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Hello!
I am learning how to edit TEM images in Fiji (Image J).
I want to highlight some specific areas of my images using colors.
Can somebody help?
Thank you!
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It will depend if will draw something manually or if you will apply an automatic routine. (and what kind of images the journal will accecpt)
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The TEM image shows the appearance of Moiré fringes with a width of 5-10 nm in the eutectic layer. When strain distribution is analyzed using GPA, strain concentration bands with similar positions to Moiré fringes appear. I would like to know if the presence of such Moire fringes will affect the strain analysis of GPA?
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Kaushik Shandilya Thank you very much for your reply, my sample has Moore's stripes in the vast majority of the area in the TEM bright field image, so it may not be practical to remove them at the time of shooting. My new question is, how do I make image corrections to remove or rather make good use of the moiré streaks for strain analysis, e.g. how does the Fourier filtering you mentioned work?
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phage tails are getting broken during precipitation and sample preparation, can you suggest a protocol?
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For many phages you can use PEG precipitation and fairly gentle centrifugation. I think high speed centrifugation may be what inactivates the tail.
However the best method would be equilibrium gradient ultracentrifugation so that you never pellet the phage.
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The existence of anti-phase domain boundaries (APBs) in polycrystalline materials is usually established by electron microscopic techniques (SEM/TEM) [1] and is also discussed in diffraction data analyses.[2]
I don’t have a good familiarity with TEM/SEM (and I’m very open to be educated here) but it doesn’t seem convincing enough to look at some microscopic images with atomic level resolution where APBs are found as a straight line (or arbitrarily curved line as in Figure 7 in ref. 1) forming a boundary/wall between the two domains in the same particles, while there is no disorderliness of any sort around and away from the APB.
The reason I’m raising this point is that particle surface is usually more disordered than any kind of defects in the bulk. In fact, it’s even well established that the surface of solid particles behave more or less like a liquid layer [3], and the smaller the particle size the thicker the liquid layer at the surface. And yet, in the TEM images of nanoparticles that I have seen in some articles there is(are) only the APB(s) visible, and no sign of the bigger unavoidable inherent surface disorder.
Is it possibly due to the fact that in TEM, the electrons pass through the particles and form an image which is influenced by the bulk of the particle? If so, why then the rest of the atomic arrangements within the domains look nearly perfect (i.e. as if it’s a single layer of pointy ordered atoms)?
And as for diffraction data, APBs affect some of the reflections selectively but usually there are different broadening contributions which make it challenging to disentangle. Nevertheless, at least the existence of planar defects like APBs is indicated in diffraction patterns.
Any input would be appreciated.
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Thank you for the detailed answer. So, in principle it's possible but challenging.
I'm actually not going to be a TEM operator in the future (I'm not planning to), so I'll not need to know all the experimental problems and considerations. I mainly wanted to know how much I can rely on the data that I see in the literature (I gave a specific reference to an article in the question).
It's good anyways to have general information about it, so thank you for your time.
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One researcher did ballistic performance of kevlar fiber impregnated with nanosilica/ polyethylene glycol shear thickening fluid. And he loaded 10, 15, 20Wt% of Nanosilica with polyethylene glycol.
After fabrication, how we can confirm the equal distribution of nanosilica over the kevlar. Whether nanosilica is spread all over the kevlar equally or not?
How I can confirm? Any SEM, TEM test required ?
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You can use SEM/EDS to check for homogeneity of Si distribution. If you expect non homogeneity on 1-100 micron scale, you may want to use line scans of Si radiation (maps are not as sencitive). If scale is in range of millimeters or cantimeters, you may want to perform spot analisis for a lot of spots or use not a micro analytical technique, like XRD.
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For the LFA, detection antibodies need to bound to gold nanoparticles. what are the analytical techniques to verify a successful conjugation? Please suggest any articles with the standard protocol.
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Assuming there were no other protein, you can do BCA on the nps and on the reaction volume to ensure that the antibody is present at a known concentration on the beads.
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I'm having trouble getting clear images of my bacteriophage under TEM. The process I've followed thus far is described below. Any help would be appreciated.
I've isolated bacteriophage active against Staphylococcus aureus bacteria from wastewater.
I grew these to high titer by infecting liquid cultures (S. aureus grown to OD600=0.1) with a low dose of bacteriophage (Multiplicity of infection = 0.1 to 0.05) and incubated until lysis was observed ~3-5 hours later.
These lysates were spun down at 5000g for 5 minutes and filtered through 0.22µm filters.
Chloroform was added (0.1 volumes / 1ml in 10ml), mixed, and left to incubate at room temp for 15-20 minutes with inversions every so often.
These were then centrifuged at 3220g for 10 minutes and care was taken to remove the lysate without touching the cell debris and chloroform layers.
We then used ultrafiltration columns (Cytiva: Vivaspoin 20, 100,000 MWCO) to resuspend the phages (trapped on the column membrane) in pure SM-buffer.
30µl of high titer phage was then fixed using paraformaldehyde and sent for TEM.
TEM images are stained with Uranyl acetate.
Please advise.
Thanks, Josh.
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I agree with Michael J. Benedik . Just a thought: it looks like the darkened area is in the region of the highest phage aggregation. Maybe it is the reason for higher charging? Please note, that the resolution in that region is also affected - drastically reduced.
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I have doped 3%, 4% and 5% of a rare earth metal oxide into a parent material. Is it possible to identify or confirm the dopant through HR-TEM even at these small dopant percentage levels? Also, will the dopants lattice spacing corresponding to the respective hkl plane also be identified?
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Thank you Tarun Kumar Dhiman and Mewin Vincent for the informations.
To check the structural modification please do the XRD study. XRD results will give you an idea about any kind of change and based on that data the HR-TEM results will help you to find out the lattice fringes and spacing. I can suggest this from my side.
Regards
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I have attached two SAED patterns for graphene based nanofillers. Can anyone guide me through the indexing of the said patterns? And also, is it possible to find out the number of graphene layers with the help of diffraction patterns?
Any help will be appreciated.
