Transmission Electron Microscopy (TEM) - Science method

This is exactly what we are doing right now! Thank you for your replies.

Dear Luo Zhi

TEM analyzes the crystal structure and nanoscopic details of cobalt carbide particles and is very effective for observing the atomic structure with high resolution. XPS is also useful for examining the surface oxidation of cobalt carbide or the bonding between cobalt and carbon atoms. XRD is widely used to determine the crystal structure and phase composition of cobalt carbide. This technique determines which phases the material is in by examining the arrangement of atoms that make up the crystal structure.

I would go with 80 kV - 120 kV.

SEM uses low-energy electrons that allow us to identify the morphology of the object of study, i.e. size, shape, roughness.

TEM uses high-energy electrons that allow us to analyze small samples of a few microns, obtaining their crystallographic structure and phase identification.

Dear Janak Paudel ,

Your university has a lab with modern STEM. Ideal place to discuss your problem in detail.

Would you please more clear and accurate about what you are expecting?

There are discrepancies regarding the toxicity of cerium oxide, such as studies that report 5 cerium(III) oxides. Cerium oxide has applications in catalysis, modulator of oxidative stress in living organisms, in biomedicine, among others. The effect of five cerium oxide (Ce) nanoparticles with different characteristics has been reported in a model organism: for example: the microalgae Pseudokirchneriella subcapitata.

Steps to follow: 1) collect capacitance and dissipation factor data for a series of frequencies

2) Use a spreadsheet (such as Microsoft Excel or Google Sheets) or data analysis software (such as Python with Matplotlib or R).

Determining the precise cellular parameters of a phase in powder diffraction is a fundamental process in the characterization of crystalline materials. Here are the steps and methods commonly used to make this determination:

1) Sample preparation

2) Obtaining diffraction standard

3) Identification of diffraction peaks

4) Determination of interplanar distances

5) Determination of crystal structure

6) Calculation of unit cell parameters

7) Adjustment and refinement. Rietveld refinement through the use of FullProf software. For simple tuning you can use Python

8) Analysis

It depends upon the level of details that you want. If you want to study the surface morphology then SEM can be good option.

If you are interested to know the internal structures TEM can be better.

But microgrid sample and operation for TEM can be quite challenging. If you are using for 1st time.

TEM is localized measurement while XRD is global measurement. Therefore, they should not be the same. Also, what method you have used to calculate the crystal size? have you used modified Williamson-Hall method or Scherrer Equation?. I recomend using Uniform Deformation Model (UDM) or Uniform Stress Deformation Model (USDM) to calculate the crystal size. Also, using synchrotron xrd will be much accurate since the crystal size is very small.

You get a number distribution from TEM. The standard error is proportional to the recipricol of the square of the number of particles counted. For 1% standard error you’ll need to measure 10000 particle; for 10% SE, you can measure 100 particles. Remember too that you are imaging a tiny fraction of the sample so showing representativeness will be difficult - in the whole history of electron microscopy no more than a few tens of grams have been imaged. Also consider you’re looking at a slice through the particle (think of slicing a cube in different directions) and interpretation is difficult. Measuring a diameter without automation is also not easy. Automatic image analysis is one possibility.

Preparing Polyaniline Emeraldine Salt (PANI-ES) Sample for TEM Analysis

Question

I need to do TEM analysis for my Polyaniline emeraldine salt form. May I know which solvent is appropriate for preparing the TEM sample?

Answer

Preparing a Polyaniline Emeraldine Salt (PANI-ES) sample for TEM analysis involves several steps to ensure proper dispersion and sample quality. Below is a detailed guide on how to achieve this:

Materials Needed

Steps to Prepare the TEM Sample

Tips and Considerations

By following these steps, you can prepare high-quality PANI-ES samples suitable for TEM analysis, enabling you to obtain clear and detailed images of your material.

Thankyou Jignesh G. Hirpara

If you mean staining sections on Synaptek large slot grids, it is not much different from staining normal square mesh grids. I used to make my own Formvar-coated slot grids and place the sections on the Formvar of the slot grids. Synaptek grids are thicker than normal mesh grids, which aids in the staining procedures ( less physical bending/damage). I never found commercial-coated grids to be very good; the support films tended to break. And always blot the grids from the sides, not face-on.

