Oiling the condenser to the slide

[75RR]

This side topic came up on the other forum - thought it worth sharing here as there seems to be some ambiguity as to when it is appropriate/sensible/reasonable to save on clean-up by not oiling the condenser to the slide. (See particularly the paragraph in italics)
Personally, I think that if one is going to oil and then clean an objective it is not much more trouble to do the same to the condenser.

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

To quote from it:
' A factor that is commonly overlooked when using oil immersion objectives of increased numerical aperture is limitations placed on the system by the substage condenser. In a situation where an oil objective of NA = 1.40 is being used to image a specimen with a substage condenser of smaller numerical aperture (1.0 for example), the lower numerical aperture of the condenser overrides that of the objective and the total NA of the system is limited to 1.0, the numerical aperture of the condenser.

Modern substage condensers often have a high degree of correction (see our section on condensers) with numerical aperture values ranging between 1.0 and 1.40. In order to effectively utilize all the benefits of oil immersion, the interface between the substage condenser front lens and the underside of the microscope slide containing the specimen should be also be immersed in oil.'
This system has been termed a Homogeneous Immersion System and it is the ideal situation to achieve maximum numerical aperture and resolution in an optical microscope. In this case, the refractive index and dispersion of the objective front lens, immersion oil, substage condenser front lens, and the mounting medium are equal or very near equal. In this ideal system, an oblique light ray can pass through the condenser lens and completely through the microscope slide, immersion oil, and mounting medium undeviated by refraction at oil-glass or mounting medium-glass interfaces.

When using high-power achromat oil immersion objectives, it is sometimes permissible to omit the step of oiling the condenser top lens. This is because the condenser aperture diaphragm must often be reduced with lesser-corrected objectives to eliminate artifacts and provide optimum imaging. The reduction in diaphragm size reduces the potential increase in numerical aperture (provided by oiling the condenser lens) so the loss in image quality under these conditions is usually negligible.


Glycerin, refractive index 1.47 may be a viable alternative as the liquid between the upper surface of the condenser top lens and the underside of the slide, with a rather easier clean up as it is water miscible. Even water, refractive index 1.33 would presumably be better than air. However the full advantages of truly homogenous immersion would then be lost.

[75RR]

This might help clarify the previous post:

Image from: FUNDAMENTALS OF LIGHT MICROSCOPY AND ELECTRONIC IMAGING
Douglas B. Murphy
http://www.biology.uoc.gr/courses/BIOL4 ... s/book.pdf

[apochronaut]

The way I see this question.


There has been quite a bit of discussion about this topic, just about anywhere microscopes or microscopy is discussed. I don't think it is debatable that in order to obtain the highest possible resolution from any objective with a potential N.A. of 1 or over, that oiling the condenser is advantageous. I agree that the effort to clean the condenser and slide of oil afterwards, is small. It is in fact much less work than cleaning the objective, which requires some magnification in order to do a thorough job. Where I need a quick assessment of a sample, I tend to use a dry .90 achromat condenser, irregardless of whether I am oiling an objective or not. If my assessment is lengthy or necessarily more detailed, I always oil a condenser with a higher N.A.

The use of alternative immersion mediums that are of a lower n , and being water soluble for cleanup, will give some satisfactory results with some oil objectives but not with others. Some objectives seem to exhibit excessive longitudinal spherical aberration internally, when not provided with the correct operating specifications, in terms of working distance. The n of the space between the front element of the objective and the coverslip, will affect the working distance. A case in point is the dramatic difference between the AO iris equipped objectives # 1028, a 50X .85 oil immersion achromat and the #1016, a 50X .80 oil immersion advanced planachro. The # 1028, despite it's higher N.A., performs extremely well, dry, with water or oil : in fact it does so well, that it is a better objective dry than several AO 45X .66 achromats. The # 1016, is a whole other story, clearly requiring oil immersion to achieve an adequate performance level.

Using an alternative immersion medium for a condenser, is a little more problematic. I have tried water but never glycerin. With water, I did not find any value in doing so, probably because dry achromats provide better imaging than an oiled abbe and there was no advantage using water with one of those, and using water on an abbe aspheric or an aplanat achromat, doesn't make any sense to me, because the cleanup of oil is so relatively easy and you have to clean up water or glycerin anyway too.

As to whether an unnoiled condenser , for example a .90 dry achromat will limit a 1.30 oil immersion objective to a functional N.A. of .90 is something my experience does not support. I know that the theory is there but there are also sources that claim that in practice, the functional N.A. of the objective would be somewhere between .90 and 1.30., which is the camp I fall into, at this point.
Using an oil immersion objective with an iris diaphragm and N.A. markings for that diaphragm on the barrel, however , should be a good enough tool in order to test the theory against practice.

[75RR]

This is probably a good place to re-post "The Clean Microscope" by Zeiss link: http://microscopy.duke.edu/downloads/Th ... scsope.pdf

It gives very useful cleaning tips.

I seem to have settled on:
air blower: I use an ear dropper (I keep it in a sealed bag)
absorbent paper: with which I remove the excess oil with by laying it on the lens and gently patting and then peeling off
Isopropanol (alcohol): which I use with cotton wool swabs which I make with long, thin wooden bamboo sticks (I blunt the ends with sandpaper)

[billbillt]

I had forgotten about this.. Thanks for re-posting...