Thank you
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Meenaketan Sethi Thank you for the reference
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Assuming that there is a special type of advanced high strength steel that has a fully martensitic microstructure and a nominal tensile strength of 1500 MPa. By changing the composition of a single alloying element, i.e. increasing the C content, we can change the morphology and mechanical properties of the steel. How can we effectively compare the changes between the two steels at a fundamental level that goes beyond the general SEM, EBSD, and TEM characterization methodologies? Are there any crystal plasticity models that can be used in this case? Is a crystal plasticity study even valid for martensitic steels?
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Oleksii Sherepenko , as I have understood this question can be divided into 2 parts, namely
1) The influence of the carbon content on morphology of martensite. The alloying has influence on the lattice parameter of both parent and product phases, therefore the interfacial configuration, and the eigenstrains are changing. So both factors, which govern the morphology of product phase (i.e. elastic energy and interfacial energy) are functions of the alloying content. The modelling methodology in this case could be some crystallographic based models, molecular dynamics/monte carlo or phase-field model, depending on the resources available. Also with different content, the parent phase can have different grain size, which also has effect on martensitic transformation.
2) The influence on mechanical properties. From my point of view, the question can be divided in several "isolated" effects
2.a) Solid-solution strengthening. With increase in amount of secondary element solid-solution phase should have increased yield strength
2.b) Morphology of martensite. The size and form of martensites should play a role in the motion of dislocations. The number of interfaces should play a major role here.
About some crystal plasticity models.
There are some models, which take into consideration Petch-Hall effect, for example
It could be used as starting point for further model development.
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Recently I synthesized a material. To specify, I loaded about 0.2%Mg on the synthesized MnO2 nanoparticles, the catalytic performance is very good. I guess that the trace amount of Mg may exist as the single-atom state on the surfaces of MnO2, but I don't know how to chatacterize the Mg. Usually in the publiactions, "heavy" metals such as Pt, Au or Ir acting as single atom catalysts will deposite on "light" supporting materials such as carbon, MOF, Al2O3 or TiO2. And STEM-HAADF will always be used to characterize these single atoms because it can detect and distinguish different elements with big difference in atomic numbers. However, in my research, the single atom Mg (I guess) is lighter than Mn, so STEM-HAADF does not work here. Can anyone give suggestions here? Thanks
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Thanks for your suggestions.
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Hello, I conducted TEM on cells pre-treated with various compounds, which induced cell stress and death. I originally measured mitochondrial size, swelling of the endoplasmic reticulum, and vacuole presence. However, I read a publication that suggested a difference in vacuole membrane based on type of cell death induced. I re-reviewed my samples, but I'm unsure how to interpret the vacuoles. Some look just like tearing, without a membrane at all.
Any insight or resources appreciated
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Double membranes (twin bilayers), are characteristic of autophagic death. Other than that you look for chromatin condensation status to see necrosis vs apoptosis, mitochondria fragmentation or ER aggregation often studded with ribosomes in calcium overload related death. Alternatively, you can refer my paper here
Which has More TEM images in supplementary materials.
let me know if you have any questions.
J.
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I used TEM equipped with tungsten gun to obtain attached image (several other too, like shown).
Side blotting technique followed by negative staining (1% PTA for 30 sec) was used. I cannot get the more clear background and there's also problem of resolution.
Please suggest how can I improve sample preparation? or any other suggestion to get better images.
The sample is polyelectrolyte complex in water.
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Everything in terms of the preparation looks good to me; you just need to underfocus a bit more. You can clearly see areas with no stain; just blank support film. So, I think everything is good except your focus. Don't use the wobbler. Focus to what looks like the best contrast using your eyes.
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I am working on hollow carbon quantum dots. During the TEM analysis, it is very difficult to get a clearer image of the crystal structure due to the extremely small size. I want to measure the exact size of the shell, hollow-core, etc.
Is there any solution to this problem? I have tested on both: holey carbon grid and the conventional grid but the results are same.
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yes, that would work. I will try that way.
Thank you for your suggestion, sir.
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Often research reports will only include only one concentration metric when characterizing nanomaterials. However, often it is necessary to convert among different concentration metrics, does an outline/rubric/methodology exist for how to rigorously perform these conversions?
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Two most important structural parameter of a molecule is size and shape. While there is many spectroscopic (like DLS, neutron scattering, fcs) and microscopic (SEM,tem, AFM, etc); for shape measurement as I know, we depend on microscopic techniques.
I want to know other than such imaging techniques are there any such techniques that can measure the shape of nanoparticle or protein?
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Yes, microscopy is essential but may not be quantitative and we often have a low contrast image for the materials that are described. Usually though we only have a 2D image of a 3 particle and 'sample preparation' (especially TEM) is a trap for the unwary. Cryo-TEM has had excellent success.
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I heard using XRD  it is possible to measure dislocations density or observe them using TEM. I will appreciate if some one explain me how I can measure dislocation density?
I also heard that density of a material can be related to dislocation density, but as I measured the density it was not that much precise to be used in this concept.
Many thanks, 
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Dear Dr. Bahmani, the determination of dislocation density in thin film with TEM method is explained in attached file, the advantages of this method is that the dislocations and their distribution were observed, however TEM is done in small volume, so that measurement statistics is limited as compared to XRD method. It is noticeable that min deformation introduced during sample preparation for accurate results.
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Hi everybody. I am trying to analyze some diffractions patterns obtained from TEM, on different parts of the cross-section. The diffraction rings are wide and diffusive because the structure is amorphous but there is still a short-range ordering and I am trying to get more quantitative data from these patterns. So, from one point of the cross-section to another the ring gets wider and more diffusive. I cannot measure its definitive diameter (to calculate d-spacing) since it is diffusive. Any recommendation? most resources and articles are for analysis of crystalline materials.
Thanks
Elham
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Dear phage experts,
I recently tried TEM analysis for my isolated phage (H. W. Ackermann, 2011 protocol), but I could not get images and results. Can anyone help me to analyze my phage by TEM?
Many thanks.
