NetNotes


electrons for getting a very fast separation of parts on specimen with a small interaction volume like hairs on insects or cells or substrate etc. Te backscattered detectors (I have three) I use to get surfaces (topography) ad hoc separated in a different color. You can also try to get more different images with different accelerating voltages and the best suited detectors for it... Also distance of the detectors to the specimen can make very different images. Tere is a PDF from a Mikrokosmos article which describes my work, but only in the German language: www.elektronenmikroskopie.info/pdf/ Mikrokosmos06-99.pdf (1 MB) I am using a digital image acquisition system from point electronic, www.pointelectronic.de, which can handle up to 8 detectors and scan up to 4 detectors in a resolution of maximum 16K × 16K pixels. Te scanning soſtware has the ability to directly mix the signals from the SEM into a colored image. For the images, have a look at: http://www.elektronenmikroskopie.info/ galerie%20-%20rem-biologie.htm for biologic specimen and http:// www.elektronenmikroskopie.info/galerie%20-%20rem-material. htm for some images from material science. Sorry, pages are only in German language up to now and the site is still in construction. Stefan Diller stefan.diller@t-online.de Fri Aug 12


EDX:


nanoporous material I am doing some SEM/EDX analysis on nanoporous alloys


containing Ag-Au-Pt with a pore size between 3–15 nm. Tis nanoporous alloy is on top of an alloy with the same elements. Te average thickness of this nanoporous layer is around 8 µm. Typically, I prepare metallographic specimens to either determine the thickness of my nanoporous alloys or to analyze its composition by doing EDX on a cross section of the sample. However, I found that because the Pt concentration is very low (~1 at.%) I have to use accelerating voltages higher than 20 kV. I have tried lower accelerating voltages obtaining very weird EDX results. I have two main concerns. One is about the effect of porosity in my results because I know that this type of analysis assumes 100% density whether you are using standardless routines or with standards. My second concern, which is related to the first one, is if there is any real concern of having an interference of the substrate in my EDX reading, mainly due to high interaction volume and high porosity. Terefore, I was wondering of there is any correction or consideration to take into account to do this kind of analysis. Adrian Vega adrian. vega@utoronto.ca Wed Aug 10 Given those elements, you may be okay using higher voltages.


You probably want to use something like Casino or some other Monte Carlo method to estimate the excitation volume. I ran simulations on pure Ag (the lightest component) and found that the excitation depth was about 1 µm at 30 kV. It was about 700 nm at 25 kV and about 500 nm at 20 kV. You should not be exciting your substrate. I am uncertain what you mean when you say you had problems with low Pt concentration calling for higher accelerating voltages. What is your Au content? If it is high, then you need to make sure that your system is deconvoluting well. It will be more challenging to pick out a small intensity for the Pt M line next to a strong intensity for the Au line; however, it should be possible. You could use the L lines around 9.5 keV, but the intensity is much lower than for the M lines. Such challenging deconvolutions will need critical review rather than simply believing the numbers. You should check the fit and examine the residuals to make sure everything is accounted for. I recommend that for all critical applications. If there is a problem with your energy calibration and the peaks shiſt, then you could really have problems with the Pt intensity. Residuals would show leſtover intensity on one side of the peak and a deficit of intensity on the other. Of course,


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that could be reflected in errors in the Pt concentration. You could also have problems if the stored peak widths are not the same as the operating conditions at the time of analysis. We have an old Oxford ISIS system. Te factory peak profiles were collected at the highest resolution setting, but we usually reduce the pulse processing time to count faster. Our peaks are a bit broader and the default profiles don’t fit the actual spectra very well. New soſtware probably takes the pulse processing time into account and adjusts the profiles. Terefore, we have collected our own profiles under the exact same conditions. Te default profiles are pretty good, but our own profiles take all possible differences between our system and the factory into account. I would recommend comparing results for L lines and M lines. At 30 kV, they should give you similar numbers if the deconvolution is good. I would also recommend testing the analyses on a sample with no Pt (maybe on a sample of pure gold). You could overlay the two spectra to see if Pt appears on a shoulder of the Au peak. You could also check the deconvolution results. If the soſtware still reports Pt in the no-Pt sample, then you should downgrade the trust in your soſtware. Ofand, I don’t know that porosity of 3–15 nm should cause problems with the analysis, provided it is homogeneously distributed. It would allow a deeper penetration of the beam and it would allow a longer path-length for absorption on the way out. Te mass absorption effects on the way in and the way out should be the same. EDS can be quite a powerful technique if done well with good soſtware. Just beware that not all soſtware is created equal. You will need to watch it carefully as you apply it to a number of problems and determine how much trust you can put into the results. Warren Straszheim wesaia@iastate.edu Wed Aug 10


Specimen Preparation: precipitate in TEM Can anyone suggest solutions (pun probably intended) to the


problem of mystery precipitate on cells prepared for TEM? Tis is 5 nm–15 nm diameter black dots that appear on cells, but not on resin sections or on support film. Te dots localize strongly to chloroplasts and myelin when osmium is involved, but not otherwise. In glutaraldehyde-only fixes the dots stay on the outside of the cells or in vacuoles. Many people have posted before about precipitates; on the basis of having gone through the Listserver archives we decided to try a series of experiments to try and get rid of the mystery 5 nm dots currently gracing all cells prepared for TEM in this lab. Our results are inconclusive, thus we are wondering if anyone else has tried anything and found a way of making the dots disappear. I have tried many combinations and permutations of local/ bought water, 3 different purities of glutaraldehyde, paraformaldehyde with and without glut, new osmium, lots of different buffers, new glassware, and plastic containers instead of glassware . . . the only thing I haven’t tried yet is the ruthenium/veronal acetate fixes . . . Anyone with time to have a look can find details of some of the experiments in the pdf here, ⟨http:// dl.dropbox.com/u/10613310/PrecipLIST-12May2011.pdf ⟩ and these are representative of other results with different buffers. Obviously the cells aren’t particularly nicely fixed - that’s another set of experiments. Any suggestions most gratefully received. Giselle Walker giselle.walker@ anatomy.otago.ac.nz Tu May 12 I got similar precipitate when I didn’t wash out the PBS enough


before block-contrasting in uranyl acetate. Also, I got this type of precipitate when I leſt my samples on osmium too long (>3 hr, and they were not the ones with the PBS not washed) If you mix PBS (or any other phosphate) with uranyl, you will get fine precipitate. I am not sure about the mechanism of precipitate forming aſter longer osmium exposure. I read one paper where the author identified intracellular


www.microscopy-today.com • 2011 November


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