Electron Microscopy - Science method

Hi, can you provide a sample image to show the increased density of cells.? DK

Luigi Marongiu You say you want to investigate the surface but you talk about staining. What type of electron microscopy do you want to use in your studies?

The PDF-5+ SAED Extension that is bundled with the ICDD database can perform single/multi-cell searches for Phase ID

There are a couple of things to consider.

The image of a (theoretical) point on the object side appears at that location where all the diverging rays, being captured by your optics, are converged into one point on the image side. Having the situation that all object points you want to image are in one plane (object plane) and this plane is perpendicular to the optical axis of your system, all these points are imaged in the image plane (also perpendicular to the optical axis and assuming that the optical system is not desgnied for off-axis imaging).

These two planes have a well defined distance to the optical system. Object points being before or behind this plane are imaged not sufficiently, as the rays before and behind the image plane are not "focussed" as in the image plane (they converge or diverge). Maybe you have a look at Figure 22.13 in the attached file.

The respective depth of field is the range in which the image appears to your eyes as 'sharp'. This depends on magnification, distance of the viewer and other things. Chapter 22.2.4 of the attached file... see "circle of confusion".

The infinite resolution is normally referred to, when talking about a (more or less theoretical) optic, where one can zoom into an object scene at any zoomlevel, still getting an image with a satisfying resolution level.

Some reading on a similar topic might be: https://www.edmundoptics.com/knowledge-center/application-notes/microscopy/understanding-infinity-corrected-objective-resolving-power-and-magnification/ (this is available in various languages.

Not sure about the zero resolution term. This might be interpreted differently in various domains. I could assume, that you have an additional fluorscence detector in your microscope? In this case it could refer to the characteristic, that the signal does not have an 'image resolution', but simply detects the amount of fluorescence coming from a certain area (steradiant). By this it would also detect signals from object aligned behind each other.

Jan

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.

There are a few potential non-destructive methods for removing metal coatings from biological samples for electron microscopy:

The key for all these methods is controlling the rate and degree of metal removal to avoid over-etching the valuable biological structures underneath. I'd recommend starting with low concentrations and gentle plasma or chemical etching first. The goal is removing the minimum amount of coating required for analysis, without compromising the integrity of the soft tissues or bone

Agree with Yannick. However, some antibodies work for both WB and imaging applications. A relatively easy way to test a primary ab for imaging is usually by light (fluorescence) microscopy, using applicable secondary ab (fluorophor conjugated). Also keep in mind that most ab do not bind after fixation with standard glutaraldehyde concentrations used for EM.

Thank you. I hadn't thought to add pictures. Now that you have, and that I am presenting this material this afternoon, here you go with scaled interaction volumes overlaid.

I hope this article helps the readers to this topic.

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.

Then I am a bit perplexed. I could imagine the enzyme might have an effect on the peptidoglycan but its it's hard to imagine how it would cross the OM to act.

You might want to do some controls to be sure it is the action of the enzyme directly on the cells or something to do with the endproduct of the reaction.

Do the cells stop growing or slow down so that you have a phenotype to observe?

Dear Hao Xiong,

This question was partly answered, in particular in the context of 4D-STEM, by our paper in 2021:

For you, the key information is that, in the aberration-free case, HAADF imaging is optimized when the probe is focused slightly below the entrance surface of the specimen.

Best wishes,

Hoel Robert

If you want to keep your phages in 4 degree Celsius, it's better if you store them in SM buffer (50mM Tris-HCl pH7. 5, 100mM NaCl, 8mM MgSO4, 0.01% Gelatin (v/w)) instead of in LB or ddH2O. I observed when I made phage dilution with water and keep it in 4 degree, the titer will decrease quickly.

For maintaining titer a longer time you can keep in -80 in glycerol.

It can be distinguished very clearly by using TEM analysis. Even I worked with SS 304 steel by laser shock peening process...https://doi.org/10.1016/j.mtcomm.2022.104200. you can refer it in TEM section. Hope this may help you.

