Microscopy plays one of the most important roles in the field of biological study. Some real-world applications range from identifying microorganisms, determining cell structures, to even diagnosing illnesses. The sectioning process, which is responsible for sample preparation, is a vital step in microscopy. Thanks to microtomes, scientists can now create thin and precise sample sections that otherwise, could never have been achieved by hand.
Due to the sheer number of microtome types and variants that are commercially available, serious research must go into the selection process before deciding on a microtome that would best be suited for your laboratory and research applications. Luckily, Laboratory App has done all the work for you and compiled everything you need to know into one efficient and easy-to-read guide. Keep reading and you’ll find several guide questions that will help match you to the best microtome type for your application. You’ll also get a rundown of safety features to consider, find tips on ensuring high-quality sections, locate the best places for microtome maintenance services, and know which suppliers to trust.
1. Determine The Application Requirements For Your Microtomes
Determining the microtome type that best fits your facility is highly dependent on what samples are being analyzed and which applications your samples will be used for. Here are some guide questions to help identify which Microtome type your laboratory needs:
1. What type of material are you cutting?
To slice materials like hard, fragile, or fatty tissue types embedded in resin or paraffin for light microscopy, consider purchasing a more general-use type of microtome, such as the Rotary Microtome. This machine is designed with a heavy build for sturdiness, uses a handwheel for cutting samples, and features an adjustable cutting angle to control the knife. Larger blocks of tissue can be cut on this microtome. Serial sections, which are commonly used for biopsies, can also be achieved on this machine.
On the other hand, several microtome types have been engineered to cut specific kinds of samples. Take a rocking microtome which was specially designed to slice paraffin-embedded biological materials like plant or animal tissue in small batches or blocks. The rocking microtome is the oldest type of microtome still in use today. Unlike a rotary microtome, it features a lightweight, simple design and is quite affordable. However, due to its lightweight structure, rocking microtomes produce vibrations that prevent users from producing very thin sections. Serial tissue sectioning can still be achieved on this machine, however.
When cutting larger biological samples like whole brains or harder materials like teeth and bone, one popular microtome of choice would be the Base Sledge Microtome, which holds specimens on a heavy base that runs back and forth against a fixed horizontal knife. Sled microtomes, as they are also sometimes called, are compatible with celloidin-embedded blocks and paraffin-embedded samples. These machines are especially useful in the fields of neuropathology and ophthalmic pathology.
Another microtome that can cut through celloidin-embedded blocks of tissue would be the standard sliding microtome. While it was designed mainly for this type of embedding material, sliding microtomes can actually also be used for paraffin-embedded samples. Most operators will use sliding and sledge microtomes interchangeably, but the main difference between the two is that sliding microtomes have a moving knife with adjustable holding clamps while sledge microtomes have a moving base.
When preparing frozen sections, be sure to use a freezing microtome or cryomicrotome to get the most efficient and best quality results. While other microtomes can be modified to slice frozen specimens, cryomicrotome are better suited for the job because they delay the thawing of samples by controlling the flow of CO2 on the knife’s edge. Freezing microtomes are capable of sectioning samples whether or not they have been fixed with preliminary embedding.
Vibrating microtomes are unique in that they are designed to cut tissue that has not been fixed, processed, or frozen. Because of this, they are widely used in enzyme histochemistry and ultrastructural histochemistry. Vibrating microtomes get their name from the vibrating razor blade that it uses to cut samples at a very slow speed while the materials are immersed in water, saline, or fixative to prevent disintegration during sectioning.
To prepare harder and more brittle specimens like decalcified bone, teeth, glass, and ceramics, use a saw microtome. Saw microtomes cut sections that are commonly embedded in resins and work by slowly moving samples along a diamond-coated saw rotating at about 600rpm.
Another machine commonly used for preparing biological tissues and other samples is the infrared laser microtome. Infrared laser microtomes are capable of cutting samples in their native state without contact, thermal damage, or the need for preparation techniques such as freezing, dehydration, or embedding. While laser microtomes allow for more precise cutting control, take note that they may be too slow for pathology applications.