One can image RNA, DNA, with TEM, but unless you have a means to label the hybrid uniquely, with an electron dense label, I do not believe it will be unique from other DNA/RNA, as seen with the TEM.

Dear Ayshwarya Ravikumar ,

you may have a look at the answers of a similar question from RG:

and also the following paper may help:

Good luck and

best regards

G.M.

Two curving lattice fringes are not suitably fitted by using two straight lines.

You could try using a Fourier transform instead, that will use more of the image and would be a better way of extracting the periodicity in the image.

Viruses; some cell lines produce (usually unidentified) viruses. I've seen this in more than one cell line.

Hey, you can follow this paper and I think it will be beneficial for you.

Chinedu Onyeke, you can interpret TEM and SEM images of carbon materials by examining pores and their sizes. In SEM, analyze surface features such as particle size and shape, while TEM allows for direct observation of internal structures. Consider the following pore size ranges: Micropores (< 2 nm) and Mesopores (2 - 50 nm).

I hope you find these helpful.Warm regards

Hey there Dmitry Bagrov!

So, about the whole negative charge thing with TEM grids during glow discharge treatment – it's a bit of an interesting phenomenon, isn't it?

Here's the deal: when we subject the grids to glow discharge, they do indeed pick up a negative charge on their surface. But why?

Well, it's not as straightforward as you Dmitry Bagrov might think. Sure, the grids are placed on the cathode during the process, which you'd Dmitry Bagrov expect to attract positively-charged ions. However, it's not just about attracting ions; it's also about the balance of charge.

During glow discharge, the bombardment of ions onto the grid surface causes the removal of contaminants and the generation of free radicals. Now, while the grids are indeed on the cathode attracting positive ions, they're also simultaneously losing electrons due to the bombardment. This loss of electrons contributes to the overall negative charge buildup on the grid surface.

So, in essence, it's not solely about attracting positive ions; it's also about losing electrons, which ultimately results in the negative charge on the grid surface.

Hope that clears up the confusion! Feel free to reach out if you Dmitry Bagrov have any more questions or if there's anything else you're Dmitry Bagrov curious about. Let's keep unraveling those scientific mysteries together!

What is "TEM/EDS image"? EDS mapping is the same thing as EDS image.

Yiqin Li I'd look at representative sampling. How did you prepare your sample for TEM? The DLS technique has looked at billions of particles simultaneously. You'd need to examine 10000 particles under the TEM to get a standard error of 1%. How many did you examine? If, for example, you took the TEM sample from the top of a container, the larger particles could have settled or be settling and you'd extract a sample with smaller particles. In the whole history of TEM not more than a few g of total sample has been measured. If your TEM sample has been produced by microtoming then you'd have a set of discs, the maximum of which would be the actual diameter of your particle (consider slicing a monodisperse distribution of spheres - what would you get?).

Dear Mubasher Ali ,

the SAED rings are like the Debye-Scherrer rings in ray diffraction.

It is the quite same mechanism of diffraction: electron versus x-rays...

Literature reports never tell you how many attempts the authors took to make the particles.

Thank you so much Sir

Michelle Darmawan

It is necessary to understand the solution to the problem using methods

1.UV-Vis. You determine the amount of absorption from the interaction of plasmons of nanoparticles in the visible region and prove, by comparison with other studies, that you have nanoparticles. If you reduced with hydrazine or borohydride, then you don’t have to dry the dispersion. If you restore with leaf extract, then there may be the presence of coloring substances that can change the plasmon band.

2.FTIR. You determine the presence of functional groups after the reaction. It is necessary to work with dispersion.

3.DLS. It is necessary to work only with a stable dispersion without agglomerates.

4.SEM/TEM. Prepare the dry film so that it is sparse and individual nanoparticles are visible. The sample will be placed in a vacuum and therefore must be dried under vacuum in air. Do not heat.

Sorry, I do not have experience with these samples. However, do you have an idea why the infiltration of resin is not complete? Maybe some air pockets are in the sample after the dissection procedure? Then application of vacuum pump might be helpful during early processing steps. Does some water or solvent from previous steps remain in the sample after resin infiltration steps are done? Longer infiltration times, infiltration on a shaker, processing using a microwave protocol, or increased temperature during the resin infiltration (without DMP initially) might be helpful, since the resin will be much less viscous at high temperatures. Switching to a different resin formulation might be helpful, e.g. a less viscous resin to improve infiltration properties, or maybe a resin that is of a hardness/property more similar to the mensicus tissue.