I use an old hot plate set at 1 for Lowicryl. Hold the bottom side on the plate for five seconds then wedge a razor blade between the resin and the disc. They pop right off With little effort. Should work for lr white too.

Google; "Gloves; Lint free, Clean-room" = stretch nylon, one size fits all. All EM supply companies have these available and they are what I've used for decades for the purpose you describe.

The Kikuchi diffraction in TEM is generated via bragg diffraction by inelastic scattering electron, and the diffraction intensities from the front crystal plane and the back plane are different, therefore, one of the Kikuchi lines is bright and the other is dark. The inelastic scattering electron beam density nearer to the central beam is stronger than others far away from the central beam, however, much of the electron beam is diffracted to the dark line direction, thus, the total intensity of the Kikuchi line nearer to the central beam is weaker than the other one.

Dear Dr. R. Peguero,

it is just a proposal: would you mind to first provide way better images than you attached to your question (which is - clearly - an interesting one...should be glad to receive your protocol for staining grids with Uranyless as well as the Pb-citrate-stain you use. I understand that you stain your grids manually instead of using an automated GRID-STAINER).

The images provided are blurry and of low resolution and cannot aid an exact interpretation by zooming in.

From what you describe I guess that not the '70%EtOH' treatment is the problem.. but we'd need more detailed information - in general - regarding the specimen processing steps you performed.

There is a possibility that the formvar film (BTW, which solvent did you use when fabricating the film =mounting your grids ?) got some altered microareas which are prone to become porous or develop holes under the ultrathin sections during processing the grids when staining them....(e.g. different surface tension forces during staining and/or washing and drying mode), but first of all:

Please provide HR-images of representative areas of 'odd appearances'. Thanking in advance,

Wolfgang H M.

Ultrastructural analyzes are sometimes quite complex and demand a deeper understanding of the cell development stage, physiological role and environmental factors. I work specifically with the plant cells ultrastructure and we generally find cells with a nucleus occupying most of the cell volume in meristematic cells with high mitotic activity. In addition, cells that have a high density of mitochondria generally require a high energy demand. Metabolically active cells will present more mitochondria, larger and with well-developed crist mitochondria. It is important to consider environmental factors, as cells subjected to different types of environmental stress can generate plastic responses that will modulate cellular ultrastructural aspects. I believe that before looking at your TEM samples you have a thorough understanding of the context in which these cells were cultured or the biochemical and physiological functioning of normal and abnormal cells. And always try to have a representative amount of samples to confirm that the ultrastructural changes are representative. I suggest 3-5 repetitions per treatment or condition. Perhaps in your case it would be interesting to use some tomography technique to help understand the 3D architecture of mitochondria between the two cell types. I hope I was able to help you in some way.

I think you are talking about cell culture and about observation of a surface of cells. In this case you need SEM. You can find "specific steps" by googling "cell culture sem protocol"

Pictures are OK. Of course, they can be improved by decreasing accelerating voltage, as was said by Denis Korneev (15 kV is too high for your task). The main problem - I do not see any nanoparticles (only 1 micron size).

PTA has been used, as Indranil has suggested, but so has uranyl acetate. You will need to look up a good method so you have a good working method.

Hello Michael Delannoy

Thanks for your comment. Actually, I am looking for a 40-nm cage-like structures that could be identified when use negative staining by electron microscopy.

But the attached pics are confusing.

Kunal Madhav

I hope you are doing well. The Scanning Electron Microscopy (SEM) technique is used to analyze the morphology of the nano/microparticles while Transmission Electron Microscopy (TEM) technique gives morphology along with material information on the atomic scale. So depending on the requirement, one can characterize the sample with SEM or TEM or Both.

Hello Iman Bastawecy

One of the main advantages of using EM for viral diagnosis is that it does not require organism-specific reagents for recognizing the pathogenic agent. Other tests involving molecular and serological methods require that a specific probe be available for virus identification. In the event of a disease caused by an unknown pathogen, it is hard to know which reagent to pick.

