Coverslip thickness

Have questions about the equipment used for macro- or micro- photography? Post those questions in this forum.

Moderators: rjlittlefield, ChrisR, Chris S., Pau

dhmiller
Posts: 256
Joined: Mon Nov 11, 2019 7:42 am
Contact:

Coverslip thickness

Post by dhmiller »

I just heard a comment that coverslip thickness can make a difference in photomacrography. I'm using a polarizing microscope to photograph crystals that have formed when I dissolve a chemical in a solution and heat it on a slide- are there any recommendations for coverslip thickness that I should be aware of? I usually just get the biggest coverslips I can find in order to cover the largest area of the slide... Thanks for any info.

Beatsy
Posts: 2102
Joined: Fri Jul 05, 2013 3:10 am
Location: Malvern, UK

Re: Coverslip thickness

Post by Beatsy »

Simplifying, it mainly depends on the N.A. of the objectives you're using, higher N.A. requires more accuracy. Higher quality (e.g. planapo vs achromat) increases the "visibility" of issues caused by incorrect coverslip thickness.

You won't really notice a variation of +/- 0.02, or even more with N.A. up to 0.5 or 0.6. But as you get higher it starts to show clearly in the details. With diatoms, using oiled lens and condenser at N.A. 1.4, I can easily see the smearing and loss of resolution (spherical aberration) if the slip is (a massive) 0.01mm too thin. It seems worse too thin than too thick by the same amount - but I don't know if that's a thing. Maybe just my setup.

The coverslip *is* the front element of the objective, but it's thickness can be approximate when (required) image resolution is moderate or low.

rjlittlefield
Site Admin
Posts: 23543
Joined: Tue Aug 01, 2006 8:34 am
Location: Richland, Washington State, USA
Contact:

Re: Coverslip thickness

Post by rjlittlefield »

Dennis, see the discussion at viewtopic.php?p=198514#198514 and in the surrounding thread.

--Rik

Macro_Cosmos
Posts: 1506
Joined: Mon Jan 15, 2018 9:23 pm
Contact:

Re: Coverslip thickness

Post by Macro_Cosmos »

Depends on NA.
Below NA 0.3, the difference isn't noticeable. Spherical aberration will increase rapidly as NA goes up and the coverslip isn't present, too thick or thin. It's visible at NA0.5, and detrimental at 0.75 and above, it'll be as if the objective is smeared with fog. Then with oil immersion, the visible spherical aberration goes down again since the refractive indices are kind of matched. This only applies to normal oil immersion, if you're using silicone oil or water, the same rule as air objectives apply.
Objective_Coverslip_Correction_G2-780.gif
For your application, here's some recommendations I have. I assume you're using a 20x at most, though some formations warrants 100x.
- Form the crystals ON the coverslip, then mount onto the slide. Don't form on the slide, then cover it with a coverslip.
- A micrometer can be used to cherrypick coverslips. 0.17mm +/- 0.03mm should be good.
Friend told me that filtered solutions give better results with less impurities such as dust particles and fibre.

dhmiller
Posts: 256
Joined: Mon Nov 11, 2019 7:42 am
Contact:

Re: Coverslip thickness

Post by dhmiller »

Thanks much for the detailed reply. I am always looking for highest resolution when photographing the crystals, and my objectives vary from 4x to 40x (will check the NA - .1 to .65, I believe). So maybe best to try different thicknesses and see what works with what.
Beatsy wrote:
Mon Jul 19, 2021 8:26 am
The coverslip *is* the front element of the objective, but it's thickness can be approximate when (required) image resolution is moderate or low.

dhmiller
Posts: 256
Joined: Mon Nov 11, 2019 7:42 am
Contact:

Re: Coverslip thickness

Post by dhmiller »

viewtopic.php?p=198514#198514 and in the surrounding thread.

From the link above: "For optimum performance, always use .17mm .."
I tried to find .17 online but I only see ".13mm - .17mm" and ".17 - .25," etc. So which might be best?

Thanks, Rik.
Last edited by dhmiller on Sat Jul 24, 2021 10:01 am, edited 2 times in total.

dhmiller
Posts: 256
Joined: Mon Nov 11, 2019 7:42 am
Contact:

Re: Coverslip thickness

Post by dhmiller »

Thanks very much, M_C. I do use 40x (.65) occasionally, but don't often know in advance what magnification I will want for any given subject. And since I am heating the solution, it would be tricky to use a coverslip from the start, rather than put the solution onto the slide and then cover.... I will try that for some solutions that I don't plan to heat though. Thanks again.