That sounds like an exciting field! Here are some steps you can take to find a postdoctoral position in microscopy and physics of microorganisms:

By following these steps and leveraging your expertise in microscopy and physics, you can increase your chances of securing a rewarding postdoctoral position in this exciting field of research.

l Perhaps this protocol list can give us more information to help solve the problem.

Stefan Baunack Thank you so much professor for the explanation. I will keep this in mind next time.

Hello,

your TIF files contain some information in tag 270 (see below).

(To read the tags you can use the ImageJ plugin here:

http://rsbweb.nih.gov/ij/plugins/tiff-tags.html)

It seems the calibration data are identical for both images:

XpixCal=233.059 YpixCal=233.059 Unit=A (The unit should read 1/A).

Using this information you can try to figure out the calibration in both images and see if the results for Al fit the published patterns for Al.

AlStandard_005_D.txt

TagNo (Tag Name) (Count TYPE ) Value

======================================================================

270 (ImageDescription) ( 108 ASCII ) I.M.A.G.E. 10/28/10 9:31 0.1 51 60 HC-DIFF 12.8 -15.2 -0. XpixCal=233.059 YpixCal=233.059 Unit=A ##fv3

Nanowire.txt

TagNo (Tag Name) (Count TYPE ) Value

======================================================================

270 (ImageDescription) ( 112 ASCII ) I.M.A.G.E. 10/27/10 11:55 0.1 51 60 HC-DIFF 748.7 333.4 -0. XpixCal=233.059 YpixCal=233.059 Unit=A ##fv3

Jason Jones It appears that you have two inquiries.

First, while utilizing serial EM in low dosage mode and alternating between focus and record, why does the beam move? Low dose mode allows focusing and tracking operations in a separate location, reducing beam exposure.However, this requires the beam to move between different areas, controlled by image shift coils that deflect the beam using a magnetic field. The amount of image shift depends on magnification, spot size, and beam intensity.

Second, how to fix the blurry images caused by the beam movement? One method is to modify the exposure duration or the camera binning to change the beam strength, while maintaining the same magnification and spot size for the focus and record regions.

when tannic acid reacts with Cu₂O its gives tannate with Cu²⁺ ions

Vladimir Dusevich Stop stalking and bullying me. You look funny in your picture.

It should be a simple task for a mechanical workshop of an university to build a similar tool or modify the existing one (if the black parts are fixed by screews). An original part would be xk€, my guess.

Vladimir Dusevich

Can you please stop stalking and bullying me on cyberspace? I am really tired of your childish behaviour. You never answer any question. You accuse everyone who is answering. I can understand your frustration. You can not even put your real poic on RG. Please stop complaining and start complaining. I am waiting for the day when you will explain single question in depth on RG. You are a menace to scientific community. Please stop stalking and commenting on me. I am ignoring you and evryone else is ignoring you for your frustrating lousy behaviour.

The phenomenon of exfoliation, particularly in layered materials like graphene or clays, involves separating layers from the bulk material, resulting in a change in the material's structure and properties.

Layer separation: Exfoliation involves separating layers within a material. If the layers were strongly bound together in the original material, their separation could result in a decrease in peak intensity due to the disruption or weakening of the crystal lattice.

Size Effects: Exfoliation can lead to the formation of smaller particles or thinner layers. These size effects can impact the diffraction patterns, potentially resulting in changes in peak intensity or shifts in peak positions.

You're welcome

Thank you sir

Jamal Nasir yes I would still confidently suggest there is excellent agreement. This is for several reasons (including more than 3 decades of experience), the main ones being that (1) we are comparing apples and oranges here - the TEM samples a relatively small number of grains, whereas XRD averages a huge number, and most important (2) the XRD-derived crystallite size is a simple calculation based on a simple model that rarely applies to real materials, and so I would suggest that if you think your XRD size error is "between 3 and 8%", then you need to think again - that would imply that the "error" for your examples, i.e., 6-8nm crystallite size, is +/- 0.2nm, which is a statistical error based on least-squares fitting parameters and is not actually the probable error in crystallite size, which is almost certainly an order of magnitude larger, i.e. +/- a few nm.

It would be helpful if you add information about the imaging modes (TEM or STEM, bright field or dark field).