EM allows an “open view” of whatever might be present, while molecular tests require knowledge about the potential agent(s) to determine the correct test(s). EM, though it may not be able to identify a virus beyond the family level, at least points the way for more specific identification by other methods such as biochemical assays for specific pathogens.

Another fact to keep in mind is that reagents do not exist for all viruses; when they grow poorly or not at all in in vitro systems, obtaining enough material to produce commercial test kits is difficult.

Finally, in cases of dual infections, molecular or antigen-based testing would likely miss the second agent.

The purpose of sequencing the whole gene of glycoprotein B will help identify novel mutations in the gene and further help to compare the viral glycoprotein envelop genome to the genomes of other herpes virus strains allowing to identify the genetic attributes of the viral glycoprotein envelop that contribute to its pathogenesis.

Best.

The JEOL Automated Data Acquisition System (JADAS) is a software system built for the latest generation of the JEOL Transmission Electron Microscopes. It is designed to partially or fully automate image acquisition for ice-embedded single particles under low dose conditions. Its built-in flexibility permits users to customize the order of various imaging operations. In this paper, we describe how JADAS is used to accurately locate and image suitable specimen areas on a grid of ice-embedded particles. We also demonstrate the utility of JADAS by imaging the epsilon 15 bacteriophage with the JEM3200FSC electron cryo-microscope, showing that sufficient images can be collected in a single 8h session to yield a subnanometer resolution structure which agrees with the previously determined structure.

The articles from Tomikawa E, Valentine WM or Jortner BS in Tox Path 2019 may help you.

Dear Iman!

Electron microspopy is not ignored as a method for imaging viruses. It is not widely used for a number of reasons. 1. Electron microscopes are not available in all laboratories; they are expensive.

2. The technique of electron microscopy itself is a rather laborious multistage process that requires a long fixation time and experience in cutting sections.

3. In a situation where it is necessary to carry out daily diagnostics of many patient samples for viral infections, the use of electron microscopy is significantly inferior in terms of the number and time of obtaining results in comparison with PCR.

An easy way to get it loose would be to pour some liquid nitrogen into the chamber of the AFS2. The metal will shrink faster than the resin, which will allow you to break it off.

To prevent this ( it happened to me as well) I always have a few ml of 100% ethanol in the chamber, this also helps cooling down the metal blocks.

best regards,

Rob

Actually beam current IS a spot size. Just not really good wording from some manufacturers.

Paul Nnarotam Onyishi

Paul, you might try (in any case: no claim for sufficient/comprehensive/excellent content!):

Edited by Viacheslav Kazmiruk

Published: March 9th 2012

DOI: 10.5772/1973 ISBN: 978-953-51-0092-8

eBook (PDF) ISBN: 978-953-51-4329-1

Copyright year: 2012

Also find:

SCANNING ELECTRON MICROSCOPY Copies of transparencies Vikram Jayaram

https://www.iitk.ac.in/acms/doc/SEMPart1.pdf;

https://www.iitk.ac.in/acms/doc/SEMPart2.pdf;

https://www.iitk.ac.in/acms/doc/SEMPart3.pdf, and

https://www.iitk.ac.in/acms/doc/SEMPart4.pdf, which contains the

==>"Introduction Welcome to the lecture training module on scanning electron microscopy. You should read these notes in conjunction with the slides of the presentation. Text passages...."

or

GOODHEW et al (Eds), 2001:

You might try as well Open Access sources like:

SpringerOpen and open access: Applied Microscopy:

https://appmicro.springeropen.com/

Archival content cf: http://www.appmicro.org/main.html

Applied Microscopy will start publishing with SpringerOpen in 2019. Content published before the end of 2018 can be found at the society website.

or:

https://www.jeol.co.jp/en/applications/pdf/sm/sem_atoz_all.pdf

------------------------------

But most Text Books (on SEM) were published subject to a charge:

e.g. (no claim to completeness!):

Author: Anjam Khursheed (NUS, Singapore)

https://doi.org/10.1142/7094 | November 2010;

SPRINGER:

