Do you put together samples for electron microscopy and wish to save time, cash, effort, and frustration?
Do you picture different folks’s samples and need recommendation you’ll be able to simply share with these getting ready them?
Electron microscopy (EM) is a robust method that contributes to revolutionary discoveries on the frontiers of science. It has large potential inside key fields comparable to epigenetics and structural genomics and well-established ones like drug discovery.
However it’s exhausting work.
Partly as a result of it exists on the interface between biology, chemistry, and physics—you’re endlessly encountering tough ideas.
One other problem is the astounding number of EM functions—every utility and pattern are totally different and require particular person therapy.
This text gives hands-on recommendation that can assist you get the absolute best knowledge out of your EM experiments.
Recommendation You Can Belief
To carry you the perfect EM pattern prep recommendation, we interviewed EM skilled Dr. Andreia Pinto who has not gone longer than a month with out touching an ultramicrotome within the final 12 years.
Andreia began her profession as a Biomedical Scientist in Histocellular Pathology with a selected curiosity in electron microscopy. She labored as an Electron microscopy specialist and in addition within the analysis of Major Ciliary Dyskinesia (PCD) in Lisbon, earlier than shifting to the Royal Brompton Hospital, London, in 2019, the place she was chargeable for coaching a deep machine studying platform to acknowledge patterns in EM pictures of cilia in diagnosing PCD. She accomplished her PhD on this subject in 2022 whereas additionally investigating new insights into SARS-CoV-2 an infection of the respiratory airway.
Andreia is at present an Superior Workflow Utility Specialist with Leica Microsystems, based mostly on the Imaging Centre of the European Molecular Biology Laboratory (EMBL), Heidelberg (Determine 1).
Prime 10 Knowledgeable Electron Microscopy Pattern Preparation Ideas
We’ve distilled Andreia’s information and recommendation into 10 hands-on ideas for EM pattern prep.
1. Deal with Pattern Preparation and Imaging Equally
We began by asking Andreia why pattern preparation is so essential in an EM workflow. She began by emphasizing that with out good pattern preparation, outcomes could be deceptive:
“It doesn’t actually matter how properly outfitted your lab is, with refined microscopes and ultrastructural imaging techniques, in case your pattern preparation is just not good… the result will likely be a disappointing failure at finest, and misleadingly incorrect at worst, as a result of you can be seeing issues that aren’t reflecting actuality. We are attempting to understand actuality, so you will need to get this step proper.”
Highly effective electron microscopes produce correct and interpretable ultrastructural knowledge—provided that samples are ready with care enough to protect essential options.
2. Perceive Your Scientific Aim
Andreia additionally advocates for cautious planning and taking the time to contemplate the ultimate goal, as it will crucially affect pattern preparation selections:
“An important factor to contemplate is all the time the ultimate goal. What does one wish to obtain in the long run?”
She additionally asks folks to contemplate potential selections earlier than getting ready samples, specifically, whether or not correlation with gentle microscopy will likely be required (e.g., for technical orientation or focusing on research). Early concerns comparable to these affect the way you put together your samples.
“Take into account solely the construction or morphology of the pattern wants preserving, or if there’s a want for spatial context, preservation of antigenicity, or any requirement to retrieve fluorescence. All these must be factored in. For every certainly one of these outcomes, there will likely be a special workflow involving totally different tools and a special talent set.”
3. Protect Your Pattern Accurately
Some of the essential selections in pattern preparation is easy methods to protect your samples. There are a number of approaches, from immobilization to embedding. Every preservation method has benefits and downsides, and the selection could restrict the downstream functions you should use. Andreia stresses the significance of rigorously selecting the preservation and embedding method you employ to make sure you can extract the information you want, for instance:
“…if one realises they wanted to label their pattern with, for instance, gold particles, however that they had already embedded the specimen in epoxy resin because it was the one system out there within the lab and the one protocol recognized… it will actually pose as a bottleneck. This process is irreversible, and it will likely be difficult to make correct, reliable labelling. So, it’s helpful to consider all of the potential wants early on.”
One other nice instance, she explains, is someone who desires to do correlative research however skipped essential patterning and mapping steps. In such instances, it will likely be “nearly inconceivable” to search out your area of curiosity, and time in EM could be a lot much less environment friendly.
Keep in mind that selections you decide to early on could restrict the kind and extent of knowledge out there while you picture your pattern, which could be significantly expensive in a CLEM workflow.
Cautious planning of your experimental design earlier than you get hold of your samples will assist make sure you use the appropriate preservation method.
“…the answer is all the time the identical; cease and undergo all the things earlier than having your samples in a harmful limbo.”