Images should be presented as uncompressed data (uncompressed TIF or PND), jpg can introduce artifacts.

I suggest - given the size of the objects - you see Moiré pattern here.

With the help of the TEM Centre of beam alignment shows the working hours of LaB6 filament.

Hello,

You don't need to fix your exosome for TEM observation. Uranyl acetate will do the job.

If this helps anyone, here was the solution we came up with.

We ordered the following parts from a liquid nitrogen supply vendor:

Once the 20 L container is out of LN2, we vent it, take off the siphon head, insert a transfer hose from a large LN2 container, and fill our 20 L container nearly to the top. We let the metal at the opening warm a bit and then attach the siphon head. We move it into our TEM lab so that it can build up pressure overnight. Then, we simply insert the transfer hose on our container into the dewar on the microscope, and the pressure pushes it up the hose. We only have to do this refilling process about once a week.

Here is a video showing the overall procedure: https://youtu.be/zrCT433gaK0?si=0jMnaafOOavsEepc

If your sample is nano-powder, a small amount of your catalyst nano-powder sample ( one or two grains) is dispersed in ethanol (10 ml). Every step is taken very carefully to avoid the contamination. Place a droplet of the catalyst nano-powder suspension on the TEM grid using a glass dropper (cleaned and washed) then allow the ethanol to evaporate (minimum 24 hours). after completing this protocol sample grid insert the sample holder and insert the TEM.

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?

I suggest reviewing the following paper:

Hi Wei,

Have you refined the structure? And do you know which atoms are replaced by Zn at the surface? And do you know which surface(s) (hkl) is/are mostly exposed in this material (I personally don't know about it)? And do you know if Zn occupies sites only at the top layer?

If you have answers to the above, then maybe the following steps can help you.

1) Calculate the area of the most common hkl plane the particle is exposed at for the unmodified material

2) Estimate the maximum possible area for the case where all the replaceable atoms are replaced by Zn (from ionic radius)

3) Use the cell parameters of the modified material to calculate the area for the same hkl.

By comparing these values, I think it would be possible to know approximately how much of the surface is covered. Unfortunately, many assumptions are made.

If there are multiple crystallographic sites that can be replaced, then maybe you need more experimental data to get the coverage for each separately.

Best regards,

Hello, it happened to me when the osmium was already degraded and once when it was also contaminated with heavy metals. The iron in the tissue, and even more so when it is non-heme iron, can generate this type of interference. Make sure that your fixation buffer is well filtered and the glutataldehyde is not precipitated or contaminated. greetings.

Hello,

here are some references about the SiO2/Si Interface:

Additionally search for "gate oxide", these are very thin oxide layers (of Si, Hf and others) used in microelectronics.

First evaluate your XRD Data, match the peaks, get theri HKL values, using an appropriate software, and then Study HRTEM data, again using the appropriate software, e.g. image J. There are many videos on utube to analyse SAED patterns and fringes using image J. This will give u hkl values also, d spacing values also. Then match these values with XRD data

Oh how we all love the occasional inconsistencies of TEM processing...

In general I do not like the results with tannic or gallic acid too much, whether applied before or after osmication. I prefer to work with reduced osmium (using buffered 1.5% Ferricyanide +1% OsO4), which results in better membrane staining.

You can also try the good old OTO method, which is a bit more time consuming, but will definitely help with increasing membrane contrast.

Dinesh Kumar Duraisamy

The antipodes of twisted fibrils cannot be determined using TEM and CEM. You will not distinguish between the left and right hands with your eyes if their morphology from above and below is the same. Also, you can't tell which antipode you see if you don't see another side by side. Alpha and beta glucose will look like white crystals under the microscope.

Anithadevi Sekar thanks for your answer, I always use the ultra sound in dispersing my sample and I obtain the same images. However, most of the published work regarding this point post images with high number of carbon dots in every photo.

@ Rajat, probably during preparation of your samples in the dry state for TEM analysis some aggregation may occur or larger particles may come during sampling for TEM as sample size is very less for TEM (only a few hundred particles ) . When you have a monodisperse size distribution, ie all particles of the same size then a size measurement by TEM may give a size similar to that measured by DLS. TEM is a number based particle size measurement whereas DLS is an intensity based one. Therefore, DLS is more accurate than TEM in terms of size measurement.