Authors: Joseph I. Goldstein, Dale E. Newbury, Joseph R. Michael, Nicholas W.M. Ritchie, John Henry J. Scott, David C. Joy

Scanning Electron Microscopy and X-Ray Microanalysis © 2018

-------------------------------------

Author: Anwar Ul-Hamid

("Provides a concise and accessible introduction to the essentials of SEM")

-----------------------------

1st Edition

Edited by Alina Bruma Copyright Year 2021

ISBN 9780367197360

Published December 21, 2020 by CRC Press

164 Pages 25 Color & 81 B/W Illustrations

& so on....(knowing that this was NOT requested by your question...(;-||)

More results perhaps in 'Deep Net....' Plain Google search does not find more results for Open Access or "free of cost" (Text Book) contents on SEM.

Best wishes and good luck!

Satadru Chakrabarty Hello dear,

How have you opened emd file ?

I have same problem right now.

Here is a table. The last column is density.

If you can lay your hands on a copy of the CRC Handbook of Chemistry and Physics, which everyone used to have, you will find tables of physical data like that for sucrose and many other substances.

Sodium cacodylate is a good buffer, no reason to replace it with something else. But OsO4 (dangerous and expensive, sometimes just transferred from TEM protocols without any reason) usually can be replaced with tannic acid and other harmless and cheap reagents:

Good luck!

Dear Anton Zvonarev thanks for asking this very interesting technical question on RG. As an inorganic chemist I'm absolutely not a specialist in this field enough to give you a qualified answer. However, I know that UranyLess is a useful alternative to the commonly used uranyl acetate. Nowadays it has become rather difficult in many labs to work with uranum salts due to more restrictive safety regulations. UranyLess contains lanthanides instead of uranium and is not radioactive:

However, it is apparently very important to strictly follow the published staining protocoll in order to get good results with UranyLess. In this context please have a look at the following potentially useful link:

Please also see the following interesting reference:

This article has been posted by the authors as public full text on RG. Thus you can freely download it as pdf file.

I hope this helps. Good luck with your experiments and best wishes, Frank Edelmann

Hello Frank Krüger, thank you for your feedback, we recently started testing the eLabFTW in the workflow at our Electron Microscope. It would be cool if we could compare/exchange our methodology. It would be useful to consider a user meeting on this topic.

Hello Renata S. Rabelo

You may please refer to the article below. It may help you better understand the topic.

DOI: 10.1016/s0022-5320(67)80150-3

Best.

Correlative Probe and Electron Microscopy

I've had good experience in the past with Sigma rabbit anti-GABA polyclonal Ab

Sorry for posing another answer. This answer may help other researchers who are looking for the same information. I have created cfg file using MATLAB, as mention before. If you want to convert cfg file to xyz, you can use the building model tool that comes with QSTEM. Best of luck

Biological systems can be filtered in all size ranges, with predictable molecular sizes and weights retained or passed through each. The manufacturer of filters can give advice.

You might consider centrifuge to remove the particles, if there is a small density difference.

Dear Kyle Kuchynsky ,

You could follow the same protocol as you do for the normal seeding of your cells.

(I always autoclaved the coverslips prior to seeding cells on them to avoid any contamination.)

You can use forceps to place the pre-autoclaved coverslips on the cell culture plate (also to take them out once you want to process the sample for TEM).

If your cells have problems to attach onto the coverslip then you can also pre-coat (45min at 37C) the coverslips with Poly-L-Lysine or Matrigel (for better attachment) prior to seeding the cells.

For the pipetting, I had optimized the cell number to a specific density of cells so that they would easily attach in every part of the wells and coverslip as monolayer.

So I did not have to pipette precisely.

Hope this helps!

Garima Chaturvedi Thank you. I will check that software.

Dear Marine Dehecq ,

we all would benefit from information on (at least a bit about "head lysate") and - most appreciated, processing of the spec's - especially resin type and STAINING the ultrathin sections.

