Instrument is a JEOL JSM-7600F (field emission electron microscope)

Short Problem: sample is not able to be removed from the experiment chamber and brought into the exchange chamber.

Lenghty relevent/irrelevent information: Sample was inserted into the sample holder in the exchange chamber. Chamber was closed, evacuated, the door opened to allow for it to be put into the experiment chamber. The rod pushed in, sample moved into the experiment chamber, everything pumped down, went to turn on the electron beam and errors popped up. The errors were for the turbo molecular pump.

Did a reboot of software, did not fix the errors. Did a hard reboot of the machine except the electron gun, and the pump worked. Then we tried to remove the specimen, lowered the rod, inserted it, pulled it back out, opened the chamber, and no sample. Software shows the sample is still in the experiment chamber. Checked the rod assembly, everything looks to function as normal. Tried to move the specimen in the experiment chamber to see if we could reorient things, still the sample doesn't come out.

We have no manual for this instrument, has anyone had something similar happen or any other ideas on how to trouble shoot?

There is a camera in the machine, but alas, it too does not work.

At the beginning of the semester, I had asked for tips on a project for a class in electron microscopy, and I got some really helpful tips and suggestions. I wanted to leave an update on how things are going!

My project is analyzing the most effective methods of capturing essential morphological features to identify microfauna in moss. I've worked largely on confocal, so far, which is amazing (because lasers). I finally was able to prepare, mount and image my first SEM sample this past week, and I'm obsessed. It's not hard for me to lose track of time and end up at the scopes for five or six hours. My other homework is definitely suffering!

Here are a couple of images I'm proud of so far!

Eutardigrade, 400x. Blue=UV 405nm, green=488nm (argon), red=515nm (argon), b&w=brightfield, final image is overlay.

A millipede (Polyxenus lagurus), preserved via glutaraldehyde/formaline and dried via HMDS. Mounted with gold sputter coat.

Thanks for the ideas and input!

Hey there fellow microscopists!

I have been tasked with purchasing a sputter coater for our SEM sample preparation. I am looking for the best and most reliable gold sputter coater manufacturers and I felt like you all would be the best people to ask!

The manufacturers I've seen online are :

• Leica microsystems (EM ACE200) <- This is the type I've been comparing the others to. We have the Leica EM CPD300 so this was the first one I looked at.
• Quorum technologies (Q150RS Plus)
• Cressington scientific (Cressington 108auto)
• VacCoat (DSR1)
• Hitachi (MC1000)
• SPI Supplies (12150-AX)

We are looking at pretty constant use, both for teaching and research. Almost exclusively biological samples ranging from bacteria in size to insects. As the samples we're working with are all very oddly shaped and are sometimes big and fuzzy, we need the rotation part to cover everything and make good conductive contact with the sample stubs. Measuring the thickness of the coating is a huge plus, but probably not a deal-breaker if it's missing. The SEM we have in the building is a JEOL JSM-6610 from 2010 if that influences the decision.

Am I missing a key player in the coating game? What are the types you all are using and like the most?

Also, separate but connected question: Argon vs. Air sputtering?

I've seen some discussion online about how it's unnecessary for most applications to have argon in the chamber while sputter coating. What are your thoughts on that? Do you think that the heat in the chamber is sufficient to damage biological samples or is it a bit exaggerated? Will the potential slightly more uneven coating have a huge impact on the types of samples I'm looking at. Keep in mind that we don't have a field emission SEM in house and if we were to view our samples in an FE-SEM we'd probably do the sample prep at that place.

Tl;dr: need sputter coater, which one to buy?

Thanks for reading!

I just inherited this sputter coaster, and it hasn’t been run in a while. This model is very old and no longer supported. The tech I talked to said to change the target so I did, but I still have the same problem. Are my mA too high? I have it set to 20 but when it’s running it reads 25. All advice is welcome (literally any sem advice I will take)

Hello, I was wondering what you could expect to pay for a used working JEOL JCM-5000 Neoscope. Also I was wondering if I would be able to find software or a manual for it, if I decided to get it. Thanks.

Our Thermo Apreo 2S will not maintain focus. I'll get it really nice and dialed in and then it drifts. This was on a non-charging sample (tin balls) but our Thermo engineer states that it's contamination in the column/pole piece. He said he had seen that before when a lot of organic samples had been analyzed, so he plans to come onsite and clean the column/pole piece.

My question is, is a plasma cleaner a necessity? Would it be capable of cleaning contamination of the pole piece/column?