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Hi Nazmul,
Unfortunatly the PFGE is not suffeciant enough, as diffrent clinical strains of the same bacteria will share the same PFGE. the best evidence in my opinion is the sequencing of the mutated gene.
Do you mutated the bacterial strain at lab or it is natural resistance appear during infection of bacterial strain with phage?
If it is natural resistance, you have to look in the literature of what gene might be changed and seguence it with simple primers. Regards
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We are studying organic micelles suspended in water or diesel and want to get images of them to see what they look like (an actual image, rather than size characteristics from something like a DLS). At a minimum, we want to see the micelles after formation, but it would be nice to observe their formation as well. Unfortunately, they are very susceptible to temperature changes so any low-temperature applications likely won't work. I know TEM is an option for this. Is anyone aware of any other options?
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If you mean surfactant micelles, then they are formed in water through hydrophobic interaction (combined by the intermolecular interaction of water with each other). In hydrocarbon solvents through the dipole-dipole interaction of hydrophilic groups. Temperature greatly affects hydrophobic interaction and crystallization; therefore, it is not correct to study cryoTEM. Better to use SAXS or SANS methods. DLS method gives original results. For comparison, see the article
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Hi all,
I have synthesized a catalyst for oxygen reduction reaction (ORR). Polyvinylpyrrolidinon was used as as source of C,N while it was pyrolyzed with FeSO4 as a source of Fe and S at 800C in N2 atmosphere using silica template. The EDS shows 0.9% Si, 0.7% S, 0.2% Fe.
Fe may be in the form of Fe3C , Fe2O3, Fe3O4, FeS2 or FeN as per initial XRD observation.
I have seen a lot of TEM images in articles but I could not find similar to my one. Please help me in understanding the TEM images (attached). I have also enclosed SEM images for clear understanding. Thanks in advance!
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Md. Nazmul Islam your question is totally different with respect to the current thread (so it's difficult to find the answer here due to different expertise). It's better to ask it as a new question in researchgate.
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I want to take the SEM and TEM images of samples I synthesized. I synthesized g-C3N4 (Graphitic Carbon Nitride). If I need to take SEM and TEM, should I disperse the obtained powder in a medium (i.e. water, IPA, etc) or can i do the SEM and TEM tests in powder form?
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Take your specimen in as a powder to your SEM/TEM technician. He/she will be able prepare needed specimens in a short time (5-30 minutes).
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I've recently imaged a pathogen in the Phylum Ascomycota and am having difficulty discerning between different organelles and possible endosymbiotic bacteria. I can't seem to find any reliable sources showing examples of fungal organelles in TEM images.
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Hi Ashton,
Chapter 2 in the book Cellular and Molecular Biology of Filamentous Fungi
(Edited by K. A. Borkovich and D. J. Ebbole ©2010 ASM Press) is dedicated
to hyphal stucture of filamentous fungi and also shows some good TEM images of fungal organelles. Take into account however that the preparation method you use greatly affects the visibility and morphology of the organelles in question.
best regards,
Rob
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I’ve been experiencing difficulties in trying to obtain TEM images of my liposomes. We don’t have a cryo-TEM here in Serbia, so I decided to opt for freeze drying, stabilization using sugars or alditols, different staining techniques etc… Today I got my first images of a newly prepared sample: I freeze dried my liposomal solution (already placed on a grid) over night, and this morning we placed it into a TEM. Any idea why the liposomes (if they’re liposomes) appear like these white shapes inside a big circular dark blurry one? The liposomes are egg phosphatidylcholine + cholesterol + the small organic aromatic molecule I’m encapsulating, in phosphate buffered saline solution. Thank you!
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Hi,
Your method does not sound straight forward, making it also hard to interpret the result. Freezedrying like this is not suited for soft matter. I'd be extremely carefull before using this. Try negative staining: Put a drop of your liposomes on a grid and let it sit for 1 minute. blot away the liquid and straight after (don't let it dry in between) add a drop of 2% Uranyl acetate solution, blot this and again without letting it dry a second drop of UAc. let this sit for 1 minute and then blot away the drop and let it dry. Good luck!
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I synthesized AuNP array using protein and DNA. I waned to do element mapping of AuNPs and their surrounding protein and DNA by EDS analysis of TEM equipment. I used protein and DNA to assemble AuNP array, so I wanted to check phosphate, sulfur and nitrogen under the assumption of protein or DNA. However, the peaks of P, S, and N were not detected due to overlapping Au of EDS kev values.(2~2.3kev) Is it really hard to confirm the element of gold particles and DNA or protein together With EDS? I could hardly find paper for element mapping of NP,protein, and DNA together. Anyone know about that issue?? who tried to do it like me?
Thanks.
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I would not recommend neither EDS or EELS. Both techniques use high energy electrons and the DNA-Protein layer on AuNP is quite small. Thus you will have to integrate the signal for a while and the probability to "burn" the organic material is high. Since EDX have a penetration depth ca. 1 µm, you'll also observe a lot of background signal coming from the substrate where the AuNps are. I believe you will have a hard time searching for papers using EDS to get info on AuNP's surface modifications.
It is way more useful to go to XPS in this case. XPS is a surface technique and you can even differentiate between different oxidation states and do some semi-quantitative analysis. You can also try Raman and look to DNA backbone signal (phosphate vibration), but it is also quite challenging to have a good signal. Try an IR laser and long integration times.
Good luck!
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I have a TEM SAD image and want to determine if the TiO2 is rutile or anatase. I thought I could do this by comparing the d-spacing ratio of different planes. However, I cannot find a database that has the d-spacing between planes.Is there a quick way to calculate, and if so, could someone show me an example for rutile, maybe for the 100 plane or something?
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you can use imageJ software to measure d value. It is availble in google for free.
Thanks
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1. The requirement is to do elemental mapping so that we can get compositions of DTS (donor) and PC71BM (acceptor) domains /intermixed regions.