My first guess would be (since there are - as you certainly know) at least two typical tangential section images of primary(?) cilia.... as one can find in some web-sources:

"The organelle is membrane-bound and contains multiple microtubules running along its length"

(cf. also - only ONE reference out of many):

"Cell Science at a Glance The primary cilium at a glance" Peter Satir, Lotte B. Pedersen, Søren T. Christensen Journal of Cell Science 2010 123: 499-503; doi: 10.1242/jcs.050377 to be found at:

or:

By: Mike Adams, Ph.D. (Dept. of Biology, Eastern Connecticut State University) © 2010 Nature Education  Citation: Adams, M. (2010) The Primary Cilium: An Orphan Organelle Finds a Home. Nature Education 3(9):54

https://www.nature.com/scitable/topicpage/the-primary-cilium-an-orphan-organelle-finds-14228249/

The electron dense structures/textures I would characterize as either (as usual!(:-)) ) a staining artifact (poor washing after staining) OR ((yes I know: NOT ALL artifacts are artifacts! (:-)) ) might reflect the presence of intensely stainable (TZ-Transition Zone)-Proteins in the basal bodies of cilia.

But unfortunately we see only 2 images out of your collection, not knowing how the images were achieved....in terms of practical handling of stains (guessing double staining UO2ac/lead citrate or eventually triple staining with e.g. Tannic acid 'ante' Uranyl acetate and Pb-citrate...)

Also cf.:

Drosophila sensory cilia lacking MKS proteins exhibit striking defects in development but only subtle defects in adults

Metta B. Pratt, Joshua S. Titlow, Ilan Davis, Amy R. Barker, Helen R. Dawe, Jordan W. Raff, Helio Roque

Journal of Cell Science 2016 129: 3732-3743; doi: 10.1242/jcs.194621

= https: @//ciliajournal.biomedcentral.com/articles/10.1186/s13630-015-0018-9

Out side my experties

Md. Shahed-Al-Mahmud

Thank You for the suggestion, I will try that.

Richard Gursky

Its an Tecnai F20, 200kV microscope. While my structures can be quite long, they are relatively thin (<100nm). What resolution did you have in Your experiments?

Thank You, it s a great paper.

Dear Stefan T. Huber ,

you are right, unfortunately there are two different definitions:

please see the 'physics' and the 'crystallography' definition of the wave vector k in:

https://en.wikipedia.org/wiki/Wave_vector.

wave vector k= 2*pi/lambda , but also and k= 1/lambda

To my opinion that is a historical issue.

Relation to the momentum is via Planck's constant h and h/(2pi)

Buongiorno Francesco,

You find a speckled background signal after the immunoEM staining with nanogold, followed by gold amplification. You might want to share one or more (details of) images where this problem occurs.

A: Do you find a good signal at places where you expect the signal? In other words, does the staining work and is the nanogold conjugated 2nd Ab work fine?

B: How do you perform the gold amplification? Self-made from AuCl₄ or commercial kit?

Hahaha, yes I am. I do have a misaligned column, and I'm struggling with the alignment procedure because I don't see anything on 60000 that is required when setting astigmatism or some another step, and so on... Thank you for your answers, I thought that it should be round even if it's a tungsten filament.

Hi Bhavana,

After referring to the attached research article it can be inferred that.

If the precipitates have a coherent strain then they will show a coffee bean contrast in the TEM. Furthermore, in diffraction mode, if the concentric rings are continuous at the interface between the precipitate and the matrix, then it indicates that the precipitates are indeed coherent. Also in this work, in the electron diffraction patterns from (OO1), strong superlattice reflections were observed, which further confirms that the precipitates of A13Sc have an ordered L12 structure. You may refer to the publication for detailed understanding.

Jeremy Linsley Well, I will not draw any references (I have two horrible deadlines approaching) so I will just tell you from memory. Potentially this is good stuff, lots of questions nobody really asked:) First of all, I remember distinctly that I tried to find out how exactly is ER/Golgi separated from nucleus and whether they are connected or not. There seem to be no works about it. Thus, the question of how the nucleus is connected to them is open. Golgi/ER never really move separately from the nucleus. I did a lot of filming of cells (time-lapse) and you can definitely see there that Golgi/ER is a structure that always is attached to the nucleus - never separated. That makes you wonder why, right?