HI everybody,

Throwaway Account for obvious reasons. I worked for JEOL for some time and thought this might be of interest to some people here. Also this should help this sub to some activity!

Feel free to ask anything you want to know about JEOL and I'll do my best to answer it (except anything that might make it possible to find out who I am, of course).

I'm looking to purchase a book to keep in my office for teaching the principles of our SEM, so I was curious what/if anyone has a favorite book they often reference?

Thanks!

Are scientists able to see the creation/growth of new skin, hair or nails with an electron microscope? If so is there video of the synthesis of new human tissue that we can pull up online?

Loveliest TEM-microscopy experts,

I would like to explore the possibilities of pushing the charge densities at the tip of cold field emission guns far beyond their structural and material limitations by applying an alternating voltage at 10^12 Hz. At this frequency I hypothesize the voltage could be increased significantly compared to previous approaches without encountering tip breakdown, facilitating achieving peak charge densities beyond previously thought possible. At this frequency the material merely does not entertain the necessary duration in time to break down. This hypothetical approach is based on research on EM radation induced plasmon oscillations in which charge densities are pushed beyond their static limit.

I would love to hear your feedback on my assumptions. I would not remotely classify myself as an expert and do not know if frequencies of alternating voltage in the TeraHz range are even workable, but I am sure that if there were to be any experts that had the answers, I would find them in this community.

Many thanks for engaging in the discussion,

Kind Regards,

Today I stayed overtime at work, and used the plasma cleaner to clean some tem samples and storage in the storage holders... It worked perfectly well, and I turned it off. However, I forgot to take a picture of the sample and tried to turn it on again and now it doesn't work. I don't know what to do!

Hello everyone, I think this is the right place to ask this, because I couldn't find this information anywhere on the Internet; I will be working with silicon nitride membrane normally used for TEM, (my specific use is for cathodoluminescence apparstus) and I would need to know the attenuation factor of a beam of electron with energy in the range 10-100keV passing trough a silicon nitride membrane with thickness in the range 200-1000nm, I would need an estimate of the transmission of electrons and if possible the energy loss, but I couldn't find any article with something like this. Any help is really appreciated.

Can anyone tell me how to turn on the magnification real setting on a TEM LVEM5? Their customer service reps aren't available and I'm on a time crunch. If I'm in TEM mode, it won't zoom, or if it will zoom it won't give me a scale bar. I need to be able to zoom and have a scale bar. Thanks

Hello all, I have already posted this in "Labrats", but maybe the question rather belongs here... I've been struggling a lot with producing nice TEM images of my tissue models in the last few months, and it's all come down to optimizing the osmolality of my fixation buffer. I finally ventured into a neighbouring lab to use their Osmometer to measure the osmolality of my cell medium and my fixation buffer:

I am using Sorensen's PB buffer pH 7.2, which according to my original, trusted protocol is made like this:

0.2 M PB buffer:

7.16 g Na2HPO4 x 12 H2O (Mw = 358 g/mol) in 100 ml water

3.12 g NaH2PO4 x 2 H2O (Mw = 156 g/mol) in 100 ml water

Mix 40 ml Na2HPO4 with 10 ml NaH2PO4 to make 0.2 M PB buffer.

Since I wanted to try higher concentrations as well, I doubled all concentrations to make 0.4 M buffer, but kept ratios the same.

Now, according to a publication from 1967, the osmolality of the buffer should scale linearly with the molarity (i.e. osmolality of 0.1 M buffer is around 200 mOsm/kg, and that of 0.2 M buffer is supposed to be around 400 mOsm/kg). I have included an image of the figure from the publication and of my own measurements here: https://drive.google.com/drive/folders/1dXLZ7RBYy8p7msF0QmOx0YmWj4hfTcwn?usp=sharing

HOWEVER, according to my own measurements at the Osmometer, the molarity / osmolality relationship of my buffer is not linear at all. I measured osmolality of 0.1 M buffer at around 220 mOsm/kg, which is in accordance with the literature. However, 0.2 M was at 280 mOsm/kg, and 0.4 M at 325 mOsm/kg. ALSO, there is a weird "spike" in the osmolality for molarities between 0.1 and 0.2 (measured 0.15 and 0.175 M, which had higher osmolalities than the 0.2 M buffer??? See image behind the link)

I am utterly confused and wondering if I'm making some systematic error. The way I understand the concept behind osmolality, diluting the buffer 1:1 with water should half the mOsm/kg ?? But this does not seem to be the case according to the measurements. Can anyone explain this? Am I not seeing something??

