x out of the way, prepare my slide with the 40x as before, then rotate in the 100x, swiveling it back and forth a few times to coalesce the drops above and below. This works great. I do not re-oil my objective every time I use another slide but I do so after a few slides, keeping my oiled slide at hand. Further, with my light turned up, I get a pinpoint of light in the center of my oil on the slide (the optical axis). This serves as a guide when I lift the slide. (I need to confirm this latter point a little more clearly.)

On the subject of viscosity, given that I am in a tropical climate around 34 C, I would do better with Type B rather than Type A. Meanwhile, I went to Wikipedia < "Oil Immersion" < External links < and found an article by none other than John J. Cargille: https://web.archive.org/web/2011091119 ... ope.shtml published in New York Microscopical Society Yearbook, 1985 from which I quote an excerpt on viscosity:

Viscosity of immersion oil is mostly a matter of individual preference of the microscopist. Low viscosity oils are more likely to creep if applied over-abundantly. The low viscosity (150 cSt) is preferred by some when the distance from the cover glass to the objective is very small. Low viscosity oils are less apt to retain small bubbles.

Higher viscosities, (approximately 1250 cSt) have been found to be the most in demand and are supplied by more microscope manufacturers than any other. The higher viscosity oils fill the larger gaps more satisfactorily and are also reusable in that a second slide can be positioned and swung into place and contact made with the oil drop remaining on the objective lens. The 1250 cSt viscosity gives more latitude with oiled condensers since it will fill a larger gap without "breaking" and making re oiling unnecessary.

"Very high viscosity" (21,000 cSt) oil is particularly useful for wide condenser gaps, long focus (low power) oil immersion objectives, horizontal or sharply inclined instruments and some micro projectors. If the manufactured viscosity is not the most suitable, blends can be made. If a blend is desirable, an immersion oil manufacturer may mix a small special lot when the material is ordered as a percentage of different types of the regular line.

A couple of points here. First, there is no right or wrong on viscosity. It depends on your individual use needs (for example, my ambient temperature) and your preference. Second, "cSt" is the value that measures viscosity. Third, you can mix oils! Regardless of what you use, the refractive index remains the same.

I checked out cargille.com -- As examples Type A = cSt 150; Type B = 1250; NVH = 21,000; OVH = 46,000. They sell a nice sampler set which I intend to purchase. They are located in the state of New Jersey, U.S. It costs USD $24.75 and includes five 0.25 oz. bottles: 2 of Type A; 1 of Type B; 1 of Type 300; 1 of Type NVH.

If you read his article, you will find oilable condensers with oil both underneath the slide and on the slide. I am assuming that this requires a special condenser. Does anyone have one of these?

[dtsh]

If you read his article, you will find oilable condensers with oil both underneath the slide and on the slide. I am assuming that this requires a special condenser. Does anyone have one of these?

Most of the high NA condensers are intnded fo use with oil, I don't think one can get the high NA without it. Do you know what model yours is? It's NA? My limited understanding of it says that it is typical to oil slide to condenser and slide to objective. I'm sure there have been setups that deviate from that, but I think that's the norm for high NA condensers. That is the case with all of mine that are 1.25 NA, only my 0.9 NA condensers are not designed for use with oil.

[linuxusr]

@dtsh

I never saw if my condenser lens was specced (NA). I will have to check. I am, however, focusing my light source from the field illuminator via moving the condenser lens in the Z direction.

[MicroBob]

I apply my immersion oil to the objective this way:

- Slide with specimen and cover slip on sage
- Focus with objective with enough free working distance to leave room for a drop
- Swing nosepiece to intermediate position between 2 ports
- Apply oil onto cover slip with dropper bottle or small wood stick or whatever is useful to reach the spot
- Swing 100:1 objective into the oil drop, once through and back in, not passing the 40:1 through the oil on the way...
- Specimen is close to focus now

The immersion oil has to fill the whole gap between front lens and cover slip and do this even after moving th slide under the objective while observing. With a bertrand lens or phase telescope you can observe the back lens of the objective. Here it shows when too little oil is applied. Since you have brand microscopes it should be easy to find out the aperture of the condenser. If it is an oil immersion condenser it can go beyond n.a. 0,95 when oiled, if it is a dry condenser it can't.

Bob

[viktor j nilsson]

I use a small syringe with a 21G needle (0.8mm x 40mm) to apply immersion oil. I find that I can have much better control over placement and quantity this way.

[Hobbyst46]

I use a small syringe with a 21G needle (0.8mm x 40mm) to apply immersion oil. I find that I can have much better control over placement and quantity this way.

There are even blunt, short (0.5" long) or long syringe needles, very inexpensive from China. I bought #27 and #23 for other purposes.

[mrsonchus]

Hi, these are the ones I use, also for application of stain, water etc around the edges of coverslips - very handy indeed.

[linuxusr]

@MicroBob

Could you please give a brief description of how I use a Bertrand lens as well as how I buy one? Also, to view surface placement of the oil, why would I want to view the back lens and not the front lens?

[linuxusr]

@MicroBob #2:

I must have a dry condenser. If I have a condenser made for oil, I'm sure it would have been noted in the specs, which it wasn't. Is there any point in applying oil to a dry condenser lens and should it even be done?

