Micrometer - mount-thickness tests

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mrsonchus
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Micrometer - mount-thickness tests

#1 Post by mrsonchus » Mon Feb 29, 2016 12:25 am

Hi all, the micrometer I ordered after following an excellent thread here, where the use of a micrometer to measure the thickness of cover-slips was discussed, has arrived!
I bought one that is metric and (nominally) reads in 0.01mm increments - it was about £20 and having used it quite a lot already I would say that it is superbly made and well-worth this price-tag. It performs the task for which I bought it to my complete satisfaction and in excess of my desired performance-level - in short, I love it!

I've used it to make some quick, easy and accurate measurements of a selection of different cover-slips of different sizes and thicknesses (specified not accidental that is), new high-quality slides and the same slides with fully-dry and permanently mounted specimens with cover-slips upon them - the results I think show the micrometer to be a superb and accurate piece of kit.

It's very easy and quick to use, consistent and accurate to about 0.002mm I would say, as it's calibrated to 0.01mm with the usual lines on the barrel, but it is easy to literally 'read between the lines' (i.e. interpolate visually) with what I would estimate to be a reliable and useful accuracy of about 0.002mm!

Here's the beauty with it's shiny-new box...
ws_700x525_DSCN3417.jpg
ws_700x525_DSCN3417.jpg (90.88 KiB) Viewed 2566 times
I've tried it out and it's very easy and quick to use.

I thought I'd try a quick run with it as follows, the intention being to mount 2 coverslips without sections just plain cover-slips mounted with 'Numount', the resinous mountant I always use. One had a wooden mini-peg placed upon it during a 26hr drying time, the other didn't - to see if this applied weight made any difference to the dried thickness of the whole thing, measured as slide+coverslip_mountant-layer between them.
Of course the hypothesis was that the mount with added weight would finish drying as a thinner and therefore more desirable mount than the one dried without weight applied....

To make the test more reliable I should measure and use 2 seperate slides, 1 for each cover-slip, making sure that each spot used on each slide was of identical thickness and similarly each cover-slip was of the same thickness - I didn't go to that length as this was as much a practice for the use of the micrometer as for this test, although at a later time I would be interested to run a similar series perhaps even using sections mounted within the mountant, anyway this is the basic test in it's imperfect but still useful I think form...

I measured a slide's thickness in 2 spots, with the intention of mounting (side-by-side) 2 cover-slips, each centered on one of these spots, a & b.

Nominally these slides are 1mm, the actual measured (2) spots along it's long-axis measured,
spot a on the left = 1.018mm, spot b on the right = 1.013mm, reasonably close to their stated 1mm, not really relevant in use but needed to know this for this comparative test.

The 2 cover-slips A on the left and B on the right measured (in their centers) 0.169mm and 0.183mm respectively. This gives a theoretical (and probable) thickness total of 1.018 + 0.169 = 1.187mm before mountant for the left side mount
and 1.013 + 0.183 = 1.196mm for the right...

Measured after 26hrs drying time, the left-hand mount with weight applied throughout, the 2 sides measured as follows,
on the left the total thickness of slide(1.018) + mountant(call this as yet unknown 'ma') + cover-slip(0.169) = 1.193mm,
so the mountant-layer (ma) must be 1.193-(1.018+0.169) = 0.006mm or 6µ, and for the right-hand mount we do the same,

On the right the total thickness of slide(1.013) + mountant(call this as yet unknown 'mb') + cover-slip(0.183) = 1.205mm,
so the mountant-layer (ma) must be 1.205-(1.013+0.183) = 0.009mm or 9µ...

So we have the mount on the left, with added weight, at a final dried thickness for the mountant only of 6µ, and the mountant only of the right-hand mount without added weight drying at a thickness of 9µ.

It's perhaps possible to tentatively suggest two things I think:
a) - the addition of weight during drying reduces the thickness of the finished dry layer of mountant.
b) - the degree of reduction may in this case at least be expressed as approx 33% (i.e. 3/9)..

Not the most disciplibed or valid of tests of course, but I think the basic principle may be accepted and worthy perhaps of a more controlled series of tests with standard sizes and multiple test-subject beyond just 2 on the same slide!