2. We hope to look at the Carbon and the Sulfur Edges by doing EFTEM
3. If we use a Cu TEM grid with the carbon film the signal from the grid might affect the actual signal
so what is the better TEM grid to use ? and if we use a grid with a carbon film is there a possibility to subtract the signal/effect from the film so that we can isolate the actual signal from the active layer ?
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So... what about graphene TEM support grids then? Not sacrifice too much contrast, also C contribution to EDS would be almost negligible?
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Hi,
I am looking to characterize virus particles within cells via TEM, and am having trouble finding good example images of Retrovirus particles within cells specically type B, C and R. Are there any good references that you could suggest?
Also any other literature or advise on characterization via virus particle morphology, or location within the cell would be helpful as well.
Thank you for your help in advance!
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Hi.I suggest the Reza Sanaye answer best for this question
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This SAED pattern is generated for a metal semiconductor hybrid nanostructure. I can see it's not single crystalline but I am not sure how to index the planes? Could you please help?
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You can try to work out the d-spacings of the strongest reflections in your electron difffraction pattern and then try to correlate those with the expected ones for your materials, however, if you have not explicitly calibrated this specific camera length you used then the values will be no more than 2-5% accurate or so (depending on the diligence of your service engineer) and so this won't help you for higher order reflections whcih are often more closely spaced and where higher accuracy may be required.
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I have been trying to look at blue silver nanoparticles (AgNPs), which are supposed to have a prism shape, on transmission electron microscopy (TEM). However, I have been seeing irregularly-shaped nanoparticles (size ~ 50 - 80 nm) for the majority of time on TEM. I do see some prism shapes but there weren't that many. These AgNPs were prepared fresh and were put on TEM grid within a few days so there should not be degradation due to aging. Also, the UV-Vis spectrum agrees with previously published papers. Just to be sure, I tried putting on AgNPs on the shiny side of the copper grid and also the dull side. I did not see the differences. Could you advise me how you would prepare AgNPs on copper grids ? Thank you!
[Silver Nanoparticles Synthesis (chemical reaction method)]
- Silver nitrate
- Trisodium citrate
- Hydrogen peroxide
- Sodium borohydride
[TEM sample preparation]
- Copper grid with formvar and carbon coated
1) 5 uL of AgNPs were put on the grid
2) Let the grid dry under air in dark
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For TEM you can actually use either side of the copper grid and it shouldn't be a problem.
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Hi everyone!
So, I have these four electron diffraction patterns. Can anyone help me for indexing these electron diffraction pattern? Or can anyone give me the step by step to index the electron diffraction pattern?
Thank you in advance.
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Please check out the link below, it will help you to understand how to do SAED indexing. As
Thomas Breuer
mentioned above, you need to identify precisely your phases (XRD can help).
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I have to do the transmission electron microscopy of clay samples. What is a good dispersing agent to be used for the same? In soil engineering as we normally use a solution of sodium hexametaphosphate and sodium carbonate to prevent coagulation of soil, can the same be used for TEM analysis?
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In order to obtain true dimension of clay particles, materials needs to be made free of all cementing agents such as organic matter, CaCO3 and free iron, aluminium oxides. And then sand (2000–50 micrometer), silt (50–2 micrometer), total clay (<2 micrometer) fractions are to be separated by the procedure of Jackson (1979).
Jackson, M.L., 1979. Soil Chemical Analysis—Advanced Course, 2nd ed. University of Wisconsin, Madison. Published by the author.
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Hi everybody,
I am starting to work with synthesis of silver nanoparticles from natural extracts. I have some pictures of TEM from my samples and I would be grateful if somebody can help me to interpretate them. The difference among them is just the incubation time for the reaction: for the two first photos, it was 1 hour and, for the other two, 24 hours.
Are the darkest spots agglomerates of nanoparticles?
What do you think about the distribution of nanoparticles?
Thank you very much in advance 
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Recommend Elif Alyamaç-Seydibeyoğlu answer
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As you know we have 2 kinds of dislocations in the dislocation accumulation procedure. Statistically Stored Dislocations (SSDs) and Geometrically Necessary Dislocations (GNDs) which either of them can developed by multiplication and strain gradient field, respectively. But in Annealing process which of them can annihilated drastically? in Recovery stage which dislocations can moved easily ?
and is this related to geometrically necessary boundaries (GNBs) and Incidental Dislocation Boundary (IDBs)?
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The answer lies in the name - GNDs are geometrically *necessary*, i.e. until the geometry of crystal is restored to a 'perfect' state, they must exist, else the crystal will become discontinuous.
The 'imperfect' state, relevant to GND, comes from bending of planes (such that some planes are stretched (tension) and some are compressed - important in single crystals) or from mismatch between crystals at interfaces (in polycrystals). So unless this imperfection is removed, the GNDs will survive. Annealing won't 'un-bend' planes (ignoring shape memory effects), and only in theory will it completely remove grain boundaries. So annealing and recovery processes primarily affect the SSDs, not the GNDs.
A possibly useful reference is below. You will find there that SSDs are treated using hardening and recovery terms (formation and annihilation), while GNDs are treated only with formation terms.
Note: An unrelated but maybe helpful thing to remember is that plastic deformation is an irreversible process - the deformation is made possible (in terms of continuity) by GNDs -if we could annihilate every GND by annealing, the irreversibility would be lost.
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Hi everyone,
I recently did a TEM on fibroblast cells and noticed that the cells, including control (normal and untreated)cells appear torn (or massive vacoules). The fixing and processing of the cell pellet was performed by a lab that offers these services. I have attached a figure for you to look at. Can anyone tell me if this is normal for skin fibroblast cells or was there an error in the processing? If so, what would you recommend doing?
