Sample mounting

Revision as of 09:56, 24 June 2009

Different samples require different kinfos of sampke mounting, with different mounting medium and different thickness coverslips.

If you get this wrong, your images will suffer from poor z resolution and loss of intensity due to spherical aberration caused by refractive index mismatch between the sample and the lens immersion medium, and incorrect sample+coverslip thickness for what the lens is corrected for.

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 the well never be "look" the same width in z anbd in xy, always a bit larger... thats just how the optics work.

remember, the image of a point is not a point, its a blob called the psf, which is larger in z and in x and y. see 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.

(the total thickness of the sample and the coverslip should be about 0.17. The lenses are corrected for that thickness) for a very thin sample, like a cell monolayer, only a few microns thick, then use a number 1.5 coverslip which is 0.17 mm,

but for a very thick histological section.... that is 200 microns, use a number 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) right, because that gives about 0.172-0.175 thickness. If you use number 1 coverslides, then that 0.15 plus 0.002-0.005 = about 0.155.... 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, or if the water immersion lens correction collar is set to 0.17 !!!)

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 0.17mm, 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.