Imaging with different coverglass thickness

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wtse23
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Imaging with different coverglass thickness

#1 Post by wtse23 » Mon Apr 20, 2020 4:50 pm

Hi,

My name is Andrew, I'm new to the forum.

I have a question about why a lab in my institution is using a #1.0 chambered coverglass for an objective lens that's meant for 0.17 thickness coverslip. I have tried using #1.5 chambered coverslip but the image never focuses.

The lab does calcium signaling with Fura-2 on an Olympus IX 70 inverted microscope set up.

Any help is much appreciated.

Best regards,
Andrew
Last edited by wtse23 on Tue Apr 21, 2020 2:10 pm, edited 1 time in total.

Tom Jones
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Re: Imaging with different coverglass thickness

#2 Post by Tom Jones » Mon Apr 20, 2020 5:12 pm

At the risk of being obvious, have you asked them?

While someone here might have a good explanation as to why you might want to,- I don't - it's only speculation as to why they are doing it.

Scarodactyl
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Re: Imaging with different coverglass thickness

#3 Post by Scarodactyl » Mon Apr 20, 2020 5:53 pm

I helped set up a camera on a microscope on a friend's academic lab. Their sccopes were old leitzes, and that one had a leitz objective, a wild, an olympus, a zeiss and a nikon. Not exactly on purpose, just from inherited equipment that was likely bodged together over time. Similar issues with their main imaging setup--it was very elaborate, using lots of high quality specialty equipment, but it turned out they'd basically just grabbed whatever lighting and optics were lying around, and cheaper more common stuff I could source easily worked way, way better.
All that to say, what happens in academic labs may not be the result of anyone making a specific decision.

Hobbyst46
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Re: Imaging with different coverglass thickness

#4 Post by Hobbyst46 » Mon Apr 20, 2020 6:06 pm

Here is a conjecture: it is non-uniform nomenclature.

0.17mm-thick coverslips are considered to be ideal for high-NA imaging. This includes fluorescence studies (like the Fura-2).
There are several mafg. of "chambered coverslips" (the term is new to me - knew them by other names).
For example, Ibidi (Germany) specs are #1.5 chambered coverslips, that are indeed 0.17mm slips.
However, it seems to me that Nunc markets #1 chambered coverslips, of thickness 0.13-0.17mm. So perhaps the "#1" here is specific to Nunc rather than part of the common thickness scale of coverslips.

Sorry, no better explanation.
Zeiss Standard GFL+Canon EOS-M10, Olympus VMZ stereo

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daruosha
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Re: Imaging with different coverglass thickness

#5 Post by daruosha » Mon Apr 20, 2020 6:19 pm

wtse23 wrote:
Mon Apr 20, 2020 4:50 pm
Hi,

My name is Andrew, I'm new to the forum.

I have a question about why a lab in my institution is using a #1.0 chambered coverslip for an objective lens that's meant for 0.17 thickness coverslip. I have tried using #1.5 chambered coverslip but the image never focuses.

The lab does calcium signaling with Fura-2 on an Olympus IX 70 inverted microscope set up.

Any help is much appreciated.

Best regards,
Andrew

Hi Andrew and welcome to the forum. Have you measured the actual thickness of the coverslip with a caliper? May be it's on the higher side of the accepted range (160-190 μm) and you have a few dozen microns distance between your specimen and coverslip filled with mounting medium. I'm just guessing. You have to do some measurements and try different coverslips batches.
Daruosh.

MicroBob
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Re: Imaging with different coverglass thickness

#6 Post by MicroBob » Mon Apr 20, 2020 8:20 pm

Hi Andrew,
apart from the cover slip thickness the further medium between cover slip and object is part of the calculation.If there is a tiny water layer cover slips of less than 0,17mm might actually offer a better image.

Apart from that: a #1 is thinner than a #1,5! So if you can't focus the problem is never the #1 cover slip.