Thank you
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Hi Paalini,
Swelling of the endocytic/exocytic vesicles often happens in chemically fixed samples (the 'vacuoles' you mentioned). The tubular extrusions do exist on fibroblasts but can also be caused by the processing. Did you grow the cells in suspension or as adherend cells? In the latter case trypsinizing or scraping the cells to get a suspension before fixation can also cause these effects.
in that case it is better to do the fixation first and then scrape the cells in 1% low gelling point agarose and pellet the cells by centrifugation. after solidification of the agarose the pellet can be cut in convenient pieces and further processed for resin embedding.
best,
Rob
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Impact
Significance
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A new approach could help materials scientists identify the appropriate molecules to use in order to synthesize target nanomaterials. The method was developed by Daniel Packwood of Kyoto University's Institute for Integrated Cell-Material Sciences (iCeMS) and Taro Hitosugi of the Tokyo Institute of Technology. It involves connecting the chemical properties of molecules with the nanostructures that form as a result of their interaction. A machine learning technique generates data that is then used to develop a diagram that categorizes different molecules according to the nano-sized shapes they form.
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Like FIB-TEM.
I am looking for a technique by which polymer composite properties remain constant.
Plz insight us
Thanks in advance.
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Dear Bhaskar;
You have three choices:
1. You can prepare a very thin composite to see sharp nanofibers in the FE-SEM images (This method is specially useful for hydrogels reinforced with nanofibers).
2. You can provide a cross section and see the dispersion of fillers in the coating matrix ( ).
3. You can used AFM analysis (topographic and phase contrast images) to investigate the nanofillers.
Good luck
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I use ethanol as a solution and ultrasonicate it for 10-15 minutes and then using a micropippet I pour few drops on carbon coated copper grids and keep it overnight in a oven at 80 deg. But I'm getting an agglomerated micrograph of the sample. And when I'm sintering the sample at a very high temperature, it's fully agglomerated (in microns). So is there any other procedure to get a nicely dispersed particles?
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An easy thing you could try is to not let it rest overnight but put the sample on the grid for half a minute or one minute and then touch the grid to a bit of filterpaper to get rid of remaining material and fluid. The drying process may be forcing the particles together as the big drop evaporates, so as long as the sonication step was sucessfull you would get a better result when blotting.
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The micro-structure of graphite in SEM is like onion pulp or corn. The main point is how long or how to decide the grain boundaries. The measurement is also very easy. To decide the grain boundaries is difficult task. Any suggestion, I will be very much grateful.
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If you could share some pic of this pulp like structure, then perhaps it is easier to answer. I feel that the tilt angle you are using is not correct. also the resolution of your "pulpy samples" needs to be improved. HR-SEM would give better results I suppose, if you can afford it.
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I am size selecting the graphene produced in the lab. While performing Raman analysis, I am finding that the larger flakes have a greater ID/ID' than the smaller flakes. Could anyone please explain why this is happening? (Please see the attached image)
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Hello Akibul Islam ,
First of all I´m so happy to find someone with the same interrogations and tell you that the graph you post it has great meaning, so if you permit me to give you my point of view take it, and construe as you consider.
Larger nanoplatelets tend to stabilize ans show more of their inner vacancy defects (something like grain boundaries) nor the edge defects. But smallers graphene nanoplatelets show more edge defects, even that ratio could be very useful to determine the size at witch you can obtain more oriented nanoplatelets.
I´d be attentive, bests
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Which aqueous medium is the best for stable and homogeneous DISPERSION of ceramic (PbTiO3) particles for TEM sample preparation ? if particle size is in 500 nm range what precautions must be follow?
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Hi Chandra Your question hints at some potential problems if you use TEM. TEM requires a thin section, typically < 100 nm. What information are you trying to get from visualization of your system? You state your particles are in the 500 nm range. Thus you'd have to prepare your samples usually by microtoming a thin section of your material in, say, an epoxy resin (and thus you'd need an organic solvent to disperse the particles). Imagine that all your particles are monodisperse (absolutely identica), identical in shape and you slice sections from them. If they're spherical to start you'll have a collection of discs ranging from virtually 0 nm up to the true diameter of the sphere (500 nm in your case). If they're cubes then you'll end up with anything from triangles to octagons. Thus you'll see an apparent distribution from a monodisperse starting material! See attached for an example.
At the sizes (500 nm) you quote, I'd be looking at laser diffraction for a quantitative particle size distribution and SEM for some guidance as to shape, degree of agglomeration etc.
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Dear All,
I will be appreciated if anyone can help me to know more about the TEM analysis of the core/shell spherical particle. An expert told me that in TEM images (in case of bright field images), when the atomic number and density of a component is higher than the other material, we observe that part darker. But, contrary to his idea, I found some literature as you can find some of them as below which made me confused.
- "A Facile One-Step Approach toward Polymer@SiO2 Core−Shell
Nanoparticles via a Surfactant-Free Miniemulsion Polymerization
Technique": DOI: 10.1021/acs.macromol.6b00038
- "Preparation, characterization and thermal properties of micro-encapsulated phase change materials": https://doi.org/10.1016/j.solmat.2011.09.020
- "Nanometre Ni and core/shell Ni/Au nanoparticles with controllable dimensions synthesized in reverse microemulsion": https://doi.org/10.1016/j.jallcom.2008.07.115
- "Properties of Core−Shell Ni−Au Nanoparticles Synthesized through a Redox-Transmetalation Method in Reverse Microemulsion": DOI: 10.1021/cm070182x
- "Preparation of porous hollow silica spheres via a layer-bylayer
process and the chromatographic performance": DOI 10.1007/s11706-017-0366-z
- "Facile One-Pot Fabrication of Hollow Porous Silica Nanoparticles": DOI: 10.1002/chem.201303656
- " Al2O3/TiO2 core/shell powder derived by novel sol–gel routes": DOI 10.1007/s10971-015-3739-8
- "Increasing the Thermal Storage Capacity of a Phase Change Material by Encapsulation: Preparation and Application in Natural Rubber": DOI: 10.1021/am200870e
So, I am wondering if there are any other parameters that can be effective in TEM analysis of core/shell structure particles along with the atomic number and density of the material? I will be appreciated If you can also introduce me some references about TEM analysis of core/shell spherical particles.
Thank you so much in advance.
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You should have no problem if your particles have diameter of 10-100 nm, their core and shell have sharp difference in density and have dimensions well above resolution limit. In other cases it could be still possible. Just go ahead and try, its not difficult task, rather easy specimen preparation and takes not much of microscope time.
