>Thanks to Guy for his very helpful and complete description of how he >measures "resolution" (a term I find very slippery, though nonetheless >necessary). Absolutely! Rayleigh resolution and FWHM are not the same thing, and various other parameters, such as the precision with which we can locate objects (recently discussed in this group) are different things entirely. The main merit of FWHM, my favoured parameter, is that it is relatively simple to measure. Also - see below - in practice one can resolve apart separate objects separated by the FWHM. > >I think that it is important to remember that the "size" of the data >recorded in this fashion is not strictly speaking the instrumental "Point >Spread Function" (PSF) that appears on all of the theoretical charts and >graphs, but the PSF convolved (read as: multiplied-in-3-dimensions) with >the ACTUAL size of the bead. Larger beads will give "worse" resolution. >Assuming that one is using 100nm beads, you are ALMOST justified in >subtracting 100 nm from your measured FWHM. I'm glad you said ALMOST, Jim! As Stefan Hell has already pointed out, for practical purposes 100nm is small enough to be regarded as a point. You are certainly NOT justified in subtracting 100nm from the measured FWHM! It is, of course, true that in principle the size of the actual bead wil have _some_ effect on the measured image, and last night I tried out some quick tests to find out how much. I ran two samples - Biorad's standard 210nm bead slide supplied with their systems and 100nm beads (thanks to Carol Cogswell for these) in Cityfluor. The lens used was a x63 Plan Apochromat NA 1.4. Lateral FWHM with the 210nm beads was ~ 300nm, Axial ~ 700nm. The vertical profile looked a bit asymmetric, implying that the mounting medium (can anyone from Biorad tell me what it is?) was not a perfect match. There were quite a few beads fairly close together, and beads 280 nm apart (centre to centre) were easily resolved. With the 100nm beads lateral FWHM was ~ 240nm and axial was 400 - 450nm (it's hard to be more precise when you're working in 180nm steps!). The profile looked reasonably symmetrical though the side lobes were quite noticeable (I had the background fairly high). So adding 110nm to the bead diameter has only increased the profile (laterally) by ~60 nm. And even that may be largely due to refractive index mismatch. This implies to me that reducing it below 100nm would have a pretty insignificant effect. >Two remaining questions: > >1. Guy, could you tell us about the rest of your system? Does it include >a "normal" fluorescence attachment or a DIC module? As I remember, on the >Optiphot, both of these impose a 1.25x magnification increase when viewed >from above and causes an inverse effect on the size of the beam in the BFP >which may explain some of the differences in resolution discussed here. Confocal microscopes didn't exist when the Optiphot was a current model! My only recent experience with 160mm tube-length Zeiss systems is on a 12-year-old Zeiss Standard, and as Jim implies, putting anything into the tube is an optical disaster. The fluorescence attachment is not properly compensated and when we once tried putting a confocal head on it (the little HBH, predecessor of the Optiscan) we had to take out the fluorescence attachment to make it work. So I wouldn't advise anyone to add confocal to an ancient microscope. We use a Zeiss Axiophot, which is an infinite tube-length system so that whatever you put into the tube makes no difference. However you don't want too much glass in the way, so I don't advise adding anything that can't be pulled out to give a clear path. And one thing which is absolutely essential is that you pull out any DIC prisms before doing confocal work. The laser beam is inherently polarised and they will completely screw up your resolution. (One friend of mine - no names, no pack drill - actually called in the company engineer because his new confocal was giving lousy resolution - and had to be gently reminded to pull out the DIC prism ...) > >2. Do you have any problem with fading? No, I don't. Both the Biorad-supplied 210nm bead slide and my Molecular Probes 100nm beads in Cityfluor seem durable enough to record a z-series at adequate sampling for a reasonable measurement. Sure, there's noise, but you measure a few beads. The real limitations are the step size we can get (too big) and the precision of the Zeiss focus drive (woeful). I hope soon to be able to try a precision x-y-z stage which should give much better sampling. Of course it may be that fading _will_ then become a problem! >As I assume the answer is yes, >what do you do about it? (Laser power levels? Zoom/pixel-size/plane >spacing? Techniques like "focus"-here/collect-data-there? >Do you "fit" the >data recorded to some sort of mathematical function in order to reduce the >effects of noise on your measurement of FWHM, or just estimate my eye? I don't, but given the limited sampling imposed by the Z-drive it would in principle seem like a good idea. However you'd probably have to smooth the data first and by then you're getting a long way from the original ... So I just eyeball it and do quite a few beads so as to get an overall picture. >Other practical suggestions? Yes, one. Doing a line scan doesn't, in practice, always quite hit the line you've selected in the X-Y image. (The ballistics of the galvo mirror, I presume). So you may need to take a few shots. The other approach is to take a complete series of sections and extract an XZ or YZ section from these. Then it's easy to get the profile you want _but_ with most software (certainly with mine) you are limited to integer interpolation so you'll have to go for the nearest figure and make a small correction to your vertical measurements. My software doesn't do linear interpolation, either - it just duplicates slices. I regard this as preferable but it's open to argument. Voxel-View does linear interpolation when it 'pads out' a volume, and you should be aware of this if you use it to generate your XZ sections. Guy Cox