Dry mounted slides (no coverslip) are hard to see

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ardeact
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Joined: Thu Jul 07, 2022 4:12 am

Dry mounted slides (no coverslip) are hard to see

#1 Post by ardeact » Thu Jul 07, 2022 4:22 am

I found that dry mounted slides, whether it be smears or dried samples, hard to focus and the resolve isn't as good as wet mounting slides using water as a medium. Of course, this has to do with Refractive index but I didn't know that not using a medium can render viewing unusable. Some sites say to use immersion oil directly on heat fixed dry slides as a medium, others say to never use oil without a cover glass. What is the correct way to view dry samples? Or is wet mounted the way to go.

Alexander
Posts: 402
Joined: Fri Jun 11, 2021 7:10 pm

Re: Dry mounted slides (no coverslip) are hard to see

#2 Post by Alexander » Thu Jul 07, 2022 5:38 am

Immersion oil directly on the sample works well as long as the sample is thrown away after inspection. Many immersion objectives are designed to work with or without a cover slip.

Dry objectives with a NA higher than about 0.5 will provide poor contrast and resolution without a cover slip. The picture will be quite blury. That's a well known issue.

apochronaut
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Joined: Fri May 15, 2015 12:15 am

Re: Dry mounted slides (no coverslip) are hard to see

#3 Post by apochronaut » Thu Jul 07, 2022 2:10 pm

These planes of interface: the object/mounting medium, the mounting medium/bottom of coverslip, the top of the coverslip/immersion medium as well as the immersion medium/front surface of the objective are areas where spherical aberration (sa) can take place prior to the image forming rays enter the objective. Spherical aberration is the result of component rays of the image not coming to focus at the same point. The sa formed at each of those zones results in a cummulative sa that needs to be corrected for by applying an equal and opposite sa in the objective, largely within the first lens interspace and second lens cell but also further downstream in some cases.
This is accomplished by the designer knowing what interfaces will take place and the refractive indices of the components. For instance , with a high refractive index mount such as balsam used with oil immersion, the sa between the object and objective will be negligable. This condition has been called homogeneous immersion. Once refractive index differences are introduced however, such as air being used as an immersion medium, sa increases and changes to the objective design must be made accordingly. Departing from an objective's design parameters will result in uncorrected sa being introduced, which will cause varying degrees of loss of contrast and resolution, resulting in a washed out , fuzzy image.

It is true that in production situations, oil immersion can be used directly on stained, dried smears but cover slip corrected oil immersion objectives are not the best for that, especially in warm conditions where the oil can puddle. A higher viscosity oil, such as that normally used with inverted microscopes will keep a higher profile meniscus and more easily replicate an oil-coverslip immersion.
Dry objectives as Alexander pointed out above, over .50 N.A. will have too much sa to be of value. The exact cuttoff point is a bit debatable because despite identical N.A.s, different objective designs are affected more or less by a change in immersion specifications. To be safe, generally .40 will be a guaranteed cutoff point. That is one of the reasons so many manufacturers of 20X objectives stick with .40 or .45 , so that a quick uncovered preparation can have good results and so that the objective can still be serviceable for uncovered incident illumination if need be.
The rules of thumb are : / - means cover or no cover o.k. : / 0 means no cover only : / .17 normally but can be an other number means you should stick to that quideline but small variances of up to .3 +- are o.k. for standard N.A. objectives and tighter for higher N.A. If the specification is a range of numbers, stick to that.

Homogeneous immersion, HI, Homog. Imm., H immer. etc. means any combination of standard glass covers and immersion oil can be used including oil only.
Many companies made/make high N.A. dry objectives for use with dried uncovered smears. While those objectives are all .90 or .95, they still provide 1000X magnification with high resolution and save time.

ardeact
Posts: 3
Joined: Thu Jul 07, 2022 4:12 am

Re: Dry mounted slides (no coverslip) are hard to see

#4 Post by ardeact » Sat Jul 09, 2022 4:32 am

apochronaut wrote:
Thu Jul 07, 2022 2:10 pm
These planes of interface: the object/mounting medium, the mounting medium/bottom of coverslip, the top of the coverslip/immersion medium as well as the immersion medium/front surface of the objective are areas where spherical aberration (sa) can take place prior to the image forming rays enter the objective. Spherical aberration is the result of component rays of the image not coming to focus at the same point. The sa formed at each of those zones results in a cummulative sa that needs to be corrected for by applying an equal and opposite sa in the objective, largely within the first lens interspace and second lens cell but also further downstream in some cases.
This is accomplished by the designer knowing what interfaces will take place and the refractive indices of the components. For instance , with a high refractive index mount such as balsam used with oil immersion, the sa between the object and objective will be negligable. This condition has been called homogeneous immersion. Once refractive index differences are introduced however, such as air being used as an immersion medium, sa increases and changes to the objective design must be made accordingly. Departing from an objective's design parameters will result in uncorrected sa being introduced, which will cause varying degrees of loss of contrast and resolution, resulting in a washed out , fuzzy image.

It is true that in production situations, oil immersion can be used directly on stained, dried smears but cover slip corrected oil immersion objectives are not the best for that, especially in warm conditions where the oil can puddle. A higher viscosity oil, such as that normally used with inverted microscopes will keep a higher profile meniscus and more easily replicate an oil-coverslip immersion.
Dry objectives as Alexander pointed out above, over .50 N.A. will have too much sa to be of value. The exact cuttoff point is a bit debatable because despite identical N.A.s, different objective designs are affected more or less by a change in immersion specifications. To be safe, generally .40 will be a guaranteed cutoff point. That is one of the reasons so many manufacturers of 20X objectives stick with .40 or .45 , so that a quick uncovered preparation can have good results and so that the objective can still be serviceable for uncovered incident illumination if need be.
The rules of thumb are : / - means cover or no cover o.k. : / 0 means no cover only : / .17 normally but can be an other number means you should stick to that quideline but small variances of up to .3 +- are o.k. for standard N.A. objectives and tighter for higher N.A. If the specification is a range of numbers, stick to that.

Homogeneous immersion, HI, Homog. Imm., H immer. etc. means any combination of standard glass covers and immersion oil can be used including oil only.
Many companies made/make high N.A. dry objectives for use with dried uncovered smears. While those objectives are all .90 or .95, they still provide 1000X magnification with high resolution and save time.
Very helpful, I didn't know about spherical aberration or following objective parameters.

Tom Jones
Posts: 336
Joined: Wed Apr 13, 2016 3:47 pm

Re: Dry mounted slides (no coverslip) are hard to see

#5 Post by Tom Jones » Sun Jul 10, 2022 4:09 pm

In clinical labs dry mounted blood smears get a thin coating of immersion oil over them prior to examination with low power objectives to reduce diffraction. Horrible images otherwise. Body fluid smears and grams stains, too. It acts just like a coverslip. Another drop is added when going up to 100x, and wiped off with a glass rod afterward.

For medium term storage we soaked the slides in xylene or Citrisolve to remove the oil. For long term preservation a regular mountant and cover slip was added.

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