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Interfaces in composites are expected to be non-planar. What attempts have been made to provide expressions for the displacement and stress fields due to dislocations and cracks that lie on a non-planar interface in three dimensions?
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Expressions for crack-tip stresses and crack extension force are now available for a model of non-planar interface crack in bi-elastic solids with oscillatory front ξ= ξ (x1, x3) spreading in x2x3-plane perpendicular to fracture propagation x1-direction. The average crack plane is Ox1x3, perpendicular to the applied tension x2- direction. The shearing stresses are applied in the x1 and x3 directions, parallel to Ox1x3. Also considered are induced shearing and normal stresses that originate from Poisson contractions in the media. Please see “Non-planar interface crack under general loading III. Dislocation, crack-tip stress and crack extension force” in our contributions in ResearchGate.
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Dear Experts,
I am trying to get information about stacking fault and dislocation based on TEM image. In the attached figure, I process the image using FFT implemented in digital micrograph DM. Are the circled locations are evidence for SF and dislocation. I really will learn a lot from your comments.
Thanks in advance
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What is the length scale here?
Is this a HRTEM image or Moire fringes?
It is not easy to have an exact answer with a non-scale image.
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when i want to try thick sectioning of my sample, I fill in the tray with water bu even with angular and axial regulations and also filling and vacuuming water , it does not wet the upper part ! and so does not approach the edge ... I do not know what is the problem...is it from the water or the glass or maybe cleanliness.... ?please advice
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Long, since no simple answer! Otherwise: apologies!
Dear Seyede:
first of all: you should provide further detail on the knife/knives you are talking about...
For Ultramicrotomy usually one uses "knives" made from glass strips in a distinct fashion by scoring and breaking, then the resulting triangular pieces containing the cutting edge have to be mounted with a kind of self-adhering tape (usually a special type of golden or silver 'Scotch' TM tape, + additional 'glueing' and 'sealing' the "hole" on the back with dental wax) to get the water-trough necessary for collecting the ultrathin sections easily. As long as one does not know about your practice and making knives one cannot help you effectively...
As a 'shot into the blue': 5 possibilities:
i) the cutting edge isn't properly forming (here it would be of essence to know if you use a "knife maker" and if yes: which one...? how your cutting edge looks like?),
ii) you're using wrong glass quality,
iii) the small (approx. 4-6 mm) ridge of your glass might have gotten "oily" or "greasy" somehow... For the latter you have not much options (CAREFUL, but thorough cleaning with p.a. acetone) OR make new knives, taking care not to contaminate again the knife's ridge...,
iv) your trough("boat")(or silver-tape) isn't glued properly (upper edge must be positioned orthographically /vertically to the edge (hope you know what I mean!) and last but not least,
v) your glass has tension ( produces and adverse surface tension for wettability*) by the trough water you fill in best with a syringe .... BUT BE AWARE that unused injection needles as well as syringes taken "freshly" out of / from plastic package often are coated with silicone oil which prior to use in ultramicrotomy must be eliminated: bathingand sonicating at least thrice in p.a. acetone....
Disclaimer: having done such work hands on at the bench by myself for more than 10 years I know about those problems and how constraining and frustrating such "presumably technical" problems can be...
Last point: you might convince your PI/Lab-Head to buy at least ONE diamond knife (you know for sure a / the source)...otherwise I would be able to tell as a former satisfied user....
*) wettability issues can be improved either by buying special "wetting solutions" or (if you have no hygienic concerns) just to spit into a small volume of distilled water, fill into clean glass or plastic bottle, shaking vigorously, apply some drops into the knife's trough fluid, let stand and react for some minute and then wash thoroughly with a. bidest. (e.g. jet-washing/flushing by means of a wash-bottle). Active principle: removal or at least reduction of surface tension by the composition of saliva (esp. enzymes like amylase and or lipase)....
If you own a DDK-diamond knife, their manual for working and taking care of the diamond knife describe the use of a low amount of acetone in the trough water / fluid (cf: http://www.ddk.com/PDFs/knifecareanduse.pdf) but NOT all manufacturers of diamond knives recommend the adding of a harsh organic solvent like acetone (cf. 100% Ethanol - section E. Trouble shooting in https://www.diatomeknives.com/diamond_knives/manual.aspx; also www.diatome.ch/Messerprospekt_01_2015_EN_LowRes.pdf; https://www.emsdiasum.com/microscopy/products/diamond/ultrathin.aspx; https://www.agarscientific.com/media/import/14-diamond_knives_pgs_270-276_date_17_06_10_web.pdf ; ).
Article Wettability of a Glass Surface in the Presence of Two Nonion...
= (Notabene: add ' pubs. ' in front of the following address:) acs.org/doi/abs/10.1021/la8008078 ; https://books.google.at/books?isbn=0323150292 p.132: Use of 0.1-1.0% non-ionic detergent such as Triton X-100 or Freon 113 (the latter as of today obsolete and perhaps forbidden too in your country), BUT DO NOT USE common laboratory detergents like Alconox , etc. (HAYAT M.A.,1986: in 'Basic Techniques For Transmission Electron Microscopy,p.132);
or:
Article Care and Use of Diamond Knives
(HAYAT and ZHANG 1998) = (Notabene: add 'www. ' in front of the following address:) sciencedirect.com/science/article/pii/S096843289800016X
Regards, W.M.(private mail: womuss@gmail.com)
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How does the TEM electron beam change when I key the dark field button on the panel?
And I do not understand the meaning of "Use Multifunction X and Y knobs to bring the Bragg reflection opposite to the one selected to the point" in the axial dark-field imaging manual.
Why move the DP twice?
Attachment is the Tecnai F20/F30 Operating Manual axial dark-field imaging operating manual.
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Hi Yanbao, sorry, I don't think you have understood dark-field imaging. The idea is that you tilt the beam and leave the objective aperture on axis. As a beam incident on a lattice plane is diffracted by Bragg's Law, and reflection and incident angles are equal in size, if your Bragg angle is +theta then your incident angle must be -theta. So, in order to 'see' dark field image with +theta you need to tilt the beam to -theta, and the angle between the two is 2xtheta!
