Amateur microscopy and professional micrometer

[Kinetochore]

Hoping to visualise stomates and chromosomes from some thick leaved plant material. Finally accepted I'll need to section them

Was gifted a working Microm HM355S microtome but a professional colleague in academic histology has emphasised the learning curve will make me very sweary. Unit is also heavy with a large footprint and a steep learning curve ( the micrometer, not the colleague )

It's good to have advance warning about all this, but it begs the question- is this professional micrometer overkill? I'll need to spend about AUD$500 on paraplast-X and disposable blades to even get it up and running

Should I purchase a quality handheld unit and carefully store or sell the giant microtometer-brick?

What are people's experiences with starting out sectioning basic things like leaf tissue?

[Hobbyst46]

Several threads on the forum through the past 3-4 years demonstrate that simple handheld, even DIY-made microtomes can produce great results after some experience.

[Kinetochore]

Several threads on the forum through the past 3-4 years demonstrate that simple handheld, even DIY-made microtomes can produce great results after some experience.

Blergh, sorry of course you're right. Wasn't thinking. Posted a few other questions here today I couldn't find answers to in the search bar, and this question fell out of my head and totally bypassed my brain

Off to the search engine

[Alexander]

"Quality handheld microtomes" are not easy to find. The market is flooded with terrible crap from China and India while the makers of quality ones stopped production decades ago. If you are lucky enough to find some used Jung or Leitz it will sell at several times the money a new one from China would cost.

The blades for a handheld do cost the same as for your big one.

Even after a long long learning curve you will never achieve cuts as thin as a real microtome will give you. Handhelds allow (after the learning curve) sections of about 50 μm while a paraffin embedded sample is easily cut to 10 μm or even less using the big monster. 50 μm are good for colorfully stained and pretty looking sections. Visualizing chromosomes is a much more demanding business. A handheld microtome and a microscope are not enough to achieve that. You will need very specific stuff for success.

[BramHuntingNematodes]

Yeah, using a big competent microtome the 10 μm setting is for course shaping of the block, then you set it to about half of that to cut the ribbon. Thinner than 5 μm is what you might call "ultramicrotomy," but in any case even your regular microtome should do about an order of magnitude thinner (and more frustrating) than the handheld.

The Microm is a relatively recent, full featured set up looks like. I am using a Sorvall JB-4, completely mechanical. One thing is that you may be able to get an adapter that allows you to use feather brand one sided razor blades. These are much easier to deal with than trying to keep a traditional microtome blade in good fettle and I have on some authority are used in working research labs. You replace them frequently and they stay sharp like that.

Glass knives are a giant pain the butt and are best for ultrathin sections.

[Kinetochore]

Wow. Thank you so much

I'd suspected chromosome counting might require finer sections than a handheld could give- this confirms it

Wish I'd known this three years ago- I was working with a plant breeding 'guru' on a major project- that's where my interest in microscopy started- he claimed to know everything about chromosome counting and had produced several unique varieties- and he was aware of none of this. We never got to see chromosomes and he wouldn't even acknowledge his current microscopes were too dirty to use

Lovely bloke, but I started to suspect he was of the generation where all his downstream post-pollination work was set up for him pre-visualisation, and that elves or other clever technical people serviced his departmental's microscopes etc regularly but he was out of that loop. Turns out I was right

One of those science-outputs-as-social-outcome things. Project tanked for other reasons

I really need 5μm sections for 3 μm chromosome visualisation? A friend just found an Olympus CH-2 + fluorescence microscope in one of his auction hauls, we may just move straight to that + DAPI once it's cleaned up and checked. Which is a different learning curve again

Have been warned by people with a few decades of experience the microtome learning curve will make me swear- a lot. Which is really handy to know in advance. I'll prepare my curse words in batches and be prepared for it to be a process

Three years of feeling useless and incompetent has just sloughed off my shoulders after learning all this. There is literally no way I could have known any of the above until I got here

Thank you again

[Chas]

Alexander, I am very curious to know what sort of things can be wrong with the handheld microtomes from China/India ?
(I have only cut a few stems with a hobby knife much like one might cut spring onions .. so 'in my imagination' I was thinking that a chinese microtome might be a step up!).
Thanks.

[Alexander]

Alexander, I am very curious to know what sort of things can be wrong with the handheld microtomes from China/India ?

They are manufactured to very low standards. They are wobbling a lot, no precision at all.

[MicroscopyUniverse]

I use several microtome brands and models.

