Is an Oil Distribution System Necessary for Optimal Oil Immersion Microscopy?

[cohkka]

For oil immersion microscopy, I understand that the oil is required to increase the numerical aperture, and thus the resolution, of the lens. I have tried looking into these microscopes and the only method I have seen of applying the oil is to place a dot of it on the lens and then lower the lens into the specimen. However, what if the lens and specimen are moving relative to each other? Wouldn't you need an oil layer of constant thickness? Also, when imaging very thin layers, wouldn't the oil be heated significantly by the light from the microscope?

Basically, my question is whether one would need to install an oil distribution system to prevent overheating of the oil and to ensure that an even layer is coating the lens. Do you know if this has been researched? I cannot find information on it anywhere!

I figure that the oil would have to be introduced in a laminar flow onto the coverslip but I'm just unsure if this is actually necessary. I would be dealing with nanoparticles, but none of which are living. Thanks for your help in advance!

 

[Andy Resnick]

For oil immersion microscopy, I understand that the oil is required to increase the numerical aperture, and thus the resolution, of the lens. I have tried looking into these microscopes and the only method I have seen of applying the oil is to place a dot of it on the lens and then lower the lens into the specimen. However, what if the lens and specimen are moving relative to each other? Wouldn't you need an oil layer of constant thickness? Also, when imaging very thin layers, wouldn't the oil be heated significantly by the light from the microscope?

Basically, my question is whether one would need to install an oil distribution system to prevent overheating of the oil and to ensure that an even layer is coating the lens. Do you know if this has been researched? I cannot find information on it anywhere!

I figure that the oil would have to be introduced in a laminar flow onto the coverslip but I'm just unsure if this is actually necessary. I would be dealing with nanoparticles, but none of which are living. Thanks for your help in advance!

Generally, immersion lenses require use of a coverslip; 'dipping' lenses are different, and are designed to be dipped directly into the (usually) aqueous medium in which the specimen resides.

If your coverslip is moving relative to the lens, then yes- you will need a oil dispensing mechanism. However, given the tight clearances (immersion lenses are often only a few tens of microns above the coverslip surface) and depth of focus (a fraction of a micron), I would wonder how you propose to maintain mechanical alignment between the objective and moving sample.

If you are imaging, for example, a fluid channel through which material flows, there's no issue- the coverslip and channel are stationary and the fluid flow is isolated from the environment via the coverslip.

It is true that the intense light at the focal plane can heat the sample (and everything else); most sources are IR filtered to minimize this effect. For critical applications (say cryogenic microscopy), the sample holder is thermally isolated from everything else (the stage motors will also heat the sample). Some immersion lenses have a correction collar to account for the thermally induced changes to the oil refractive index.

 

[cohkka]

Thanks for the reply! I'm a little confused though; what do you mean by maintaining mechanical alignment between the objective and moving sample? The specimen would be a layer of gel spun onto a substrate, so there is no flow, with a coverslip on top. Do you mean that in moving the specimen in the x-y plane, the focal point would be affected?

 

[Andy Resnick]

Thanks for the reply! I'm a little confused though; what do you mean by maintaining mechanical alignment between the objective and moving sample? The specimen would be a layer of gel spun onto a substrate, so there is no flow, with a coverslip on top. Do you mean that in moving the specimen in the x-y plane, the focal point would be affected?

Sorry- yes. Moving the sample in x-y will generally change the distance between the lens and sample due to misalignment of the sample plane (and directions of motion) with the optical axis of the microscope.

 

[cohkka]

Sorry- yes. Moving the sample in x-y will generally change the distance between the lens and sample due to misalignment of the sample plane (and directions of motion) with the optical axis of the microscope.

Ah okay, thanks! I hadn't thought of that so will have to take that into consideration!