What is the real optimal cover slip thickness?

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farnsy
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What is the real optimal cover slip thickness?

#1 Post by farnsy » Wed May 12, 2021 9:30 pm

I have a new microscope on the way and it has some high NA dry objectives without correction collars. I have a high-precision micrometer and am willing to go through my cover slips to find the ones with the best thickness, but I'm not actually sure what that thickness is--I see contradictory information around.

Objectives almost always say they are optimized for .17mm. This article suggests that we need to buy #1.5H cover slips and maybe even look through those for ones that are closest to .17. Ok, that's one answer that I have seen around: go for the thickness that is listed on your objective.

In other locations we read that because the specimen is often not right up against the cover slip, it's better to use a thinner cover slip. I guess that's the reason #1 thickness cover slips are so common.

But in the latter case, how much thinner should the cover slip be? Is it only distance that matters? So for example, if the specimen is .04mm from the cover slip, would the optimal cover slip be .13mm? Or does the fact that the specimen is suspended in water, and not glass, make a difference in the calculation? I'm not actually sure, optically, what the math is here that makes cover slip thickness so important.

Can you help clear this up?

MicroBob
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Re: What is the real optimal cover slip thickness?

#2 Post by MicroBob » Thu May 13, 2021 5:14 pm

farnsy wrote:
Wed May 12, 2021 9:30 pm
Or does the fact that the specimen is suspended in water, and not glass, make a difference in the calculation?
Hi,
from my understanding you could add up cover slip and mountant layer when both would have the same refractive index. With the waters lower refractive index the calculation would change a bit. Since there are other factors that contribute I would suggest to test it yourself. You have the means to measure the cover slips so you can make test slides to find out what cover slip gives the best image for you specimen and your actual objectives (they can be off a little or the manufacturer has already calculated in a water layer for you). This will also give you a better idea of the theoretical and practical side of this aspect. Sometimes things do work while theory sais no. :lol:

Bob

BramHuntingNematodes
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Re: What is the real optimal cover slip thickness?

#3 Post by BramHuntingNematodes » Thu May 13, 2021 6:19 pm

Bausch and Lomb objectives are all calibrated for coverslips of .18mm, which aren't really manufactured, perhaps taking into account the mountant before the subject.
1942 Bausch and Lomb Series T Dynoptic, Custom Illumination

wabutter
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Re: What is the real optimal cover slip thickness?

#4 Post by wabutter » Mon May 17, 2021 3:44 am

Successful imaging on a biological microscope is highly dependent on the coverslip and the thickness of the mounting media. Modern objectives are generally corrected to a 0.17mm thickness. The closest coverslip class is the. # 1.5 with a thickness of 0.16 to 0.19. The most commonly sold coverslip is the #1 coverslip that has a nominal thickness of 0.13 to 0.16. Typically #1 coverslip are significantly less expensive and provide accommodation for a less precise mounting media thickness than typically happens in a clinical environment. The #1.5 coverslip demands a rigorous mounting process in order to take advantage of the optimized thickness for the objective design.

farnsy
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Re: What is the real optimal cover slip thickness?

#5 Post by farnsy » Mon May 17, 2021 4:17 am

Anyone know what the math is on this? Like, how much thinner than 0.17 should a coverslip be for a specimen that is .01mm away from the cover slip in the water? Sounds like a problem someone would know the answer to.

BramHuntingNematodes
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Re: What is the real optimal cover slip thickness?

#6 Post by BramHuntingNematodes » Mon May 17, 2021 4:56 am

farnsy wrote:
Mon May 17, 2021 4:17 am
Anyone know what the math is on this? Like, how much thinner than 0.17 should a coverslip be for a specimen that is .01mm away from the cover slip in the water? Sounds like a problem someone would know the answer to.
The refractive index of water is around 1.3, lower than the coverslip. Using the maths described http://www.microscopy-uk.org.uk/mag/ind ... rslip.html in this link, it seems like water creates disturbances with less thickness.
1942 Bausch and Lomb Series T Dynoptic, Custom Illumination

apochronaut
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Re: What is the real optimal cover slip thickness?

#7 Post by apochronaut » Mon May 17, 2021 3:44 pm

To use an aqueous sample such as pond water as an example, an acceptable evenly spread drop will be about 40 microns deep. Using a nominal cover slip thickness of .17mm or 170 microns as a target thickness, that means that a .13 cover slip plus the .04 sample will approximate the target cover/sample thickness. This is complicated a bit by the difference in refractive index between the cover slip and the sample and whether the cover slips are of a known refractive index. My personal opinion is that cheap coverslips are more likely of a cheaper grade of glass and have a lower r: around 1.47 or so. High quality borosilicate coverslips should be closer to 1.52. This is no doubt taken into account by the manufacturers when they provide idealized specs, that the coverslips should be of a known quality.
So since water represents a slightly thinner slice than the equivalent thickness of borosilicate glass due to it's lower r, a .04 aqueous sample actually behaves pretty close to about 87% the thickness, as if it were glass.
In the example above , with a 40 micron aqueous sample thickness , it would behave about like .35 microns of glass , so approx. a .135 cover, when viewing a .40 mm sample is required.

I visually tested this with high N.A. objectives at one time, using objectives of between .70 and 1.3 N.A., dry and oil but all planapo.. Generally, I found that at an N.A of .80 a .2mm variance either way was acceptable. You could see a slight difference in resolution at .2mm. The effect increased in severity proportional to the N.A. At 1.30, using a .15mm coverslip with an average aqueous sample caused a noticeable drop in resolution. .13 was better.

I have mic'ed all of my better coverslips using the thicker and generic ones for any microscopy at N.A.'s less than .70, usually. With a highly corrected objective such as a 25X .65 planapo, I use precise objectives too but with a 40X .66 achromat , generic. For the highly colour corrected and high N.A. objectives, I use between .12 and .15 coverslips, tending to thinner if I am doing oil immersion over 1.25. There are some .08 to .12 coverslips, separately boxed, for use with samples where there is debris or where I suspect that larger specimens lurk but in those cases, seldom are objectives over 20X used.

If precision is really necessary, a calculation of the required volume of water under such and such a square area coverslip to achieve a specific sample thickness can be easily made and delivered via pipette but after a while a microscopist gets a pretty good feeling for the droplet size and will select a coverslip within .10 of the required thickness , usually.

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