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how i can calculate Lattice parameter from TEM image using software Gatan. i want to measure the matrix lattice and the precipitate 
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Hi Volker Klemm,
How to interpret lattice parameters from this image as it is having no. of closed peaks and and of variable heights.
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Hi, researchers,
I confronted some difficulty in understanding the convergence semi-angle on the condition of over-focus.
The convergence angle in the condition of focus like that Fig.1 shows. however, the over-focus condition seems different, as Fig.2 shows.
The defination of canvergence angle under focus/overfocus is the same? How can we explain or understand that?
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Hi Xu,
I can´t be sure if we are right when talking about convergence angle in overfocus condition. In my opinion we may talk about convergent beam up to crossover. Overcoming crossover on Fig.2, the electron beam become divergent.
The main reason why the 2nd condenser lens is set up into overfocus, is that we would like to reach the most parallel electron beam with optic axis at specimen plane as possible. The focused or underfocused 2nd condenser lens in TEM gives undesired convergence. It is not so clear from Fig.2, but in real TEM after 2nd condenser lens there is upper objective lens, which acts as convex lens and produce (almost) parallel beam from incoming divergent beam. The specimen is then illuminated by parallel beam.
Overfocused 2nd condenser lens create source image above image plane. So the convergence (or divergence) angle is defined by diameter of this crossover above image plane and its distance from specimen. In underfocused case the convergence angle is defined by condenser lens aperture.
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Hi,
I had a 3D model to study myelination in vitro. having TEM pictures of my cells being myelinated by schwann cells, I wanted to analyse my pictures, but I don't know how.
I'll be glad if you guys tell me so.
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Dear Hanie,
I suggest you analyze you TEMs in the same manner that others use in the remyelination field. For example, this zebrafish paper measures myelin thickness, calculates the g-ratio, and describes the how compact the myelin appears:
Here are a few other papers and reviews, such as
These publications should give you some idea of the method of analysis.
Good Luck!
Jill
Note: For each of the papers above, I TRIED to link to the PubMed Central version of the paper, which is free to read. Unfortunately, RG automatically converted my links to ones that link to the papers archived on RG. I am NOT trying to violate publisher copyright agreements!
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When i took the TEM images of CuS-Ag hybrid nanoparticles, i noticed that some nanoparticles have a vibrative bubble inside.
Does anyone can tell me why this happen?
This nanoparticle synthesized in aqueous manner.
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I think your sample was not well dried.You can try  with washing with ethanol using centrifuge and make suspension in the ethanol. if Still bubbling persist,you can go through the paper referred by Mr. Jinming Guo,due to some core-shell interaction.
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I got the TEM image of my sample containing threading dislocations. So I need to calculate the density. 
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Dear Hossein,
first of all, for exact determination of dislocation density you need another instrument than TEM microscope. If you are happy with approximative determination you can use TEM, but you have to know diffraction conditions, material characteristics and thickness of your sample at the place of your interest. ImageJ is not solution of the problem.
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Pretty simple: by HRTEM, you 'see' the structure of your object which, in your case, is at least partly crystalline. In fact, what you really see, as Erico rightly pointed out, is the result of interference from your electron beam and crystal structure (any good textbook on HRTEM will explain that). So you're visualizing the crystal in real space (the object space), i.e. you 'see' the electron density distribution which is stronger at the atomic position. From this, you can directly measure lattice spacings etc. if you are in the right orientation with respect to the electron beam.
If you take the FFT^2 (i.e. you loose the phase but keep the modulus of the FFT) of your HRTEM image, what you basically get is the frequency distribution of the intensity of your object, i.e. a pattern looking like the XRD one (in reciprocal/Fourier space). That is because XRD is (by theory) the square of the 3D Fourier transform of your electron density (see any introductory textbook of XRD).
So remember, the FFT(HRTEM image)^2 = XRD image.
Now, SAED means selected area electron diffraction. What you do there, is that you image the electron diffraction of your object (by imaging the Fourier plane) and then select a specific diffraction peak which is imaged for each scan point. In this way, only the scan position of your object which is in the correct orientation will give you some intensity.
So all of those provide you complementary information, you can't really say that one makes more sense than the other. The only 'detail' you should remember, is that in HRTEM, you're only visualizing a (very, very, very) small portion of your sample (in the 100 nm range), while the beam size in lab source XRD is huge (in the mm range). If your sample is homogeneous, both would give the same information (assuming you have a fully crystalline sample). If your sample is heterogeneous, then you are getting a statistical average by XRD. So for practical reasons, you mayt want to use one or the other.
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We are planning to do a pre-embedding immunoreaction for EM. Although there are many alternatives we will try the DAB reaction on HRP-conjugated Ab, followed by silver precipitation (Gallyas, 1982) and gold substitution: GSSP-protocol.
One of the key chemicals of the GSSP-procedure is gold chloride (AuCl3). We don’t have that anymore. What we do have is the Nanoprobe GoldEnhance for EM plus (GEEM+).  Do you know if this GEEM-solution can be used to replace the silver-precipitate?
Thanks for your time
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I've never tried this one but as above in principle it should work! Only way to find out is to try it! You could also try tetramethylbenzidine instead of DAB! Said to be less toxic than DAB (little evidence) and generates crystals that shine brightly in polarised light illumination! 
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From my understanding, I need to focus the beam on sample by reduce the intensity. Then, click diffraction mode, and then increases intensity knob. For some reason, I still don't see any kikuchi line. Anyone tell me if my operation of TEM is right? 