Good practice anyway for my micrometer, which I'm already using to sort my cover-slips into the 0.17mm for which my 'scope's objectives are labelled as calibrated... Every little helps with results! :D

Measuring further into the micrometer's usable range to see how it returns to zero from here... 4.953mm measured here..
ws_700x525_DSCN3433.jpg
ws_700x525_DSCN3433.jpg (75.88 KiB) Viewed 2566 times
Here it is calibrated for zero using the nice little 'spanner' to align the collar with the micrometer at zero when closed with the slipping-ratchet as when used to measure..
ws_700x525_DSCN3421.jpg
ws_700x525_DSCN3421.jpg (77.17 KiB) Viewed 2566 times
A nice and useful addition to my kit! :D
John B

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Re: Micrometer - mount-thickness tests

#2 Post by zzffnn » Mon Feb 29, 2016 12:51 am

How thick was your mountant layer, immediately after applying (before any drying occurs)? 6-9 microns is thinner than I expect, but then I have never measured a dried mountant myself (I think your measurements are accurate).

I like your very controlled comparison, by the way. I don't think a professional scientist can do much better.
Last edited by zzffnn on Mon Feb 29, 2016 1:36 am, edited 3 times in total.
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Re: Micrometer - mount-thickness tests

#3 Post by 75RR » Mon Feb 29, 2016 1:26 am

Congratulations on your new micrometer!
Good idea for a test.
Was wondering how you measured out the mountant, was it by the number of drops?
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Re: Micrometer - mount-thickness tests

#4 Post by mrsonchus » Mon Feb 29, 2016 1:40 am

zzffnn wrote:How thick was your mountant layer, immediately after applying (before any drying occurs)? 6-9 microns is thinner than I expect, but then I have never measured a dried mountant myself (I think your measurements are accurate).
Hi zz', hmm - not sure but what I can tell you is that I thin the Numount with Histoclear (it's solvent) to get it to flow nicely without being too thin that is, too much solvent will leave 'gaps' that look like bubbles as it evaporates, too thick and the mount will surely have bubbles akin to those found with the dreaded gelatin mounts!
Getting it just right is a matter of practice and judgement but I notice that as long as the mountant flows 'in straight fronts' and doesn't literally 'meet itself coming back' and therefore encircling an area and trapping bubbles everything goes perfectly.

Basically with the mountant at just the right viscosity and carefully applied as a drop or drops, regardless of your method such as the 'double-drop' or the single suspended-drop, or the end-of-the-slide-line or my favourite for large sections such as whole-ovary LSs - the line down the middle of the cover-slip along it's long-axis, the result will be good.

As my technique has improved I've learned to get right down to the level of just-enough (i.e. the minimum) mountant to do the job, and now am able to make very thin mounts with no 'over-spill' at all...

Remember that these measurements are without a section, further and more disciplined tests will give more valuable information I think - when I get a chance I'll run several slides along in this fashion of testing. :D

An interesting exercise. :) Thanks zz' for your interest.
John B

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Re: Micrometer - mount-thickness tests

#5 Post by mrsonchus » Mon Feb 29, 2016 1:52 am

75RR wrote:Congratulations on your new micrometer!
Good idea for a test.
Was wondering how you measured out the mountant, was it by the number of drops?
Yes, I have several different 'dropping sticks' (erm.. they are the snapped-off ends of paint-brushes of varying thicknesses.. :oops: ) each of which gives me different-sized drops, and above all, consistently so I find.

The important thing for the tests was the consistency of drop-size rather than the actual volume but it's possible of course to get a rough quantitative estimate of drop-size with the measurement and averaging (weight or volume will do) of multiple drops, assuming reasonable consistency of drop is able to be presumed.

p.s. sometimes a paintbrush-end is 'eaten' by the solvent - especially certain plastics and painted woods, I once ended up with a half-full pot of Numount with black streaks running through it like a French paperweight! I - ahem - disposed quietly of it and learned an embarrassing lesson, one of many! :)
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Re: Micrometer - mount-thickness tests

#6 Post by zzffnn » Mon Feb 29, 2016 2:35 am

75RR,

I don't know how it goes with John B's mountant/solvent. One (natural hanging) drop of water is usually around 50 microliter or 0.05 ml.
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Re: Micrometer - mount-thickness tests

#7 Post by 75RR » Mon Feb 29, 2016 3:30 pm

I don't know how it goes with John B's mountant/solvent. One (natural hanging) drop of water is usually around 50 microliter or 0.05 ml.
Thanks zzffnn, that does ring a faint bell.