In my opinion, the microtomes are a similar case than microscopes. If you can find a 2nd hand / used microtome
in good conditions from serious brands (such as Reichert, Leitz, Leica, etc) you will obtain great results and final
learning curve is shorter due to their good precision. Other brands (from China, India, ..) as mentioned in this forum thread that don't have a high level of quality and precision, they will do you no good. Just to frustrate you after spend money.

I have a Reichert microtome with a lot of years (about 25-30) that works very fine.

Concerning blades, if you buy a microtome that does not have a blade included, today is frequent to purchase an adaptor for use disposable blades. This was my case.

[ddy5]

Although it's suggested in some of the comments, maybe it's worth mentioning tissue preparation. Choice of fixative and methods for embedding can have a major impact on your sectioning success. There are many good procedures published, of course, but at least in my experience with a range of animal (mostly invertebrate) tissues, each procedure needs a bit of tweaking for a particular situation.

Once you get over the initial learning hump, getting 5-10 µm sections in paraffin using a professional microtome like the Microm isn't too hard. Working with stained tissue sections can definitely be fascinating and satisfying.

Cheers, David

[Sure Squintsalot]

Although it's suggested in some of the comments, maybe it's worth mentioning tissue preparation. Choice of fixative and methods for embedding can have a major impact on your sectioning success. There are many good procedures published, of course, but at least in my experience with a range of animal (mostly invertebrate) tissues, each procedure needs a bit of tweaking for a particular situation.

Once you get over the initial learning hump, getting 5-10 µm sections in paraffin using a professional microtome like the Microm isn't too hard. Working with stained tissue sections can definitely be fascinating and satisfying.

Cheers, David

It'd be nice to find a guidebook detailing sample prep for amateurs; a guide that includes relatively non-toxic materials and techniques.

[ddy5]

Yes, that would certainly be great. I just did a quick scan on the web and found plenty of protocols for plant tissue. All were geared toward work in a lab, however.

Most fixatives use formalin or paraformaldehyde, but there are alcohol-based options. Working with paraffin requires an organic solvent like xylene - there is no way around that. There is one resin (JB-4) that we tried for cutting 5 µm sections on a rotary microtome with a steel knife. It doesn't require xylene, but the resin components themselves are toxic. People use gelatin for frozen sections, but that's a very different cutting technique requiring different equipment.

In short, I don't personally know of any way to do paraffin sectioning without using chemicals that pose some safety issues.

Cheers, David

[Sure Squintsalot]

Yes, that would certainly be great. I just did a quick scan on the web and found plenty of protocols for plant tissue. All were geared toward work in a lab, however.

Most fixatives use formalin or paraformaldehyde, but there are alcohol-based options. Working with paraffin requires an organic solvent like xylene - there is no way around that. There is one resin (JB-4) that we tried for cutting 5 µm sections on a rotary microtome with a steel knife. It doesn't require xylene, but the resin components themselves are toxic. People use gelatin for frozen sections, but that's a very different cutting technique requiring different equipment.

In short, I don't personally know of any way to do paraffin sectioning without using chemicals that pose some safety issues.

Almost everything found for sale in a regular hardware store can be safely handled with basic safety protocols, common sense, and the safety items for sale in those very stores. I draw the line at boutique solvents, heavy metals in solutions, and those weird gases used by the chip-making industry...that's some scary stuff.

If anyone knows of some impregnation protocols for the advanced amateur for making plant and invertebrate sections, please pass them along here!

[Kinetochore]

Although it's suggested in some of the comments, maybe it's worth mentioning tissue preparation. Choice of fixative and methods for embedding can have a major impact on your sectioning success. There are many good procedures published, of course, but at least in my experience with a range of animal (mostly invertebrate) tissues, each procedure needs a bit of tweaking for a particular situation.

Thank you, yes, staining and embedding definitely a major consideration and another serious learning curve. Good job I was warned it will be frustrating, so I'm ready for that

i'll be mostly sectioning plants for some time and was recommended to use Paraplast x-tra as the embedding compound by a colleague. Had to order a job lot as smaller quantities aren't available retail in Australia- it has still to arrive. Hope it works for the purpose as there is rather a lot of it

I hate xylene. Exposure to it causes serious mood swings- I was exposed to a lot of solvents in another career early in life. If I can find a workaround that avoids any serious solvent exposure even while wearing PPE I shall

Almost cursing the bloke who told me microscopy would be easy, it's led me down a very frustrating and expensive professional rabbithole for 4 years while we pursued some professional goals that- in hindsight- may not be worth the chase. Now I'm here I feel more comfortable- the quality of advice here is vastly superior to anywhere else online- and I have a better idea what's in front of me

There's still something immensely satisfying acquiring an entirely new set of technical skills at my age too