NetNotes
problems for TEM preparation. It can be very easy to work with also depending on what features you want to see. T e easiest method is to wash the hair to remove external lipids and dirt, place the hairs across a small frame made of plastic, or thread hairs through a narrow plastic tube, embed in LR-White (epoxy is ok too), trim, section with a diamond knife to about 100 nm/gold sections and section stain with uranyl acetate and lead citrate (slightly extended stain times compared to normal) and you can see most features. T e resin will not penetrate the hair. But the hair will sit inside the resin. T ere are oſt en problems with folding (you can reduce this with thicker sections) and sometimes problems at the edges of the fi bers where the fi ber has swollen with the water in the knife boat while the resin has not. If you want to see the intermediate fi laments that make up most of the cortex of the hair, you have to do something more complicated involving repeated treatments of reduction to open up disulfi des to attach stain to and use osmium. Or there is also a silver nitrate method that allows you to see the fi laments, but at the expense of seeing various other structures. I’ll send a separate email to you with a paper that colleagues and I put together with all these methods. Harland, D. P., Vernon, J. A., Walls, R. J., & Woods, J. L. (2011). Transmission electron microscopy staining methods for the cortex of human hair: a modifi ed osmium method and comparison with other stains. Journal of Microscopy , 243(2), 184-196. doi: 10.1111/j.1365-2818.2011.03493.x Kind regards Duane Harland
duane.harland@agresearch.co.nz Fri Mar 13
Specimen Preparation: removing Kapton tape
We have a very valuable sample that was wrapped in Kapton tape for analysis by synchrotron and micro-CT. T e problem now is that we want to remove the tape and sticky residue to prepare the sample for FIB and TEM. Does anyone have any recipes (chemical or otherwise) for getting the polymers off cleanly? We could bake/burn the sample (it’s refractory ceramic) but we don’t want to do this unless necessary. Chad Parish
parishcm@ornl.gov Fri Mar 27
I had good luck removing Kapton tape residue from thin
(~250 µm) semiconductor samples by soaking in warm (~40°C, covered beaker under fume hood) acetone overnight and then gently rubbing with a Q-tip soaked in acetone on a fl at piece of Tefl on plastic. Valery Ray
vray@partbeamsystech.com Fri Mar 27
Specimen Preparation: yeast
I always have problems embedding/infi ltrating yeast! I have tried
diff erent resins, vacuum steps, etc. Does anyone have an embedding protocol/resin that works? Sue Van Horn
susan.vanhorn@stonybrook. edu Fri Apr 17 T ere are many resources in the web concerning the tricky task to properly embed yeast cells for ultrathin sectioning. My favorite ones, as of today: Mary’s Manual (Boulder, CO, USA): http://bio3d.
colorado.edu/docs/mmanual.pdf , Giddings, T. H., Jr., O’Toole, E. T., Morphew, M., Mastronarde, D. N., McIntosh, J. R., and Winey, M. (2001). “Using rapid freeze and freeze-substitution for the preparation of yeast cells for electron microscopy and three-dimensional analysis,” Methods Cell Biol. 67, 27–42 (yes, Mary is one of the co-authors). Also, Kent L McDonald, “Out with the old and in with the new: rapid specimen preparation procedures for electron microscopy of sectioned biological material,” Protoplasma (2014) 251:429–448 DOI 10.1007/ s00709-013-0575-y. T is article is quite helpful as it shows that some of the paradigms of “old” embedding protocols are clearly outdated, if not to say “wrong”. It also depends on your equipment, the specifi c question, and so on. Reinhard Rachel reinhard.rachel@biologie.
uni-regensburg.de Fri Apr 17
68
Not sure what type of yeast you work with. We work with budding yeast and have been had good luck with microwave radiation, especially for stationary yeast, of which the cell wall is very tough.
http://www.ncbi.nlm.nih.gov/pubmed/17156022 . For log-phase cells, extended infi ltration (at least one overnight) works fi ne using Spurr. I believe the low viscosity of Spurr helps. Best luck and let me know if you need a reprint. Zhaojie Zhang
zzhang@uwyo.edu Fri Apr 17
X-ray Microanalysis : NIST DTSA-II Iona released
NIST DTSA-II has recently been updated to version Iona. Download for free from
http://www.cstl.nist.gov/div837/837.02/epq/ dtsa2/
index.html . DTSA-II provides a host of tools for quantitative EDS microanalysis including quantifi cation, simulation and measurement planning. Iona has a host of improvements both large and small which are detailed in the release notes on the web site. Further details on quantitative analysis with NIST DTSA-II are available in Newbury & Ritchie’s J. Mat Sci. article “Performing elemental microanalysis with high accuracy and high precision by scanning electron microscopy/silicon driſt detector energy-dispersive X-ray spectrometry (SEM/SDD-EDS)” (free for download from
http://link.springer.com/article/10.1007/ s10853-014-8685-2 ). T is article demonstrates the potential of the modern EDS detector to perform reliable, quantitatively accurate compositional measurements even for some very challenging samples. Nicholas Ritchie
nicholas.ritchie@nist.gov Tue Apr 21
Image Processing: exporting spectrum image slices
I’m trying to export data from a 2D EELS spectrum image in Digital Micrograph and I’m not sure what the best approach is. Here is my problem: 1. I have a 2D EELS map/SI of a thin fi lm interface, where x is some width parallel to the interface, y is some width perpen- dicular to the interface, and z is the EELS energy range (400 700 eV). 2. I would like to integrate all the spectra in plane (x-direction) to improve signal-to-noise (T is would essentially leave me with an EELS line scan parallel to y). 3. I would then like to export slices at specifi ed integration windows normal to the plane (y-direction) to text fi les. T e only way I can currently do this is by drawing an ROI onto the SI, which generates an individual spectrum integrated across x. I then have to export this and drag the ROI, repeating ad nauseam until the entire y length of the scan is traversed. Is there a simpler and faster way to do this? Please let me know if you need more clarifi cation. Steven R. Spurgeon steven.
spurgeon@pnnl.gov Mon Mar 9
If you use MATLAB you can import DM3 fi les. Here’s what might be a useful link (I have not used this particular one but it looks simple):
http://www.mathworks.com/matlabcentral/fi leexchange/ 29351-dm3-import-for-gat an-digital-micrograph ImageJ will also
open.DM3 fi les directly; I do all my analyses in that package. T ere are plugins that can be used for this as well, that you can try on spectrum images. Once you are in one of those programs you can easily write scripts to do any arbitrary operation on the data. Larry Scipioni
les@zsgenetics.com Tue Mar 10
I would suggest that you use a multivariate statistical analysis (MSA) approach such as using AXSIA (Automated eXpert Spectrum Image Analysis) soſt ware or the MSA plug-in for DM that does Principal Component Analysis. T e AXSIA soſt ware uses Matlab and the SIMSIMAN module that comes with the AXSIA soſt ware would allow you to extract your line profi le easily. It would also be available in Matlab for any manipulation or export that you need. T e MSA plug-in would allow you to reconstruct your data to improve your signal to noise for the profi le. In both cases, you must be careful to align your EELS spectra in energy throughout the spectrum image
www.microscopy-today.com • 2015 July
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