2. What is your required micron range for sectioning
Sample thickness is crucial in microscopy quantification. If a sample too thick, the details may not be as clear. On the the other hand, if the sample is too thin, it might fall apart. That is why it is important to select a microtome that can provide you with the appropriate sectioning range for your application.
Routine tissue normally calls for a cut of 3-5 µm in thickness while biopsy tissue is usually cut at 2-3 µm. In these cases, rocking microtomes, rotary microtomes, and sledge microtomes can do the job for you, depending on your preference. Rocking microtomes can slice samples as thick as 2µm to 24µm in steps of 2 microns. Both rotary and sledge microtomes can cut sections up to 60 μm in thickness. However, rotary microtomes can slice samples as thin as 0.5µm in manual operations. Meanwhile, sledge microtomes can only cut sections as thin as 1µm.
In cases where brain and central nervous system (CNS) tissue or tissue for amyloid diagnosis is being studied, the lineup for appropriate microtomes changes slightly. Because these tissues are required to be 8-10 µm thick, you will need to use either a rocking microtome, rotary microtome, sledge microtome, laser microtome, freezing microtome, or vibrating microtome. The first three have already been discussed, so let’s proceed with the latter three.
An infrared laser microtome can achieve 5 to 100 μm slices, but this will depend on the material being cut. Freezing microtomes are commonly built with a micron range of 5 to 40μm and can produce precise cuts in graduations of 5µm instead of 1µm, partly because of the sample’s frozen state.
The cut thickness for vibrating microtomes may vary according to sample type. Fixed samples can be cut as thin as 10µm and above while fresh samples can be cut as thin as 30µm and above.
For other applications that require thicker cut samples, you may also use rocking microtomes, rotary microtomes, sledge microtomes, infrared laser microtomes, freezing microtomes and vibrating microtomes. In addition, saw microtomes can slice materials into 20µm sections or higher.
At the other end of the spectrum, applications that require extremely thin slices like serial block-face scanning electron microscopy (SBFSEM), light optical microscopy, and transmission electron microscopy (TEM) will need a special kind of microtome, known as an ultra microtome. Ultra microtomes slice tissue or industrial materials embedded in hard resin and can produce slices as in as 0.005 to 1 µm. TEM usually requires cuts of 0.04 to 0.1µm while SBFSEM requires 0.03 to 0.05µm slices.
3. Do you prefer a rotary or a motorized model?
Laboratories that perform sectioning procedures utilize either manual, semi-automated, or fully automated microtomes to section tissue and other samples.
Manual microtomes consist of two handwheels: one to move the specimen feed or block and the other to control the block movement and create sections. This microtome type gives users the most control and is preferred over the other two microtome types when dealing with more sensitive tissues like small biopsies. Among the three types, manual microtomes tend to cost the least.
Semi-automated microtomes feature one motorized handwheel that maneuvers the specimen feed through digital controls in the form of either a hand pad or a mounted display. Users still manually operate the cutting strokes of the machine.
Fully automated microtomes replace both handwheels on a manual microtome with motorized functionalities so users can not only control the advancement of the feed but adjust the speed of the sectioning process as well. Because both handwheels are motorized, automated microtomes do tend to be priced more than the first two types. However, the cost may be justified by a lab’s return on investment as automated microtomes produce the most consistent slides at a faster, uniform speed which reduces the need for recuts.
In addition, automated microtomes reduce the risks of repetitive motion injuries (RMIs) brought about by the constant manipulation of the handwheels on manual microtomes. For those who prefer more personal control of the slices however, a semi-automatic microtome should still help reduce the chances of RMI.
4. What’s your estimated budget?
Prices for microtomes are different for manual, semi-automatic, or fully automatic machines. Motorized microtomes are priced higher than the manual ones. Furthermore, price ranges for each microtome type also vary from one supplier to another.