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Dear Zefeng Yu,
if you would like to observe Kikuchi lines, then you have to set on standard selected area or convergent beam electron diffraction pattern. The problem is that Kikuchi lines are not always visible. Kikuchi lines are produced mostly by incoherently and inelastically scattered electrons. At smaller sample thickness the Kikuchi lines are not present because dynamical scattering, which generate Kikuchi lines, is insufficient. More precisely, Kikuchi lines are produced but have extremely low intensity which makes them invisible. As the sample thickness increase (and all three Laue conditions are satisfied) magnitude of dynamical scattering increase as well and Kikuchi lines becomes visible. In very thick crystals the absorption effect dominate and make Kikuchi lines invisible again. So, for observation of Kikuchi lines you need to turn your TEM to diffraction mode and have the sample with appropriate thickness. The best sample thickness is approximately around middle of transparency thickness. You can influence this thickness by accelerating voltage or orientation of crystal along zone axis.
In case you would like to observe Kikuchi bands, which creates a pattern known as Kikuchi map, then use the procedure mentioned by Michaela. Kikuchi bands are not so sensitive to sample thickness as Kikuchi lines.
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Researcher in Nanoparticles, Nanomaterials...
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Astigmatism plays a very crucial role in conventional TEM whether its from condenser, objective or diffraction. Sample quality (thickness, dispersion, roughness, etc) is also a limiting factor. One imp. thing is mechanical noise and vibration during imaging time. At higher magnifications, even your voice could affect the quality of images. In case of convention TEM, sample should be stable under beam (80 kV or 200 kV, depends which you are using) and also it should be dry completely. 
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At the bottom of the SAED image, there is a scale marked 2 1/nm, can someone just clarify what is it, why 2 1/nm marked, and how it is used while calculation??? I have attached the reference image.
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Hi Mathias,
image b and c are obtained from SAED (selected area electron diffraction) experiments, performed with a TEM. In SAED experiments, electron diffraction pattern are obtained from the area selected by an aperture in the image plane of the microscope. In a TEM you can optain both: imaging and diffraction data.
Kind regards,
Levin
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For my HRTEM I have applied the Fourier transform and the inverse FFT.
From that results what can I extract as information? Can I define the crystal structure, the lattice parameter? How can I do that?
Best regards  
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@ M. BENDOUMIA, Please attach your HRTEM image. To know the crystal structure and lattice parameter, first you need to identify what your material and the possible crystal. To know these, you can investigate your material using XRD. Secondly, you may need several diffraction patterns (FFT images) in order to get enough data for lattice parameter.calculation.
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I am working on a crystal, and collected both electron diffraction pattern (ED) and XRD pattern. The strange thing is, one ring (low index) in ED can not be assigned to any peaks in XRD. How does this happen? is there any difference in diffraction by using electron beam or x-ray?
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here's one page of the book I find by googling the multiple electron diffraction. It is an example of symmetrically forbidden reflections shows up in ED at certain condition.
I'm sure  you can find detailed discussion in any TEM textbook.
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from this image this is electron diffraction pattern in bright rings means angle in x-ray what is the program to convert to XRD pattern as image two ?
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thank you
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I was given some liver tissue samples for viewing using scanning and transmission electron microscopes. The samples were kept in McDowell's solution at 4 degree Celcius for several months up to a year.
I wondered if the samples were already over-fixated by this time?
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I would prefer to have them osmicated and then keep them in 70% Ethanol as long as you need before processing. see this reference  https://www.diacomp.org/shared/document.aspx?id=70&docType=Protocol
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I´d like to ask, what can be an origin of the dark (electron-dense) objects (whole first picture) and in the second picture (chloroplast), it is present in the very left side, right side and above chloroplast. These dark objects are appearing during contrasting the sections (uranylacetate and lead citrate). Very often it covers all structures in the cells/tissueas and it is impossible to obtain good photos. We observed also non-contrasted samples and in this case there are no dark objects. I´ve tried also different concentrations od lead citrate (0.2 - 2 %; pH 12-13), but the objects are still present. I use 2% uranylacetate (pH cca 4) (contrasting for 35 min.), then 6x washing by MiliQ water (room temperature, afted boiling, to avoid CO2) and then contrasting by lead citrate for 2.5 min. We are sure these disturbing objects are appearing during contrasting process, but we don´t know how to avoid them. Thanks.
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Thank you for the recommendation. As I can see, there´s no other possibility, than preparation the new specimens onto pure copper grids. Anyway, thank you!
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Dear all,
Does anybody know a good protocol for electron diffraction using TEM for proteins? I would like to try it.
Thank you!
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Hi! I didn't mention that I would like to do it for amyloids! Is it still possible? Thanks!
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Dear Bidham,
yes, I will. Can you remind me around June/July ? My address is :
With best regards
Petr
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I exported my EBSD data (acquired using TSL OIM Data Collection S/W) in .ang format and saved it as a separate file from the .osc file. However, reopening the .ang file in TSL OIM Analysis gives the error: "Unable to open the file: Invalid symmetry". The .osc file works smoothly though.
Why is it so? And the same .ang files also don't work in MTEX where I need to post-process my data for many important results.
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I have not worked with the .ang format, but I generally use the .ctf format to import data into Mtex. works fine for me. There might be some issues related to conversion between formats because of which you get the error message.
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The TEM micrographs of a martensitic stainless steel after hot deformation is illustrated in attached files, what the parallel lines in the attached TEM micrographs could be implied? 
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Diffraction patterns are required to determine what the local crystal structure of the parallel features are, and will also determine an orientation relationship.  Generally speaking, your features look to be martensitic laths, but they could be something else?.  To call them martensite is a very vague term and can be better defined with proper TEM characterization. Chemical mapping is also required to determine the phase.  
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What I have done to observe particles' size is the SEM.
To be specific, I put small amount of samples onto a carbon tape and observed them through the SEM. Then, I used Gatan program to calibrate the image of my samples. Here, I attached the SEM image to show you how I measured particles' size. I'm little bit doubtful on this method because of how precisely it can be in terms of sizes and distribution of particles' size.
Thank you for your help.
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If you are going to check particle size through SEM then it will be a difficult task. Because the size of the particle you are considering may be in aggregated form of tiny particles. However, if you want to calculate particles size through SEM then use ImageJ software. But I will suggest you, check particles size of your particles through DLS or TEM.