In Labrats, someone suggested the measurements might be off if the buffer molarity is close to the maximum solubility, but this should not be the case in the 0.2 and 0.3 M buffer range.

Maser MD, Powell TE 3rd, Philpott CW. Relationships among pH, osmolality, and concentration of fixative solutions. Stain Technol. 1967 Jul;42(4):175-82. doi: 10.3109/10520296709115005.

I had another thread, didn't explain myself correctly, and learned some things.

Brief enigmatic intro: I've (some would say foolishly 🙃) dedicated my life to physically doing something about all the needless suffering in this world, as many of you probably have. I own a small medical business and would like to take micrographs of the before and after results; an electron microscope version of the photos commonly seen on medical business websites--for example botox, lip injections, bbls, etc. Except I cure people and their pets of diseases, not plastic surgery. (I'm being purposefully vague to avoid personal commentary, just as I wouldn't expect you to tell me what businesses you own, how many doctorates you have, what's your net worth, etc.) So with that said, money isn't an issue. The logic is that if the public was able to see the before and after of dead common pathogens, that would enhance the power of common before and after photos (such as a foot with and then without a plantar wart, without surgery).

Here are some example photos I'd like to take:

https://theconversation.com/chickenpox-and-shingles-virus-lying-dormant-in-your-neurons-can-reactivate-and-increase-your-risk-of-stroke-new-research-identified-a-potential-culprit-194627

https://www.researchgate.net/figure/Electron-micrographs-of-varicella-zoster-viral-particles-a-The-electron-micrograph_fig2_353040817

In the other thread, someone said it'd take 50 billion to create a basic EM facility. I understand EM is more challenging than regular microscopy but I'm ignorant of the requirements to produce such photographs, ideally 10-400 nm specimens. There are electron microscopes on Ebay for a range of prices https://www.ebay.com/itm/235633822567 this one is $17k. This one is $63k https://www.ebay.com/itm/325828386918

Ignorantly, initially I figured just as you can buy a microscope or elaborate telescope and have what you need to perform astronomy or microscopy, EM was just a much longer set-up and learning curve--a longer process (*ba dum tss*). Taking a few years to build a small lab for SEM is not impossible, but from what I've read would be an extraordinarily large amount of work. At first I thought it may be similar to how professionals build restaurants, gas stations, dental clinics, casinos, skate parks, and all sorts of things--there'd be many steps, but definitely doable. For instance if Bill Gates/Elon/Lil Wayne woke up one day and decided he wanted to take EM photos, I thought they'd be able to do so with the dedication (lil wayne pun I didn't mean to make).

If the EMs for sale can't produce images like those in the links, what are they good for? In the other thread someone mentioned they require massive amounts of added tools, "high-pressure freezer, freeze-substitution machine, a fume hood, a microtome and several highly toxic chemicals that are probably regulated wherever you live (OsO4, lead acetate, uranyl acetate, etc ...)" Others didn't say it'd be 50 billlion, but implied it'd be an elaborate list of things required, similar to how a dental clinic requires things for sophisticated tech, laughing gas, etc.

Would any of the above photographs be able to made for under 5 million?

Please don't be offended by my ignorance of the intricacies of electron microscopy--that's why I'm asking. I have yet to find youtube videos or articles on this topic specifically, and the videos I have seen make the use of SEM look condensed and replicable.

Hey all! New to taking SEM images working on a small project at undergrad level for uni on Beetle eye morphology and done some SEM photos. To start with the machine was being temperamental and I was told that is due till for servicing so these were the best I could get pictures. But wondering if anyone can guide me with the best way to just slightly clean up pictures if possible, I have little photoshop experience but hoping to mainly clear up the lines of brighter stripes on the left hand side if someone can offer some tips. I have access to adobe photoshop and clip studio.

Hello!

This is a naieve question, but can TEM grids be coated like a sample on a normal stub? We have a Leica ACE600 sputter/carbon thread coater. I have a research group that is studying non-conductive nanoparticles and they're impossible to find. They use a stain to try and alleviate that, but it just piles and piles on the grids. Do they make adapters/holders for grids in coater systems?

Thanks!

Looking for stitching errors in an EBL pattern, I’m having trouble with stig at high resolution. My current parameters are 10KV, 20na current, and I’ve messed a lot with beam/apperature allignment, can’t seem to get the “ramp” effect off the edge of my structures. Can’t post images bc company won’t let me. Scope is a hitachi 8230. Could I be too low in KV to image this? Still new to EM so any help is appreciated.