4. Push the Boundaries of Your Data
Sensible abilities are indispensable, however what theoretical and background information might allow researchers to arrange higher samples? In Andreia’s expertise, cultivating an understanding of the relative scale of proteins, organelles, cells, and organisms is helpful:
“…It’s essential to have a great spatial notion of organic techniques in 2D and 3D.”
And there’s no avoiding them —chemistry and physics. They may not strike as central to microscopists’ skillset working in life sciences, however she factors out their advantages.
“It’s essential to grasp how temperature and stress adjustments could work together together with your pattern and why. Concerning chemistry: understanding how stains, dyes, and tags can impression your pattern will allow you to make higher pattern prep selections.”
This not often comes simply, nevertheless.
“, for me, and since I’m a biomedical scientist… the trickiest theoretical level to understand is physics, however increasing my information here’s a worthwhile funding.”
5. Enhance Your Pattern Guide Dealing with by Utilizing Dummy Samples
Following up on this, we requested Andreia what she thinks is probably the most useful sensible talent for microscopists to accumulate. The reply is evident:
“…we must always by no means underestimate the ability of our personal palms as the 2 most dependable items of apparatus. Begin with good dealing with and manipulation, then you’ll conquer the world.”
What steps, then, does Andreia encourage to get higher at manually dealing with samples and enhance coordination and dexterity (Determine 2)? Once more, she’s very clear—observe!:
“Manipulate mock samples, for example, or much less treasured ones. Do it and do it and yet again. Switch grids from drop to drop till you’re feeling snug sufficient. Simply observe as a lot as potential. That is so easy, but it surely makes an enormous distinction.”
“When you’re training, play with the way you place important consumables. Plus, you realize you’ll have to examine tiny drops of clear liquid, so how do you want your lighting? Do you recognise in case your materials is already broken? Are your tweezers sharp sufficient, are they the appropriate kind for this dealing with? Apply and enhance by the minute.”
Then, while you work up your precise pattern, you’re much less more likely to mess up, and it’ll prevent time in the long term:
“Be assured together with your pattern dealing with as a result of while you attain the top of the day, you might need three grids remaining, and they are going to be very treasured to you! In the event you lose them as a result of you aren’t assured sufficient with pattern dealing with, you can be very, very pissed off with your self. Put within the time to be proficient in pattern preparation and dealing with, and it’ll repay in your EM workflow.”
You’ll be able to apply this philosophy to the ultramicrotome (Determine 3):
“In case you are working with the ultramicrotome, this is identical. Simply work with glass knives or an previous diamond knife as a lot as you’ll be able to and want, till you’re feeling assured sufficient to maneuver to your treasured diamond knife.“
However, and regardless of the commotion round diamonds, don’t shrink back from the diamond knife; it’s just about inevitable that you’ll use one on your EM work. As a substitute, begin sluggish, construct cautious confidence, and think about having someone observe you at first and providing you with good, educated ideas.
6. Don’t Underestimate the Worth of Small Tools
Given Andreia’s lengthy profession in electron microscopy, we needed to search out out what she considers probably the most thrilling developments within the area.
Maybe unsurprisingly for somebody with such expertise of assorted pattern varieties, it’s not the superior microscopes or arcane theoretical developments that excite her most, however the small, generally improvised objects designed to resolve easy points that make the distinction between success and failure:
“For me, what I discover thrilling, are the little issues…should I think about a canopy field to govern my samples beneath low humidity circumstances? Or, for example, ought to I think about an antistatic gadget that may make my sectioning enhance in so some ways? Small preparation concerns, however huge impression!”
You could be fortunate sufficient to have entry to state-of-the-art imaging gadgets that require a complete division to run them, but it surely’s the comparatively easy strategies and tools in your preparation workflow that enhance the standard of your pattern and the richness of your knowledge manifold.
Invisible and barely perceptible circumstances can do hurt to your pattern, as Andreia factors out.
“You don’t even know what’s getting in your pattern from the second you immobilize them till you load them within the microscope. Underestimate relative humidity and temperature, after which later, while you go to the electron microscope… you can be unhappy.”
Equally, freezing grids is a straightforward course of. However getting the particulars of it barely incorrect can drastically have an effect on your knowledge, its high quality, and validity.
Having an automatic and configurable grid plunger you can configure relying on the kind of pattern and remaining aim removes a variety of uncertainty and variance in pattern prep high quality and the standard of your outcomes. For instance, utilizing a great plunge freezer, comparable to Leica’s EM GP2 system, it takes a few minutes to mount and plunge freeze a grid reliably.
Her enthusiasm for tools that eliminates irritating pattern prep points is infectious and relatable:
“It goes from you sitting at an ultramicrotome for 3 hours, pulling your hair out, or then again having a pleasant session, ending earlier, going dwelling, feeling nice, simply due to one thing easy you probably did within the preparation. If all goes properly, with all the things arrange appropriately, then you’ll be able to extract an insane quantity of knowledge all through so many workflows, from typical TEM right down to single particle evaluation. How is that this not superb?”