ModelZ
Posts: 71
Joined: Sun Nov 01, 2020 4:34 am
Location: Northern Europe

Re: Coverslip thickness

Post by ModelZ »

To avoid micrometering every cover glass in a batch... there is apparently an emerging ISO standard 1.5H which guarantees 0.17-0.18mm. It seems to have come with superresolution microcopy stuff but everyone could benefit (at hi mag). Not as easy to find but several outlets exits, ThorLabs, Zeiss etc. Anyone invested in these, worth the premium?

Beatsy
Posts: 2102
Joined: Fri Jul 05, 2013 3:10 am
Location: Malvern, UK

Re: Coverslip thickness

Post by Beatsy »

Yes, I have the Zeiss high accuracy slips. They only had square ones back when I bought them and I have to cut my own round ones from them (diatom mounts).

I got these after a long and frustrating run measuring several batches of normal #1.5 round slips and finding none at exactly 0.17 thick. I still have a few hundred of the Zeiss ones left, but got fed up cutting them and...

.. looked around for others and lucked into a batch of 4000 X 13mm round slips. Very cheap. I forget the brand, will post when I'm back at my PC. More than half are *exactly* the correct thickness and the variants are close enough to be good for most anything too.

Lifetime supply secured :-D

rjlittlefield
Site Admin
Posts: 23543
Joined: Tue Aug 01, 2006 8:34 am
Location: Richland, Washington State, USA
Contact:

Re: Coverslip thickness

Post by rjlittlefield »

dhmiller wrote:
Sat Jul 24, 2021 9:37 am
I tried to find .17 online but I only see ".13mm - .17mm" and ".17 - .25," etc. So which might be best?
Look for No. 1.5 coverslips. www.tedpella.com lists quite a few models as 0.16 - 0.19 mm. That range would be fine for anything you're doing.

--Rik

Macro_Cosmos
Posts: 1506
Joined: Mon Jan 15, 2018 9:23 pm
Contact:

Re: Coverslip thickness

Post by Macro_Cosmos »

dhmiller wrote:
Sat Jul 24, 2021 9:37 am
viewtopic.php?p=198514#198514 and in the surrounding thread.

From the link above: "For optimum performance, always use .17mm .."
I tried to find .17 online but I only see ".13mm - .17mm" and ".17 - .25," etc. So which might be best?

Thanks, Rik.
I'd like to contest the claim of "for optimum performance, always use .17mm". Always is a very strong word. Modern objectives feature a correction collar (corr or korr for German brands), exactly .17mm might not be the best since the water film has a thickness and samples might be placed anywhere within the depths of the waterfilm. I personally lean "slightly thinner" than .17mm, and thinner for permanent preparations. With a correction collar, I believe .13mm is great, reduced the refractive index mismatch... I could be wrong of course and correction collar objectives are very expensive. The rule certainly holds with regards to older objectives and ironically less "premium ones" and premium ones with very high NA for confocal imaging (many of them use special exotic coverslips, such as sapphire).

Zeiss makes high precision coverslips (#1.5H as others have mentioned), .17mm+/- .005mm. I measured 100 of those and got consistently below .17mm to 0.17mm, none were for example, .1703mm, 100% of them were within the rated range. Maybe my Mitutoyo micrometer is off? Anyhow, it's a seal of confidence.

Thorlabs offers high precision circular ones, expensive... and I'm sure it's just a white label.
https://www.thorlabs.com/newgrouppage9. ... pn=CG15XH1

Marienfeld offers #1.5H coverslips too, however, it seems like they won't sell just a small box of 100, it's 1000 of them (10 boxes). They do offer the less precise ones in smaller quantities. The same seems to apply to Schott who makes excellent glass stuff and Azer Scientific.
https://www.marienfeld-superior.com/pre ... l-5-m.html

Found this on duck.com (google)
$$$$ but in small quantities.
http://biosciencetools.com/catalog/Coverslips.htm
  • for objectives with high numerical aperture and resolution
  • accurate thickness of 0.170 mm with tolerance reduced to ± 0.005 mm
  • non-corroding borosilicate glass
  • refractive index ne: 1.524-1.527 at 546.07 nm
  • Abbe coefficient: ve=55
recommended for the following objectives:
  • dry objective: N.A. 0.7
  • water immersion: N.A. 1.0
  • glycerol immersion: N.A. 1.2
  • oil immersion: N.A. 1.3
  • developed in cooperation with Zeiss and Schott
Finally, Zeiss.
https://www.micro-shop.zeiss.com/en/de/ ... erformance,