Comparing to Et₃N (liquid), the Me₃N at normal conditions is a gas - so Et₃N is simply easier to use in the lab. I suppose if you have a chloroform solution of either of them, both can be used, as long as your "drug" is soluble ...

I have gotten somewhat useful results out of this once, in that I could at least identify viral capsids in infected cells, but it obviously depends on what information you exactly need for your analysis.

The treatment is harsh and thus the ultrastructural preservation is ... bad. Expect the lipids to be pretty much gone, and the protease treatment doesn't help either ;)

No. Way too irregular and too large. By the way, adenoviruses are non enveloped, they do not feature membranes.

Very importantly, please note that these objects are out of focus. A strong defocus will lead to the kind of black and white lines around an object as is seen in your image. I'm pretty certain, that once in focus these "membranes" will disappear. I'd strongly recommend you get proper training by an experienced EM operator if this is something you plan to do regularly.

Ankur Verma You need a thin section for electrons to pass through in TEM typically < 100 nm. In a powder, there are no independent, discrete, separate particles < 100 nm, so you'll not get suitable transmission. Normal route is to disperse in an epoxy resin and microtomes off thin slices suitable for viewing.

I have the same problem but with carbon quantum dots, were you successful in your search?

Yep. Something is not good here. It would be easier to answer your question if you provide the protocol which you used for sample preparation. In general, there are a few possible causes:

1. The film on the grids is too thick. What kind of grids did you use?

2. The concentration of UA was too low or staining time was too short. The most common parameters for viruses: 1% water solution of UA, 20-30 sec staining time;

3. The surface of the film is too hydrophobic and the stain was just ran away, when you lifted the grid from the droplet. In such a case some weird "semi-stained" virions can be found. Sometimes it can be done intentionally to reach positive staining - just wash the grid on the droplet of water or weak buffer after the staining. To avoid this effect, you can use a standard glow discharge treatment of the grids or just a fresh thin carbon coating (a few nanometer) right before the sample preparation.

Typically I use a simple UA negative staining protocol with ultrathin formvar-carbon grids: https://www.mdpi.com/1999-4915/11/10/955

Good luck!

Dear friend Anusmita Chakravorty

Thanks for your clarification.

There are several physico-chemical material characterizations that can be done on heterostructure-based materials and devices like HEMTs and SBDs in the nm range. Some of these characterizations include luminescence properties (https://pubs.rsc.org/en/content/articlelanding/2022/tc/d1tc06033c), and another article in Science magazine, my favorite optical band gap structure, semiconducting ability, light-matter interactions, mechanical strength, and surface area ( ).

Now, you may want to ask what specific characterization you should explore.

Recent progress in 2D material van der Waals heterostructure-based .... https://pubs.rsc.org/en/content/articlelanding/2022/tc/d1tc06033c.

2D Nanoscale Heterostructured Materials | ScienceDirect. https://www.sciencedirect.com/book/9780128176788/2d-nanoscale-heterostructured-materials.

2D materials and van der Waals heterostructures | Science. https://www.science.org/doi/10.1126/science.aac9439.

kV - voltage, like in "accelerating voltage of electron microscope"

keV - energy, used mostly in spectroscopy (EDS). Horizontal axis of a spectrum should be marked as keV; unfortunately many people mark it as kV. It's wrong, but so widely used...

The characteristic feature of the diffraction pattern of a twinned sample is due to the different orientations of the twin domains. This results in a splitting of the diffraction spot into two or more spots with different intensities and positions. This is because each twin domain contributes to the diffraction pattern with its own crystal orientation. The number and intensity of the twinned spots depend on the degree of twinning, and the crystal structure of the sample.

For bacteria culture + antibiotic SEM works better (and much easier to use).

Hi Saied Saeid Saei dehkordi,

If you are talking about Metal-organic framework analysis then TEM as it differentiates phase by density gradient. Law density (Organic) bright, high density (metal) dark.

Giovanni Zummo

I repeat that the correlation function is related to Brownian motion and not to light scattering. Using light scattering, the Brownian motion of a solid nanoparticle in time is studied and the diffusion coefficient is obtained. For a deeper understanding, you need to read the author of the DLS

Pecora, R. Eds. Dynamic light scattering: applications of photon correlation spectroscopy. Springer, Plenum Press: New York, 1985, 420 p. DOI: 10.1007/978-1-4613-2389-1