NetNotes


STEM is definitely the next item on the menu. Te trick about


inverting the HAADF image is a good one if you don’t have a BF detector! I’m all too familiar with samples blowing up under the beam. A thin coating of carbon on the exit surface (or sometimes both sides, in my experience) usually solves it as you indicate. If only the carbon coater was in the same building as the TEM. Tanks for the advice, Chris Winkler crwinkler@ncsu.edu


Hi Henk, I’m not sure about the kind of microscope Phil was


referring to, but as for FEI microscopes such as the Tecnai the diffraction plane is at the selected aperture plane only when in low mag mode (objective lens off). I guess in Phil’s TEM it might be something similar. Chris, I have used a JEOL TEM a few times, so I am not familiar with it. But yes, there might be a way of switching off the objective in low magnification mode. You will get a better contrast. Another possibility that might work, as pointed out by Henk, is to use the ADF or HAADF detector to do dark field imaging and invert the image contrast. I’ve done (inverted contrast) ADF imaging on non-stained tissues and it works (with that sample I could see absolutely nothing in TEM mode). Another thing you can do is BSE-SEM imaging of the sectioned embedded block’s surface. Of course, you need to carbon coat


its


surface before. Use the right SEM settings to reduce the interaction volume. Maybe you can try lower kV, lower WD. Good luck. Erico Freitas ericotadeu@ufmg.br


I have a Hitachi 7700. Te FOV (field-of-view), aka selected


area aperture, works at all mags, not just in low mag. I suspect the same is true for any TEM with a SA (selected area - let’s expand the abbreviations in the emails). Just do your imaging as normal, and if you need a contrast bump without losing resolution, put in the SA/FOV aperture. It’s just adding another aperture, not changing any imaging parameters or any electronics. You do lose field of view at mags <10,000 or so, depending on the aperture size, but that’s it. Assuming you have a manually variable aperture and not some computer-controlled thing that only works when and how the computer thinks it should. If that doesn’t exist, it’s coming. Phil Oshel oshel1pe@cmich.edu


Hi Chris, what TEM do you have? If it is a FEI/TermoFisher you


could try imaging the samples in low magnification (LM) mode. In LM the objective lenses are off (you will see the strength value around 6%), and in that case the diffraction lenses act as the objective lens (you may even want to use the selected area aperture). In FEI machines you will have better contrast in LM mode. I know the magnification is not that high in LM mode, but you may still benefit from using the GIF camera that will give extra magnification. Best wishes. Erico Freitas ericotadeu@ufmg.br


I can confirm BSE-SEM imaging can really be advantageous


even on ultrathin sections. A thin carbon coating is a must, as Erico mentioned in his message. In BSE imaging mode one can play with accelerating voltage, concentric back-scattered detector rings and beam deceleration settings. With an optimal setting, you can image a really huge section on a TEM grid with almost no interference of TEM grid bars. Some years ago we used such approaches for imaging of whole ultrathin sections through mice teeth. We were searching for places with Tomes’ processes in ameloblasts and we were successful. Oldřich Benada benada@biomed.cas.cz


Tank you to all for your responses and advice! I want 70 to


summarize the responses I received regarding my query on how to improve contrast on low contrast samples in a materials science TEM:


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CO A U TH O R A M M I N O AN N O T A T E MT T A N V S BO O B G RA L IS S U DO I


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42 43 47 50 51 55 56 62 45 48 BS N 67 71 68


MI C R O S 57


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ET I M D O N E G I V I N SECT I O N W E B S I TE RE V I E W T O N Y E L L AR S O N R A N G E W E B


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G U H R S A 66


69 70 72 75 73 76 www.microscopy-today.com • 2020 November 52 53 54 49 C O P 58 59 60 61 28 29 T


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I L K P Y


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E D I T O R 21


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J O U RN AL O


A F 34 35 36 T S C I N A


1) Buy a Bio TEM with a large gap objective optimized for high contrast.


2) Adjust staining protocols--check pH levels, switch to radioactive stains (EH&S now makes this difficult in some universities), supplement non-radioactive stains with lead citrate, tannic acid, or other agents, and so forth. One responder linked me to the following protocol that worked wonders for her: https://www. sciencedirect.com/science/article/pii/S1047847714002378


3) Try to minimize contribution from the grid by moving to ultrathin carbon or plasma cleaning thicker carbon to thin it down.


4) Acquire EFTEM SI and generate jump-ratio maps around the plasmons peak, carbon peaks, etc.


5) Switch to STEM and see if contrast is improved when using ADF or BF detectors. If you only have an ADF detector, invert the image contrast to give a pseudo BF image (more acceptable to people used to TEM). Similarly, switch to the SEM and try STEM or BSE imaging at 30kV.


6) Send a sample to Delong Instruments and see how it looks at 5, 15, and/or 25kV. Also, try working at lower voltages like 40 or 60kV.


7) Work in low mag (LM) and use the selected area aperture to further improve contrast. LM contrast will be inherently higher as the objective lens is turned nearly off.


8) Sum/stack several images to improve SNR. For thin sections with little contrast and no outstanding features for correlation, this will be tricky.


9) Replace one of the objective apertures with a phase plate.


10) For JEOL and Hitachi microscopes, use the field limiting aperture located below the objective to improve contrast.


Tanks again to everyone for all the help. We’ll be trying STEM in the TEM and SEM next. Chris Winkler crwinkler@ncsu.edu


Crossword Puzzle Answers See puzzle on page 60.


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