Large volume EM imaging technologies share many pipeline components, yet different labs use seemingly different approaches for the same tasks. Its all good: here’s why.
Large volume EM imaging technologies share many pipeline components, whether they are VolumeScopes, 3View installations, FIB-SEM systems or serial sectioning SEM/TEM. For me, that was a key conclusion from the recent workshop in Cleveland, Ohio, for (prospective) serial blockface imaging (SBFI) users and system managers. Nevertheless, it is clear that different labs do use different approaches for the same tasks – e.g. sample preparation. This can lead to a certain amount of confusion among both new and experienced users.
To a large extent, sample prep workflows reflect the resources that different SBFI users have on hand. EM cores have a lot of hardware available and skilled personnel to wield it. By applying standardized protocols to each sample, they can minimize wasted SBFI scope time.
For new adopters in non-EM labs, however, a huge benefit of SBFI systems is that the cutting tool is embedded in the SEM chamber, and computers control the tedious cutting process. Using only superglue, razorblades and some silver paint, samples can be trimmed, faced off and imaging started in a couple of hours.
Which route you take does make a difference, sometimes. Precision preparative ultramicrotomy allows development of specific protocols built around trimming tolerances, and permits quality control validation by TEM plus identification of target areas within a sample prior to running a long SBFI acquisition job. For finicky correlative LM-EM studies, machine-trimming and sampling by LM or TEM may save a lot of time and heartache. For easily-charged samples, such as cell monolayers, machine-trimming them small and flat and sputter coating may make the difference between routine imaging and hard work.
On the other hand, when there is ample, well-stained tissue, there is probably little difference in image quality obtained, regardless of the approach during mounting. Moreover, reducing the time taken during prep benefits quantitative SBFI applications, allowing more samples to be run in a shorter time. Another practical limit in new-to-EM labs is the added expense of external ultramicrotomes and sputter coaters. Buying them – and learning how to use them – may simply make the threshold for adoption too high. For most of those folk, steady hands, and collaborative interactions with core EM labs for tricky samples, may see them highly productive.
While the focus here is on sample prep, the same perspective will help you interpret many aspects of large volume imaging practice. EM-trained people may make use of a wide selection of approaches. New-to-EM users will be productive using simple approaches. Smart people will probably take the fastest route to answer their research questions convincingly, be it discovery of new cell mechanisms or testing of potential therapeutics.
(Image:G.Kidd.Chatting on Philadelphia sidewalk)