Bob

MichaelG.
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Re: Imaging with different coverglass thickness

#7 Post by MichaelG. » Mon Apr 20, 2020 8:45 pm

To be honest, Andrew ... I had never heard of a #1 chambered coverslip :oops:

So, I looked here for the spec.
https://www.thermofisher.com/order/cata ... 61#/155361
... and also saw the price

I assume, and sincerely hope, that your colleagues know exactly what they are doing.

MichaelG.

.
Edit: it’s worth downloading the drawings from that page ^^^ all is explained.
Last edited by MichaelG. on Mon Apr 20, 2020 8:54 pm, edited 2 times in total.
Too many 'projects'

Hobbyst46
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Re: Imaging with different coverglass thickness

#8 Post by Hobbyst46 » Mon Apr 20, 2020 8:48 pm

MicroBob wrote:
Mon Apr 20, 2020 8:20 pm
apart from the cover slip thickness the further medium between cover slip and object is part of the calculation.If there is a tiny water layer cover slips of less than 0,17mm might actually offer a better image.
These are chambers, live cells grow within, so the water layer is not very thin, but probably several mm.
Michael: they are probably expensive, I cannot see a price, but such chambers (from any brand) are the best and most convenient means of studying live cells in culture, on top of an inverted microscope.
Zeiss Standard GFL+Canon EOS-M10, Olympus VMZ stereo

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daruosha
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Re: Imaging with different coverglass thickness

#9 Post by daruosha » Mon Apr 20, 2020 9:01 pm

MichaelG. wrote:
Mon Apr 20, 2020 8:45 pm
To be honest, Andrew ... I had never heard of a #1 chambered coverslip :oops:

So, I looked here for the spec.
https://www.thermofisher.com/order/cata ... 61#/155361
... and also saw the price

I assume, and sincerely hope, that your colleagues know exactly what they are doing.

MichaelG.

.
Edit: it’s worth downloading the drawings from that page ^^^ all is explained.
The prices are eye watering. Are they really worth this much? 4-well Chambered Coverglass w/ non-removable wells, 1000 USD for 96 pieces!
Daruosh.

MichaelG.
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Re: Imaging with different coverglass thickness

#10 Post by MichaelG. » Mon Apr 20, 2020 9:03 pm

Hobbyst46 wrote:
Mon Apr 20, 2020 8:48 pm
Michael: they are probably expensive, I cannot see a price, but such chambers (from any brand) are the best and most convenient means of studying live cells in culture, on top of an inverted microscope.
Typically just under 10 GBP each, in packs of 16 or 96

I have withdrawn my smilie of surprise, because that doesn’t actually seem unrealistic.

MichaelG.
Last edited by MichaelG. on Mon Apr 20, 2020 9:06 pm, edited 1 time in total.
Too many 'projects'

Hobbyst46
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Re: Imaging with different coverglass thickness

#11 Post by Hobbyst46 » Mon Apr 20, 2020 9:06 pm

daruosha wrote:
Mon Apr 20, 2020 9:01 pm
The prices are eye watering. Are they really worth this much? 4-well Chambered Coverglass w/ non-removable wells, 1000 USD for 96 pieces!
That means about 10$ apiece. From the point of view of a researcher, who grows cells within the chamber, perhaps under 4 different sets of conditions, and say for 1-3 days, then runs an experiment including imaging for an hour or more, it is not a high price.
BTW, and contrary to the mfg. purposes, at least some of these chambers can be recycled, that is, rinsed with proper cleaning solutions, then dried and reused. So the actual price is lower.
Zeiss Standard GFL+Canon EOS-M10, Olympus VMZ stereo

wtse23
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Re: Imaging with different coverglass thickness

#12 Post by wtse23 » Tue Apr 21, 2020 2:07 pm

Hobbyst46 wrote:
Mon Apr 20, 2020 6:06 pm
Here is a conjecture: it is non-uniform nomenclature.

0.17mm-thick coverslips are considered to be ideal for high-NA imaging. This includes fluorescence studies (like the Fura-2).
There are several mafg. of "chambered coverslips" (the term is new to me - knew them by other names).
For example, Ibidi (Germany) specs are #1.5 chambered coverslips, that are indeed 0.17mm slips.
However, it seems to me that Nunc markets #1 chambered coverslips, of thickness 0.13-0.17mm. So perhaps the "#1" here is specific to Nunc rather than part of the common thickness scale of coverslips.