I'm told they don't really give them out often, but am very jealous of some colleagues who have a couple. Have any of you succeeded in getting one?

I will be performing SEM for the first time and I'm purchasing the tools I need for my yeast biofilm analysis. I've been reading some authors using "thermanox" steril cover glasses and some authors being very elusive citing just "glass" or "plastic" cover for the 12-24 wells plate. As I seek for price/brand options, there are some very cheap ones and some expensive ones. But I cannot tell the difference. All of them seems to be 12-13mm diameter and 0.13-0.17mm of thickness. What should I be looking for?

Hi,

Is there a reason a carbon (conductive) sample would look white (showing a charging effect)? I thought that the charging effect would occur only because a carbon coating did not cover part of the sample. However, if the entire sample is carbon, shouldn't it look crisp without charging?

Teach me, seniors,

We have multiple projects where we are needing to look for critical rare earth elements such as Lanthanum and more importantly Germanium and Gallium. The Ge can be associated inside of coal particles, in the organic matrix, or outside of the coal. Depending on where it is located changes what color backscatter I need to be looking for. At times the Ge could nice and bright while other times it shows up as light gray. We have a lot a projects coming in and I need to be looking at the samples either polished in cross section or on stick tape to see if I can find any Ge and the concentration. We may have ICP-MS results to know that a sample has 500 ppm of Ge but the question also is, is it 500 ppm evenly dispersed throughout the whole sample or 500 ppm in a couple of particles with high concentration.

Long story short, with the projects coming in I am supposed to develop a protocol on the SEM (we are running IXRF software on a 840 JEOL) to try to quickly scan a sample to determine if there is Ge than go back to that spot to try to get a nice picture and more EDS information. I would be running at 20 kv for this work. I am looking for other ideas on how I could accomplish this task in a quicker method that spending hours and hours taking lots of photos and eds spectrum hoping to find something.

Hey! Our lab recently purchased a used FE SEM that had severe rust in the specimen chamber, lower lining tubes, and aperture holder. We dismantled the system and sent these parts out for electropolishing, but we avoided dismantling the upper column because aligning these components seems to be much more challenging. We inspected the gun through the getter pumps, and the steel Wehnelt cylinder looks brand new (no rust or corrosion). Do you think it’d be worthwhile to disassemble the rest of the gun to check for rust? We intend to run a full system vent and bake out before firing the gun.

I am looking at ways to improve our sample prep procedure. Many a years ago the company I worked for was able to get a mirror like finish on all of the samples, that knowledge has since been lost.

Our basic procedure includes getting sample into epoxy, some various grit sand paper, cleaning once the sand paper is done with Toluene (another chemical was once used but it has since been discontinued due to greenhouse issues), some polishing with a Dimond paste, more toluene cleans and finally after drying overnight it is carbon coated.

We are processing material such as coal, clay, deposits, minerals, ect. For many of our samples we can dry it in a drying oven for a bit before having it go into epoxy. For coal we need a color contrast between the coal, minerals, clays, and the matrix it is in. Our original procedure for this was placing the coal into carnuba wax (the hardest natural wax) and after it sits topping off the mold with epoxy. This combo creates a nice black background with the wax, the coal is dark gray, clay light gray and minerals are nice and bright.

Here is the problem though, carnuba wax is softer so it polishes off faster than the coal and the Toluene eats away at the wax during the cleaning sonication steps. I have tried ethanol for the cleaning which does not eat away at the wax as much but it has show a higher chance of charging in the scope even after carbon coating.

We have thought about trying to change the chemical for the cleaning and/or trying to find a carbon doping method or carbon impregnated epoxy. Do any of you know of anything out there? We use cold/room temp prep methods for epoxy. I have found a couple epoxies out there that are supposed to help reduce charging but either they are a hot epoxy prep method with pressure or they include elements such as Ni or Cu where we see in our samples at times.

I have been planning to look at the distinctive features of sub cellular compartmentalization by using electron microscopy of rare population of cells (10,000 -50,000 FACS sorted cells).How can one prepare sample for such experiment. Does anyone have any papers they have got ?

Looking to buy a new FESEM in the next year or so and I would like to see what is out there. Does anyone know of any expos where I could see different manufactures? Looking to stay in the US if that helps.

I should add that I want to demo multiple SEMs in person, which is why I was asking for trade shows or conferences.