BillT

[kinase]

I'm having this issue. I have an oil 40x and 100x both with an NA of around 1.4 and the condensers NA is like .5. I need to see If we can get a better condenser. It's hard to find info about condensers on the manufacturers site, anybody know where they sell one? It's for very new scopes. I think I might need to get a NA 1.4 or so condenser for an upright and put it on inverted...

I was talking to an olympus rep today and he said they made a custom solution for a guy who uses two identical objectives, one as the objective, and the other as the condensor, both silicon oiled to the slide for transmitted light and obviously your objective functions as your condenser for fluorescence microscopy. But for transmitted light, he uses two objectives to make the system.

[75RR]

Hi kinase,

is this for one of the University microscopes? What make and model?

I would have thought there would be an off the self condenser that would suit you.
Was the rep you were talking to from the same manufacturer as the microscope you referred to?
Did he say there wasn't one?

.5 NA seems very low, is that a special condenser for a specific illumination technique?
Are the objectives from that microscope or did you borrow them from another?

Agree that some websites are so very badly designed that trying to find things can be a frustrating experience.

[gekko]

I think I might need to get a NA 1.4 or so condenser for an upright and put it on inverted...

Apologies for butting in but I am having a hard time visualizing this. Are you using the inverted scope for observing prepared slides and putting the slide upside down on the stage? [Edit:] Or is the object between two cover glasses if you use a high NA objective as a condenser?

[apochronaut]

I'm having this issue. I have an oil 40x and 100x both with an NA of around 1.4 and the condensers NA is like .5. I need to see If we can get a better condenser. It's hard to find info about condensers on the manufacturers site, anybody know where they sell one? It's for very new scopes. I think I might need to get a NA 1.4 or so condenser for an upright and put it on inverted...

I was talking to an olympus rep today and he said they made a custom solution for a guy who uses two identical objectives, one as the objective, and the other as the condensor, both silicon oiled to the slide for transmitted light and obviously your objective functions as your condenser for fluorescence microscopy. But for transmitted light, he uses two objectives to make the system.

Both Baker ( probably later , Vickers too) and Spencer/AO made a condenser body that had an RMS thread for the condenser. I am most familiar with the Spencer model. It normally came with a 1.3 N.A. achromat in it but one could use an objective for the condenser. Baker also made a cardioid DF condenser nose to fit such a body.
viewtopic.php?f=5&t=2951

[kinase]

Actually we got in contact with someone who has a 1.4NA condenser, even better than what I was hoping for. It's an old Zeiss inverted microscope, I think an Axiovert 100M. I have a 40x and 100x of NA~ 1.4 for it so that condenser should work great as long as it still has DIC on it. The current .5 one has DIC and phase but I think it's just made to work with any objective you put on there. Anyway, problem solved, hopefully I'll be able to get some super high resolution images to show you guys.

Truthfully though I think this old microscope is going to break soon and last I heard Zeiss doesn't want to attempt to repair it. I would prefer to try and trade in the good 40x and 100x objectives we have and get a high NA oil 40X and 60X for our Olympus IX73. It's way easier to use and everything is still factory aligned and all. Plus the fluorescence source is LED's instead of a mercury bulb and I also have DIC and phase. But that would cost more in the short term, I think it'd save money in the long run. Olympus has these cool silicon oil objectives that seem to work quite well for deep tissue imaging, not that I usually do that.

I usually use a glass bottom dish, a small petri dish with a coverslip for a bottom. But yeah If I make a slide, it's usually with something that's fixed and embedded in resin or epoxy with DAPI and a coverslip, you just put it upside down on the inverted scope. I guess I could use a water dipping objective as a condenser and have it dip into the water in the dish and that would also work quite well but I've only ever met one person with a water dipping objective and they were an electrophysiologist. Now they have some crazy microscopes.

Also apparently the objectives are hand made, some guy aligns all the glass by using a tiny hammer and rod and gently taps everything into place. They're master craftsmen.

[gekko]

I usually use a glass bottom dish, a small petri dish with a coverslip for a bottom. I guess I could use a water dipping objective as a condenser and have it dip into the water in the dish and that would also work quite well but I've only ever met one person with a water dipping objective and they were an electrophysiologist. Now they have some crazy microscopes.

I am not up-to-date with what is available, but I find it hard to visualize a condenser with NA of 0.9 used with a petri dish without it virtually dipping into the water (I guess they could make one but it would probably cost a whole lot**). I guess a water-immersion objective (Lomo are very common, there are Zeiss ones also, but I'm not sure what the NA is) could be used. Will you have to modify the light collector optics to get the full illumination available? Interesting problem, and please do post the solution that you come to.

** and may need a counterweight arrangement to prevent it from crashing into your petri dish .

[kinase]

I'm not quite so sure how this is all going to work. The olympus rep said we could certainly do it with the IX73 we have but I think the PI is going to opt for trying to get the condenser for the Zeiss because that'll be way cheaper in the short run. I know one person who bought a high NA condenser for an upright and just mounted it upside down on an inverted scope and their setup works. And we have a 3d printer so I'm sure I can figure something out.

But now I realize why people don't often have this issue and why I've never really seen anybody with high NA condensers. It's because the only reason biomedical sciences people use microscopes is to check cells, look at H&E stains, or fluorescence. The first requires only that you can see that there's a cell there. The second requires reasonable resolution at low mags and accurate colors, and the third uses the objective as the condenser so there's little reason to have a high NA condenser.

One day I'll be in charge of ordering a lab scope...