Sorry, no better explanation.
Yes, they were using Nunc #1 for a long time. This product has been delayed for the foreseeable future. The lab would like to know if it's possible to use 1.5 thickness instead. There was someone from another lab that used 1.5 thickness for neuronal cells and we couldn't get the cells in focus. The stage maxed out just before it would come into focus. Unfortunately, I am limited to only 1 objective for Fura-2. I'm trying to figure out a solution and explanation as to why this is happening.

wtse23
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Joined: Fri Apr 17, 2020 5:59 pm

Re: Imaging with different coverglass thickness

#13 Post by wtse23 » Tue Apr 21, 2020 2:10 pm

Tom Jones wrote:
Mon Apr 20, 2020 5:12 pm
At the risk of being obvious, have you asked them?

While someone here might have a good explanation as to why you might want to,- I don't - it's only speculation as to why they are doing it.
Yes, I have asked them. The lab has been using this specific product for years. Unfortunately, I was never told the reason before why this was the case when I took over managing this system. I'm spending time now during the quarantine to figure the system out.

wtse23
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Re: Imaging with different coverglass thickness

#14 Post by wtse23 » Tue Apr 21, 2020 2:10 pm

wtse23 wrote:
Mon Apr 20, 2020 4:50 pm
Hi,

My name is Andrew, I'm new to the forum.

I have a question about why a lab in my institution is using a #1.0 chambered coverglass for an objective lens that's meant for 0.17 thickness. I have tried using #1.5 chambered coverslip but the image never focuses.

The lab does calcium signaling with Fura-2 on an Olympus IX 70 inverted microscope set up.

Any help is much appreciated.

Best regards,
Andrew

wtse23
Posts: 8
Joined: Fri Apr 17, 2020 5:59 pm

Re: Imaging with different coverglass thickness

#15 Post by wtse23 » Tue Apr 21, 2020 2:11 pm

MichaelG. wrote:
Mon Apr 20, 2020 8:45 pm
To be honest, Andrew ... I had never heard of a #1 chambered coverslip :oops:

So, I looked here for the spec.
https://www.thermofisher.com/order/cata ... 61#/155361
... and also saw the price

I assume, and sincerely hope, that your colleagues know exactly what they are doing.

MichaelG.

.
Edit: it’s worth downloading the drawings from that page ^^^ all is explained.
Sorry, I meant coverglass.

wtse23
Posts: 8
Joined: Fri Apr 17, 2020 5:59 pm

Re: Imaging with different coverglass thickness

#16 Post by wtse23 » Tue Apr 21, 2020 2:13 pm

MicroBob wrote:
Mon Apr 20, 2020 8:20 pm
Hi Andrew,
apart from the cover slip thickness the further medium between cover slip and object is part of the calculation.If there is a tiny water layer cover slips of less than 0,17mm might actually offer a better image.

Apart from that: a #1 is thinner than a #1,5! So if you can't focus the problem is never the #1 cover slip.

Bob
Hi Bob,

There is a thin water layer that covers the cells in the chamber to do the live-cell imaging.

wtse23
Posts: 8
Joined: Fri Apr 17, 2020 5:59 pm

Re: Imaging with different coverglass thickness

#17 Post by wtse23 » Tue Apr 21, 2020 2:21 pm

Thank you for the warm welcome everyone.

I have taken everyone's comments into consideration and it is much appreciated. I recently had to learn how to set up calcium imaging in the institute and I'm having trouble figuring out the minutia of the system.

Perhaps to elaborate more on my question. I don't have any issues with imaging fixed histology slides. The standard 1.5 thickness coverslip works fine on the system.

However, when I use the 1.5 thickness coverglass chambers it doesn't work for the Fura-2 imaging. I have also tested the 1.5 thickness chambers on a Zeiss system and I can image the cells perfectly. Unfortunately, my Olympus system is the only one that can do the calcium imaging with Fura-2.