Spend some time comparing prices amongst distributors to get an average ballpark price. Once you have that, go over your lab’s budget and decide just how much you are willing to spend on your new microtome purchase. Spending on a brand new microtome should be no problem for those with ample funding. However, considering the additional costs that are needed for new knives, blades, and accessories, those with tighter budgets might want choose alternative options.
Used and fully recertified microtomes are excellent choices for lab upgrades that still help you achieve high-quality slices but are lighter on the pocket. However, be careful choosing a used equipment distributor to buy from. Some companies might only be offering you scratch, dent, or cosmetic-level refurbished items without actually updating the insides of the machine.
If you already own a microtome but would like to upgrade to a better one, you could sell your current model to gain extra cash for a brand new microtome. Alternatively, try asking your supplier about any trade-up programs they might have that allow you to exchange your current microtome for the model you want at a discounted price.
If you’re still on the fence about a particular microtome, try looking for a rental that you can use to help you gauge its performance before committing to a purchase.
2.Fixed Knives vs. Disposable Blades
Once you have decided on your microtome type, the next step would be choosing a cutting tool to use. The choice of microtome knife makes a huge impact on productivity and sample quality. Microtome knives were designed for specific microtome types and were made with different materials to withstand several degrees of tissue and embedding media hardness. For example, paraffin-wax embedded tissues are meant to be cut by steel knives, while resin-embedded tissues are best cut with glass knives.
Fixed knives come in steel, non-corrosive metal, tungsten carbide, glass, diamond, and sapphire and cost less in the long run compared to the total costs of disposable blades. They are best suited for harder materials and especially bone. The edges on these knives must be maintained through frequent sharpening, honing, and stropping.
Disposable blades on the other hand are made of steel whose edges are either left uncoated or covered with either polymer, titanium nitride (TiN), teflon®, or ceramic for better lubrication and preserved sharpness. Disposable blades make it easier to produce thin and good quality sections, but they don’t perform as well as fixed knives when it comes to sectioning harder tissues. Disposable blades do not need any form of maintenance, but they do cost more than fixed knives.
3. Understanding Knife Profiles
Apart from the knife material, users must also ensure that they select the right knife profile to cut their samples with. Knives are classified according to how they look when viewed at a profile. Here is a breakdown of all 4 of these classifications and recommendations for usage:
- Bioconcave - Features two hollow ground surfaces and is extremely sharp. It is often used with rocking microtomes to cut celloidin embedded materials or foam compounds.
- Plano Concave - Used to cut nitrocellulose and paraffin-embedded tissues and comes in varying degrees of concavity.
- Wedge - Originally designed for slicing frozen sections thanks to its added rigidity, but can be used for cutting all kinds of sections for any microtome.
- Plane/Tool Edge Shaped - Used mainly to cut hard tissue like undecalcified bone and other samples embedded in synthetic resin and paraffin.
4. Safety Features and Tips
To reduce the risk of injury within your lab, make sure that your microtome has several safety features in place like multiple safety locks on handwheels and a blade guard to cover the edges when not in use.
Other features to look out for would be an emergency stop button, an object head balance that can be adjusted according to object clamp size and weight to prevent dropping onto the knife, and enough space for insertion or removal of the blade from its holder.
For motorized rotary microtomes, check to see if the handwheel handle can be centered to reduce the chances of injury while using the motorized mode.
For manual rotary microtomes, be sure to bring the coarse feed wheel as close to you as possible for ergonomic comfort over a longer period of time.
5. Microtome Warranties and Aftercare Services
When comparing microtome equipment suppliers, always ask if there are qualified, manufacturer-compliant engineers around to repair the microtome when the need arises. Also ensure that the microtome you are purchasing comes with a fair warranty period or multi-year service agreement options so you don’t have to keep worrying about time-consuming product returns.
Lastly, ensure that your microtome provider also supplies the appropriate parts in case you need to have it serviced back to factory settings. Many third-party equipment repair service providers have limited availability to parts, which may cause downtime and affect your laboratory’s overall productivity.