Sample mounting
(lined spherical aberration to wikipedia page) |
(Good start! I did some changes, but it's still quite confusing. Schemes/photos of objectives, coverslip boxes etc. would help to get the principle.) |
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| − | Different samples require different | + | Different samples require different ways of sample mounting, with different mounting medium and coverslips of different thickness. |
If you get this wrong, your images will suffer from poor z resolution and loss of intensity due to [http://en.wikipedia.org/wiki/Spherical_aberration spherical aberration] | If you get this wrong, your images will suffer from poor z resolution and loss of intensity due to [http://en.wikipedia.org/wiki/Spherical_aberration spherical aberration] | ||
| − | caused by refractive index mismatch between the sample and the lens immersion medium | + | caused by (A) refractive index mismatch between the sample and the lens immersion medium and (B) incorrect sample+coverslip thickness for what the lens is corrected. |
| − | and incorrect sample+coverslip thickness for what the lens is corrected | + | |
| − | There is no substitute for getting the optical properties of the sample matched to the lens you are using. | + | There is no substitute for getting the optical properties of the sample matched to the lens you are using. If you do that, the green things will look rounder in z... but they will never "look" isotropic (= same width in z and xy), always a bit "stretched" in z ... thats just how the optics work. |
| − | If you do that, the green things will look rounder in z... but | + | |
| − | thats just how the optics work. | + | |
| − | + | Remember, the image of a point is not a point, its a blob called the Point Spread Function (PSF), which is larger in z and in x and y. See [http://support.svi.nl/wiki/PointSpreadFunction PSF at SVI homepage]. | |
| − | + | ||
| − | http://support.svi.nl/wiki/PointSpreadFunction | + | |
| − | + | What does that mean for you? | |
| − | 1) Use coverslips that are exactly always the same thickness (in your case those nice #1 which are always 0.15 mm. | + | 1) Use coverslips that are exactly always the same thickness (in your case those nice #1 which are always 0.15 mm). |
| − | ( | + | (The total thickness of the sample and the coverslip should be about 0.17 mm - the lenses are corrected for that thickness) for a very thin sample, like a cell monolayer, only a few microns thick, then use a #1.5 coverslip which is 0.17 mm, but for a very thick histological section that is 200 microns, use a #1 coverslip that is 0.15 mm. So than section plus coverslip = again 0.17 mm.) |
| − | for a very thin sample, like a cell monolayer, only a few microns thick, then use a | + | |
| − | + | If your tissue sections or samples are about 20 microns ...... which coverslip should you use .......? Probably the number #1.5 (0.17mm), because that gives about 0.172-0.175 mm thickness. If you use #1 coverslides, then that 0.15 mm plus 0.002-0.005 mm = about 0.155 mm .... which is very far from 0.17 mm. And in that case the images will be more blurred in z than they should be (if using an oil lens corrected for 0.17 mm, or if the water immersion lens correction collar is set to 0.17 mm !!!) | |
| − | + | ||
| − | + | ||
| − | If your tissue sections or samples are about 20 microns...... which coverslip should you use.......? | + | |
| − | + | ||
| − | If you use | + | |
| − | + | ||
Get the idea? | Get the idea? | ||
| − | 2) For a watery sample, embed in 2% low melt agarose in buffer or 50:50 glycerol : buffer.... use a water objective | + | 2) For a watery sample, embed in 2% low melt agarose in buffer or 50:50 glycerol : buffer.... use a water objective. The correction collar allows you to correct for the coverslide thickness, from 0.15 to 0.19 mm !! So if you use that properly you can use #1 or #1.5 cover slides. But you need to get that right ..... or else you get? Yes! Blurry images in z direction! |
| − | The correction collar allows you to correct for the coverslide thickness, from 0.15 to 0.19 mm !! | + | |
| − | So if you use that properly you can use #1 or #1.5 cover slides. But you need to get that right..... | + | |
| − | or else you get | + | |
| − | 3) | + | 3) For dehydrated samples, use a hard setting medium like Mowiol that has a refractive index like glass ...... use an oil objective. Aim for a sample plus coverslip thickness of close to 0.17 mm, since the objective are expecting that to be the case ... but its less critical in this case since the RI of the glass and the sample is almost the same. |
| − | Aim for a sample plus coverslip thickness of close to 0. | + | |
| − | but its less critical in this case since the RI of the glass and the sample is almost the same. | + | |
Revision as of 13:13, 24 June 2009
Different samples require different ways of sample mounting, with different mounting medium and coverslips of different thickness.
If you get this wrong, your images will suffer from poor z resolution and loss of intensity due to spherical aberration caused by (A) refractive index mismatch between the sample and the lens immersion medium and (B) incorrect sample+coverslip thickness for what the lens is corrected.
There is no substitute for getting the optical properties of the sample matched to the lens you are using. If you do that, the green things will look rounder in z... but they will never "look" isotropic (= same width in z and xy), always a bit "stretched" in z ... thats just how the optics work.
Remember, the image of a point is not a point, its a blob called the Point Spread Function (PSF), which is larger in z and in x and y. See PSF at SVI homepage.
What does that mean for you?
1) Use coverslips that are exactly always the same thickness (in your case those nice #1 which are always 0.15 mm).
(The total thickness of the sample and the coverslip should be about 0.17 mm - the lenses are corrected for that thickness) for a very thin sample, like a cell monolayer, only a few microns thick, then use a #1.5 coverslip which is 0.17 mm, but for a very thick histological section that is 200 microns, use a #1 coverslip that is 0.15 mm. So than section plus coverslip = again 0.17 mm.)
If your tissue sections or samples are about 20 microns ...... which coverslip should you use .......? Probably the number #1.5 (0.17mm), because that gives about 0.172-0.175 mm thickness. If you use #1 coverslides, then that 0.15 mm plus 0.002-0.005 mm = about 0.155 mm .... which is very far from 0.17 mm. And in that case the images will be more blurred in z than they should be (if using an oil lens corrected for 0.17 mm, or if the water immersion lens correction collar is set to 0.17 mm !!!)
Get the idea?
2) For a watery sample, embed in 2% low melt agarose in buffer or 50:50 glycerol : buffer.... use a water objective. The correction collar allows you to correct for the coverslide thickness, from 0.15 to 0.19 mm !! So if you use that properly you can use #1 or #1.5 cover slides. But you need to get that right ..... or else you get? Yes! Blurry images in z direction!
3) For dehydrated samples, use a hard setting medium like Mowiol that has a refractive index like glass ...... use an oil objective. Aim for a sample plus coverslip thickness of close to 0.17 mm, since the objective are expecting that to be the case ... but its less critical in this case since the RI of the glass and the sample is almost the same.