You'd be counting the coverslips you use since some are more than a dollar each... :?

jmc
Posts: 238
Joined: Fri Apr 17, 2020 3:14 am

Re: Coverslip thickness

Post by jmc »

Macro_Cosmos wrote:
Mon Jul 26, 2021 7:27 am
Finally, Zeiss.
https://www.micro-shop.zeiss.com/en/de/ ... erformance,

You'd be counting the coverslips you use since some are more than a dollar each... :?
A dollar each - heavenly. My fused silica ones are about 15-20GBP each depending on size. I've had to become very adept at cleaning them. Carefully. Thankfully at 0.35mm thick they are a bit more robust.
Jonathan Crowther

Beatsy
Posts: 2102
Joined: Fri Jul 05, 2013 3:10 am
Location: Malvern, UK

Re: Coverslip thickness

Post by Beatsy »

jmc wrote:
Wed Jul 28, 2021 2:05 am
Macro_Cosmos wrote:
Mon Jul 26, 2021 7:27 am
Finally, Zeiss.
https://www.micro-shop.zeiss.com/en/de/ ... erformance,

You'd be counting the coverslips you use since some are more than a dollar each... :?
A dollar each - heavenly. My fused silica ones are about 15-20GBP each depending on size. I've had to become very adept at cleaning them. Carefully. Thankfully at 0.35mm thick they are a bit more robust.
15-20GBP!? You BUY 'em!? Looxury lad! Back in t' old days, we 'ad to get oop at four in t' mornin, walk ten mile t' mica pit, dig us own mica aaht, cart it ten mile back 'ome, then cleave us own coverslips from that. Yoong-uns today - doon't know 'alf o' it! :D :D

jmc
Posts: 238
Joined: Fri Apr 17, 2020 3:14 am

Re: Coverslip thickness

Post by jmc »

Beatsy wrote:
Wed Jul 28, 2021 2:20 am
jmc wrote:
Wed Jul 28, 2021 2:05 am
Macro_Cosmos wrote:
Mon Jul 26, 2021 7:27 am
Finally, Zeiss.
https://www.micro-shop.zeiss.com/en/de/ ... erformance,

You'd be counting the coverslips you use since some are more than a dollar each... :?
A dollar each - heavenly. My fused silica ones are about 15-20GBP each depending on size. I've had to become very adept at cleaning them. Carefully. Thankfully at 0.35mm thick they are a bit more robust.
15-20GBP!? You BUY 'em!? Looxury lad! Back in t' old days, we 'ad to get oop at four in t' mornin, walk ten mile t' mica pit, dig us own mica aaht, cart it ten mile back 'ome, then cleave us own coverslips from that. Yoong-uns today - doon't know 'alf o' it! :D :D
Yeah, I buy 'em, but not many of 'em..... When you add on the quartz or fused silica slides it becomes rather pricey. Quartz seems to be a bit less than fused silica, but even so, it's eye-watering.
Jonathan Crowther

Beatsy
Posts: 2102
Joined: Fri Jul 05, 2013 3:10 am
Location: Malvern, UK

Re: Coverslip thickness

Post by Beatsy »

jmc wrote:
Wed Jul 28, 2021 2:27 am
...
Yeah, I buy 'em, but not many of 'em..... When you add on the quartz or fused silica slides it becomes rather pricey. Quartz seems to be a bit less than fused silica, but even so, it's eye-watering.
With (potential) consumables like cover slips, you certainly could rack up some serious running costs without careful cleaning and re-use.

I don't need anything exotic so I'm rather more profligate with my slips these days. I generally only re-use them within a session, not across different sessions, and that's mostly out in the field (e.g. surveying pond samples or algae scrapings before collection). Then the slips are binned - just like all my single-use ones are. I always clean slips first, even if fresh from a box of (allegedly) pre-cleaned ones, so re-use wouldn't be a problem per-se. But fresh from the box ones are usually quicker and easier to clean than used ones with dried-on goop attached.

It's just time-saving really (aka laziness) because I bought lots of large, cheap job-lots over the years and have accumulated a lifetime supply to dip into (at my current usage rates anyway).

Post Reply Previous topicNext topic