This is a quote from wiki: https://en.wikipedia.org/wiki/Drop_%28unit%29
It was noted that the size of a drop can vary considerably depending on the viscosity and specific gravity of the fluid, as well as the size and shape of the vessel from which it is poured.

In mrsonchus case I suppose the key is "the size and shape of the vessel from which it is poured" re: snapped-off ends of paint-brushes!
I suppose a steady hand and practice helps as well.
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Re: Micrometer - mount-thickness tests

#8 Post by mrsonchus » Mon Feb 29, 2016 4:23 pm

Hmm... a 'vessel' pouring drops, sounds a little ambiguous to be. However I'd imagine the major factors in the case of my Numount may include, although not exclusively perhaps;

*temperature of that which is 'dropped' - here the Numount formulation
*temperature of any 'drop-forming' last-contact delivery device or even 'vessel' - here ambient room temperature = brush-end temperature
*viscosity - a function of the mountant/(added)solvent (Histoclear) ratio - here unknown but not of course unknowable

I would suggest using density to measure volume via weight of course but the difficulty lies with the measurement of volume I may think, as any meniscus would also need to be of known properties and like a 'drop' this will vary and therefore make the simplest 'level in a graduated vessel' method invalid for measurements of this scale.

Perhaps a method may be to over-fill a small bottle with a fluid of known density such as DIW and via a set of the very accurate scales such as the ones I use for stains etc to derive a volume for the bottle's contents with the idealized/standardized lid (cover-slip).
Measure and compare the weight of this volume of mountant and DIW to arrive at a density, relative or actual, for the mountant.
With this information the 'x number of drops weigh y grams' so 1 drop will on average weigh.... etc

I bet there's a simpler way! :D
John B

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Re: Micrometer - mount-thickness tests

#9 Post by gekko » Mon Feb 29, 2016 11:00 pm

Interesting tests. Do you think that it would also be interesting (instructive) to compare the results obtained from the compressed and uncompressed slide using a high NA dry objective?

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Re: Micrometer - mount-thickness tests

#10 Post by mrsonchus » Tue Mar 01, 2016 12:31 am

gekko wrote:Interesting tests. Do you think that it would also be interesting (instructive) to compare the results obtained from the compressed and uncompressed slide using a high NA dry objective?
Hi Gekko, most definitely, but a lot of factors must be made identical over a series of several examples of each option, the nature of the tissue itself also being a factor - a lot of work for a non-too-conclusive outcome I suspect. My approach now is to presume that 'thinner is better' and that a cover-slip of exactly 0.17mm as per the objectives' barrels markings will give me about as much optimisation as I can realistically attempt to achieve.
Also I suspect that I will at some stage opt for a couple of dry, high NA objectives with correction-collars, although before then I'll probably want to 'upgrade' my 2mp Toupcam also - all this will take time and money.

So, I'm measuring cover-slips for high-magnification slides such as those prepared to show nuclear-detail or even mitosis/meiosis stages (only known what these terms mean for 48hrs! :D :oops: ) and using the 'out of tolerance' ones for my 'overview of morphology' slides such as some complete ovary, anther, leaf, stem sections stained for high-level morphological detail rather than the real 'close-in' even intracellular details.

An interesting test though - I'll measure them again in a week or so to see if they've changed some more....

Thanks for your interest Gekko old fellow. :)
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Re: Micrometer - mount-thickness tests

#11 Post by gekko » Tue Mar 01, 2016 12:37 am

You are, of course, right that ideally one must have "everything else" the same, but I was thinking it terms of generaly quality of the image (e.g., spherical aberration), but I know you are right. In any case, all your images are first rate as it is.

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Re: Micrometer - mount-thickness tests

#12 Post by mrsonchus » Tue Mar 01, 2016 12:57 am

Yes but I agree with you there Gekko - it can't hurt to take a look at a few and just consider 'how they look' - I'll prepare several of my next batch in this way and basically just see what happens, better than nothing! :D
I'll put up some pictures when I do this and we can have a good ponder... :)
John B

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