Perhaps what Bob said about a layer of liquid over the cells would justify the lab's use of 1.0 thickness?

Hobbyst46
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Re: Imaging with different coverglass thickness

#18 Post by Hobbyst46 » Tue Apr 21, 2020 3:43 pm

wtse23 wrote:
Tue Apr 21, 2020 2:21 pm
Thank you for the warm welcome everyone.

I have taken everyone's comments into consideration and it is much appreciated. I recently had to learn how to set up calcium imaging in the institute and I'm having trouble figuring out the minutia of the system.

Perhaps to elaborate more on my question. I don't have any issues with imaging fixed histology slides. The standard 1.5 thickness coverslip works fine on the system.

However, when I use the 1.5 thickness coverglass chambers it doesn't work for the Fura-2 imaging. I have also tested the 1.5 thickness chambers on a Zeiss system and I can image the cells perfectly. Unfortunately, my Olympus system is the only one that can do the calcium imaging with Fura-2.

Perhaps what Bob said about a layer of liquid over the cells would justify the lab's use of 1.0 thickness?
As much as I can guess, the cells are grown on the top side of the coverslip bottom of the chamber; they adhere to the glass, and the fluorescence originates from the foremost layer of the cell, that is, the cell layer that is adjacent to the coverslip (or, if it is a confocal, there is some penetration into the cell, say a few micrometers ?) ?
If the description is correct, there is no reason that a #1.5 coverslip should cause any problem.
If, however, the cells are detached and mobile within the layer of medium within the chamber, then the thickness of the layer of medium matters; is that the case ??

Are you using an immersion objective ? if so, perhaps on the IX-70 oil leaks out from the space beneath the coverslip, for some reason ?

Another question: Fura is fluorescence microscopy. Yet, perhaps you inspect the cells first with DIC (or phase contrast) then switch to excitation ? does the focusing issue appear in both DIC and fluorescence ?
Zeiss Standard GFL+Canon EOS-M10, Olympus VMZ stereo

wtse23
Posts: 8
Joined: Fri Apr 17, 2020 5:59 pm

Re: Imaging with different coverglass thickness

#19 Post by wtse23 » Tue Apr 21, 2020 4:49 pm

Hobbyst46 wrote:
Tue Apr 21, 2020 3:43 pm
wtse23 wrote:
Tue Apr 21, 2020 2:21 pm
Thank you for the warm welcome everyone.

I have taken everyone's comments into consideration and it is much appreciated. I recently had to learn how to set up calcium imaging in the institute and I'm having trouble figuring out the minutia of the system.

Perhaps to elaborate more on my question. I don't have any issues with imaging fixed histology slides. The standard 1.5 thickness coverslip works fine on the system.

However, when I use the 1.5 thickness coverglass chambers it doesn't work for the Fura-2 imaging. I have also tested the 1.5 thickness chambers on a Zeiss system and I can image the cells perfectly. Unfortunately, my Olympus system is the only one that can do the calcium imaging with Fura-2.

Perhaps what Bob said about a layer of liquid over the cells would justify the lab's use of 1.0 thickness?
As much as I can guess, the cells are grown on the top side of the coverslip bottom of the chamber; they adhere to the glass, and the fluorescence originates from the foremost layer of the cell, that is, the cell layer that is adjacent to the coverslip (or, if it is a confocal, there is some penetration into the cell, say a few micrometers ?) ?
If the description is correct, there is no reason that a #1.5 coverslip should cause any problem.
If, however, the cells are detached and mobile within the layer of medium within the chamber, then the thickness of the layer of medium matters; is that the case ??

Are you using an immersion objective ? if so, perhaps on the IX-70 oil leaks out from the space beneath the coverslip, for some reason ?

Another question: Fura is fluorescence microscopy. Yet, perhaps you inspect the cells first with DIC (or phase contrast) then switch to excitation ? does the focusing issue appear in both DIC and fluorescence ?

The cells are adherent and we do inspect the cells using phase contrast then switch to fluorescence. The focusing appears in both if I use 1.5 coverglass. I don't have any issues when I image with a Zeiss Axio Observer system.

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