[linuxusr]

I apply my immersion oil to the objective this way:

- Slide with specimen and cover slip on sage
- Focus with objective with enough free working distance to leave room for a drop
- Swing nosepiece to intermediate position between 2 ports
- Apply oil onto cover slip with dropper bottle or small wood stick or whatever is useful to reach the spot
- Swing 100:1 objective into the oil drop, once through and back in, not passing the 40:1 through the oil on the way...
- Specimen is close to focus now

The immersion oil has to fill the whole gap between front lens and cover slip and do this even after moving th slide under the objective while observing. With a bertrand lens or phase telescope you can observe the back lens of the objective. Here it shows when too little oil is applied. Since you have brand microscopes it should be easy to find out the aperture of the condenser. If it is an oil immersion condenser it can go beyond n.a. 0,95 when oiled, if it is a dry condenser it can't.

Bob

I just found the "Reply with Quote" icon!! Please see my responses below "@MicroBob"

[75RR]

@MicroBob #2:

I must have a dry condenser. If I have a condenser made for oil, I'm sure it would have been noted in the specs, which it wasn't. Is there any point in applying oil to a dry condenser lens and should it even be done?

Dry condensers are not sealed and therefore must not be oiled!

Dry condensers go up to 0.9 NA while most oil condensers start at 1.25 NA

Can you provide a photo of your condenser?

[linuxusr]

Most of the high NA condensers are intnded fo use with oil, I don't think one can get the high NA without it. Do you know what model yours is? It's NA? My limited understanding of it says that it is typical to oil slide to condenser and slide to objective. I'm sure there have been setups that deviate from that, but I think that's the norm for high NA condensers. That is the case with all of mine that are 1.25 NA, only my 0.9 NA condensers are not designed for use with oil.

I re-checked my condenser and find that it is 0.9 -- dry.

[linuxusr]

@MicroBob

Could you please give a brief description of how I use a Bertrand lens as well as how I buy one? Also, to view surface placement of the oil, why would I want to view the back lens and not the front lens?

Repeat for MicroBob or anyone else!

[75RR]

@MicroBob

Could you please give a brief description of how I use a Bertrand lens as well as how I buy one? Also, to view surface placement of the oil, why would I want to view the back lens and not the front lens?

Repeat for MicroBob or anyone else!

A Bertrand lens is also known as a Phase Telescope

For the Objective Rear Focal Plane see this video: https://www.youtube.com/watch?v=60_jgZtyR6U

and the Köhler diagram (from Fundamentals of light microscopy and electronic imaging by Douglas B. Murphy) below:
.

[linuxusr]

First of all, press upwards, lightly, with the tip of your finger, on the end (bottom point when installed on the nosepiece) of the 100x objective. If it moves up easily, it is spring loaded (99.99999% it is).

I checked, placing a lens tissue over my finger. I was surprised to see that what retracted was an interior cylinder within the objective barrel. It is this much smaller diameter cylinder (within the objective barrel) that must include the lenses. I also noted that this cylinder is marked with a second white ring which I had not seen before. Also, my 40x is spring-loaded. I would assume that most 40x's are not . . .

[Hobbyst46]

First of all, press upwards, lightly, with the tip of your finger, on the end (bottom point when installed on the nosepiece) of the 100x objective. If it moves up easily, it is spring loaded (99.99999% it is).

I checked, placing a lens tissue over my finger. I was surprised to see that what retracted was an interior cylinder within the objective barrel. It is this much smaller diameter cylinder (within the objective barrel) that must include the lenses. I also noted that this cylinder is marked with a second white ring which I had not seen before. Also, my 40x is spring-loaded. I would assume that most 40x's are not . . .

On the contrary, most, if not all, are. Also, even lower magnification objectives are spring loaded if their working distance is shallow - for example if they are apochromats.

[MicroBob]

Could you please give a brief description of how I use a Bertrand lens as well as how I buy one? Also, to view surface placement of the oil, why would I want to view the back lens and not the front lens?

Hi, some microscopes (like my 1960s Zeiss Photomicroscope 1) have an included Bertrand lens, often combined with a magnification changer. When I select this lens in its revolvolving holder I can focus it right through the lens stack of the objetive. When the condenser aperture is the same as the objective's aperture the back lens is fully flooded with light. When the condenser aperture is reduced it shows in form of a dark border around the lit center of the back lens. With your 100:1 objective of perhaps an n.a. of 1,25 and your 0,9 condenser you will always have such a dark border, which in not bad. Usually one closes the condenser aperture to a little below the objectives aperture to improve contrast. The lit area should be well centered and evenly round. With too little oil it will lokk different. The name phase telescope comes from the way it enlarges the image of the back lens for better visual assesment. So if your microscope doesn't have an included Bertrand lens you will need a phase telescope. These are fairly basic things and can be from every brand as long as it fits your eyepiece tubes. If you buy one with RMS diameter and have 30mm tubes an adapter could be used. Once you have some experience you can simply peer into the tube after removing th eyepiece with bare eyes and get a fairly good idea about how good the microscope is set up.

Bob

[linuxusr]

@75RR

Evennett is great. He explains simply, slowly, and confidently. I have downloaded the video. Thanks.

[linuxusr]

@MicroBob

I think I'll start looking in my eye tube while adjusting various parameters and see if I can infer what I am looking at.