Unpacking the Daunting Task of Stereology for Electron Microscopy
Electron microscopy provides fantastic detail and resolution, with brain electron microscopy allowing visualisation of neurons and their individual synaptic connections. You may find yourself needing to count these neurons or connections, which can easily go into the billions. But counting these one-by-one isn’t really feasible. This article will introduce you to stereology as a counting tool for electron microscopy, and provides tips for optimizing your approach to get the best results possible, all while using your time efficiently.
If you can’t count the total number of objects, the next approach might be to determine the density of a small sample, then multiply by the volume of the entire region. However, this approach introduces bias based on object size, shape, and distribution. Enter design-based stereology, a labor-intensive but unbiased technique for quantification. You can use stereology to measure length and size of regions or objects, but this article will focus on stereology as a counting tool, particularly for transmission electron microscopy. Check out a previous article for useful techniques for scanning electron microscopy.
The Physical Dissector
Stereology uses a dissector, which is a counting frame to study a volume of tissue. Stereology with light microscopy can be performed using an optical disector. This uses different focal planes through a depth of tissue for counting objects. With electron microscopy however, it is only possible to view one plane at a time. This means that multiple sections of tissue are needed to view multiple planes. In electron microscopy, each section is referred to as a physical disector.
Start With a Pilot Project
The number of disectors you need will depend on what you are photographing. Larger objects (e.g. cells that are few and far between) will require more sections to obtain high enough counts for analysis. With smaller objects (e.g. mitochondria) your counts will rise more quickly, thus fewer sections will generally be sufficient. Regardless, it is best to spread out your sampling across the region of interest. Determine how many disectors will be necessary by starting out with a pilot project.
Collecting serial ultrathin sections requires an incredibly skilled hand. You can check out one of our earlier articles for expert sectioning tips. Sections for stereology need not only to be thin for viewing in an electron microscope, but should also be thinner than the approximate diameter of your objects; otherwise you might miss some objects between sections. Because each section is a disector, it is imperative that you keep the sections in order and that none are missing. So, before you jump on an ultramicrotome with a precious sample, practice your sectioning technique! Producing flat, intact sections oriented the same way will save loads of time and frustration for the task ahead.
Once the sections are cut, they are placed on tiny discs with a transparent membrane called grids that will go into the microscope for viewing. At this point, it can be hard to orientate the sections. Cut your block of tissue in an unsymmetrical shape, such as a trapezoid, or notch one corner. This will help you to orientate the sample properly, and will make it easier to verify that all sections are aligned in the same way.
Selecting an Area
Systematic and random sampling is critical to achieving unbiased counts. Choose both the starting point for sectioning within the tissue block and the starting location for counting within the section randomly. Ideally, you will count in an area that is photographable in all of the disectors. It can save time in the long run to identify this area in all of your serial sections before starting to photograph. You would hate to take the first two photos only to find that the next three sections have gaping holes or folds obstructing your area of interest!
Finding Your Target
Randomly searching through a section can be like trying to find Waldo in a sea of lookalikes. Develop a system to navigate each section and locate your area of interest. If your microscope has a tracing function, try outlining each section to determine your location within the section. It can also be helpful to identify large and noteworthy landmarks nearby – an unusually shaped cell nearby, or a particularly large blood vessel – to draw you into the region. The more you do this, the faster you will become at navigating through a section of tissue at 50,000 times magnification and pinpointing your target.
A lot goes into designing an experiment using unbiased stereology and you will need to set time aside to plan things out. Luckily, there are great comprehensive stereology resources available to walk you through everything you might need to know. Stereological counting can be a painfully time-intensive task but produces robust and reliable electron microscopy data. Happy counting!