7. Issue Pattern Prep into Your Funds
We needed to find what Andreia would say to anybody buying a brand new microscope.
“Thrilling! Benefit from the course of, however don’t neglect to contemplate – will there be sufficient funds left for correct pattern preparation funding? As a result of if there’s not, you will need to rethink, as a result of this needs to be equally essential.”
Easy sufficient—put money into pattern preparation and depart sufficient cash for the ‘smaller’ components chargeable for the standard of your pattern. This time is the perfect time to make these selections, and it might show tough to regulate an extended and complex preparation protocol later.
Andreia additionally stresses that equipping a lab thoughtfully will result in higher knowledge in the long term by declaring that pattern prep tools exists to make life simpler and make work extra reproducible by decreasing human errors and variability.
8. Arrange Your Lab Sensibly and Ask for Recommendation
Consideration of pattern preparation budgets is just not the one piece of recommendation Andreia would give to anybody buying new EM tools. She recommends reaching out to others which have already gone via the method of buying new tools.
“…earlier than going forward first with the massive buy, make a plan and ask for recommendation from labs which can be well-equipped and went via the method already… simply attain out to friends, to colleagues which have actually well-established labs that work very well and ask them for recommendation.”
In addition to contacting your colleagues, get in contact with the tools producers immediately for specialist recommendation.
And as a part of your plan, ask your self three questions:
i. What Tools Do You Want?
First, think about what tools you want on your functions, and perceive the state-of-the-art. For instance, do you want a high-pressure freezer on your pattern prep? In the event you’re learning a dynamic organic life cycle course of, you then would in all probability profit from one. Many prime amenities are utilizing this method, however examine their publications to grasp if this workflow is best for you.
ii. The place Will You Put Your Tools?
As soon as you realize what you want, think about the place you’ll place all the things.
Keep away from inserting your shiny new ultramicrotome wherever it could get knocked, and seek the advice of with the specialists about choices to scale back vibration, for example. Andreia councils in opposition to inserting workbenches immediately beneath vents that may blow aerosolized sediment on all the things and fascinated by what kind of waste you’ll produce, and putting in the mandatory hoods and waste streams.
iii. Can You Modify Your Lab?
Must you set up particular flooring safety the place you plan to deal with liquid nitrogen?
Is one finish of your lab extra humid than the opposite? Maybe you will have external-facing partitions that appeal to condensation.
To keep away from demotivation and pointless delays, and naturally, to make sure everybody’s security, bear in mind to plan and equip your lab in a way compliant together with your institute’s well being and security guidelines.
9. Know When to Take the Lengthy Means Round
We needed to be taught if Andreia had a selected instance of the place a single pattern prep step made the distinction between success and failure. Whereas she didn’t, she was eager to remind us of the larger image.
Purpose to construct a working understanding of how the tools in your lab works and saves you time, and know which steps are price spending the additional time to develop into an skilled.
“…you all the time should be capable to depend on your self. The tools will all the time carry extra benefits and pace and reproducibility—all the things. However by no means jeopardize your individual talent set.”
Whereas it’s exhausting to embrace doing work the good distance round, even the abject failures of experiments contribute to your bedrock of data. Irrespective of how skilled, it is a exhausting lesson all those that observe specialist abilities should be taught, Andreia included.
“…one will develop the abilities by doing it and failing and doing it once more from scratch and failing one other 20 instances if crucial. The skillset will likely be constructed… And once I say failing 20 instances, that is nonetheless not a waste.”
10. Be Conscious of Your Studying Curve
And what recommendation would Andreia give to her youthful self and younger microscopists?
She advises that all of us hold our skilled growth in thoughts and stay conscious of our must be taught.
“In the event you’ve mastered all of the devices round you, are you able to show and share what you realize? Or, do you could look additional afield to maintain up with the newest strategies and analysis? There’s a massive world electron microscopy neighborhood who embrace each educating and studying. Utilise the neighborhood to each train and be taught. You by no means know what the subsequent step is or how your abilities could be useful.”
Andreia talks concerning the totally different philosophies she thought of in her growth:
“Do you wish to be the lab guru, or would you like a brand new problem? Whether or not at your facility or additional afield, the pattern preparation abilities you be taught will likely be important. All the time remember the fact that for high quality imaging, particularly in electron microscopy and on the correlative workflows, it’s essential that you just think about pattern preparation!”
Get One-to-one Microscopy Utility Help
Hopefully, Andreia’s skilled pattern prep recommendation allows you to put together beautiful EM samples that provide the microscopy knowledge you want.
In the event you want one-to-one microscopy utility help, contact Leica immediately and get recommendation from the specialists.