So, GFP could get there from the nucleus.

On the other hand, why GFP accumulates in the nucleus? To answer this question, you need to read some papers about the hydrophobic properties of the nuclear pore complex. I think this really has to do something with the hydrophobicity of the molecules overall. An interesting start would be https://jcs.biologists.org/content/125/21/4979

See, some molecules can be just more likely to pass into the nucleus thanks to their hydrophobicity properties - not to some "homing signal". And that may be the reason GFP goes to nucleus.

Disclaimer: I have never collected specimens during expeditions, and I have very limited experience with plants.

But you did not get any answers yet, so just two cents of advise.

Properly fixed specimens, i.e. if they were timely placed as a small pieces into fixative (glut), can be stored if fixative in refrigerator (means about 4C) for prolonged time, 1 month is not a problem. So, you can keep specimens in a cold box (insulated box with some ice).

If your Alkene is specific then adding OsO4 will oxidise d ENE bond to fool and hopefully give you a denser stain

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.

Hi Yaroslav,

anything with a temperature higher than -138C will devitrify your sample.

what you are looking for is the cryo-glue first described by Karsten Richter

(https://doi.org/10.1111/j.1365-2818.1994.tb03436.x)

which consists of ethanol (100%) and 2‐propanol in a 2:3 ratio.

you apply this into a holder or cryo-microtome pin (the slotted type) using a wire loop at -140 to -145. At this temperature the glue is still viscous and you can slide the grid in using pre-cooled tweezers. next you lower the temperature of the ultramicrotome to -160 to -165, now the glue will be rigid allowing you to trim and section. be careful to remove most of the glue when trimming because it will have different sectioning properties than the rest of the sample. Also I would recommend sectioning with the grid in a vertical position (same sectioning properties in during the entire stroke) and not to make a to high blockface (this will induce curling of the sections).

The copper of the grid bar will not be that much of an issue (if you section thinner than 50nm) i've had decent results with sectioning gold plated copper grids after HPF, though if you can do the whole thing using gold grids it will section a lot easier.

best of luck!

Rob

A traditional stain with affinity for wood lignins in EM is KMnO4, potassium permanganate. This has been used in EM since the 1930s. Attached is a link covering some of the available stains for wood in EM.

The problem was completely solved with the new freezer. Wohlwend is the best! :-)

Thanks Thomas for your response.

You may want to investigate the morphologies of the TEM grids you use. There are various types and some of them are porous. Ideally, you want to use substrates with flat surfaces to facilitate self-assembly.

Hello Saleem,

In my opinion, you need to use freshly amplified phage particles for performing TEM. Usually, people have purified virus by CsCl density gradient using general centrifugation. You may use 0.75% Uranyl formate for the negative staining. If you need further information, you may follow this article instructions. I hope this information may help you.

When thinking quantification, always take pictures at the same magnification.

If you are interested, I have a semi-automated macro for ImageJ designed for bacillus cereus, made some years ago, to quantify bacteria and exosporium surface (using threshold), then number and length of pili (using polyline). it works with one bacterium per image.

The macro and its notice are in french, but you are in Quebec, Canada, shouldn't be a problem.

If you can just get the dark circles detected using 'identify primary objects' you can then tell it to fill in the space inside each object. And/or, you can use a mask on your image or a copy of the image as a different approach. If you search for a CellProfiler forum you can probably find a good pipeline or instructions how to make one. Good luck. -you could always 'identify primary objects manually' and draw circles around them all too, but that would obviously be much more involved.

Take care while using mass device as it is destructive for samples,however, chemical formula can be known from it if you know the molecules formed the polymer.

Are you sure? OsO4 is not proper agent for NS. Why don't you use common NS agents like uranyl acetate or Phosphotungstic acid?

OsO4 reacts with lipids and proteins providing positive contrast. It is a common reagent for resin embedding preparation, not for NS.

Good luck!