In biomedical research, success is often determined by details measured in nanometres. The Core Facility at the Institute of Molecular Genetics of the Czech Academy of Sciences (IMG CAS) operates state-of-the-art instrumentation that continues to push the limits of cellular imaging. Through close collaboration with the Czech electron microscopy manufacturer Tescan, the team has successfully performed the first cryo lift-out experiment in the Czech Republic. This breakthrough opens new opportunities for studying cellular ultrastructure in its native context and enables researchers to target specific regions of interest that have previously been difficult to access using high-resolution cryo-electron microscopy. We spoke with Markéta Dalecká, M.Sc., and Dominik Pinkas, Ph.D., about the opportunities and challenges of electron microscopy in the life sciences.
Dominik Pinkas:
We operate exclusively as a contract research facility - our laboratory functions as a shared research infrastructure within the Czech-BioImaging national infrastructure and the Euro-BioImaging European network. The facility consists of four specialists led by Vlada Filimonenko, Ph.D., and is equipped with a comprehensive portfolio of instrumentation centred around three major systems: two transmission electron microscopes (TEMs) and one focused ion beam scanning electron microscope (FIB-SEM).
Markéta Dalecká:
Our primary mission is to support the academic community so that individual research groups do not need to invest in their own microscopy equipment. Many researchers only require occasional imaging, making it impractical to maintain expensive instrumentation. At the same time, we also work with a considerable number of industrial customers who require high-resolution microscopy to validate products and verify whether they meet the claimed specifications.
Dominik Pinkas:
Before acquiring the microscope, our facility did not have a FIB-SEM system at all. The entire story began with the loan of an identical instrument provided by Tescan during which we worked closely with their R&D department.
This collaboration proved to be a genuine win-win arrangement. For our laboratory, it offered a unique opportunity to expand our portfolio of advanced microscopy techniques without major upfront investment. For Tescan, it provided direct access to a real-world service laboratory where developers could observe how their microscope performed in demanding biological applications and how it integrated with upstream and downstream sample preparation workflows.
Throughout this evaluation period, both teams combined their expertise. It went far beyond simply testing a new instrument. From the very beginning, IMG researchers and Tescan jointly developed entirely new methodologies - including a unique cryo lift-out workflow - while validating the first prototypes of cryo nanomanipulators and the integration of fluorescence microscopy.
Markéta Dalecká:
For me, the FIB-SEM is primarily an imaging platform. My main focus is serial FIB tomography. We mill away an extremely thin layer - typically around five nanometres - image the freshly exposed surface, and then repeat the process. By alternating between milling and imaging, we reconstruct the sample volume in three dimensions.
Because Dominik and I use the microscope for different purposes and bring complementary expertise, we are able to exploit virtually the full potential of the instrument.
Dominik Pinkas:
My background is in transmission electron microscopy. For many years, I did not consider the FIB-SEM a microscope in the traditional sense. Instead, it served as an exceptionally precise preparation tool for TEM lamellae. Transmission electron microscopy requires ultrathin specimens that allow electrons to pass through the sample.
Only after Markéta joined the laboratory did we begin to fully exploit the imaging capabilities of the FIB-SEM, which significantly expanded the scope of our research.
Dominik Pinkas:
The gallium ion beam (Ga-FIB) and the integration of a cryogenic nanomanipulator were the decisive factors. Although plasma FIB systems excel at rapidly removing large material volumes, gallium remains unmatched for the preparation of delicate biological nanostructures because it produces a much finer beam profile.
Markéta Dalecká:
Thanks to Tescan's second-generation prototype cryo nanomanipulator, we were able to introduce the first cryo lift-out in the Czech Republic. We can extract a precisely targeted frozen volume from biological material, attach it to a TEM grid by cryogenic redeposition of sputtered copper, and subsequently analyse it in the TEM while preserving the specimen in its fully vitrified native state.
Markéta Dalecká:
One of my favourite projects involved cells infected by bacteria that dramatically remodelled the mitochondrial network. Fluorescence microscopy clearly showed major structural changes, but the resolution was insufficient to understand what was actually happening.
Using FIB-SEM tomography, we reconstructed the entire mitochondrial network in three dimensions. We then correlated these data with fluorescence images acquired from living cells. This revealed that damaged vesicle-like structures - which no longer resembled mitochondria - were in fact remnants of mitochondria. Without correlative imaging, we could never have reached that conclusion with confidence.
Dominik Pinkas:
For me, it was a project involving Caenorhabditis elegans. We needed to isolate a structure only a few hundred nanometres in size within a specific cell nucleus, of which only a handful exist in the entire organism.
Using fluorescence-guided navigation integrated into the microscope software, we successfully located the target nucleus, prepared a cryogenic lamella, and transferred it into the TEM. The entire workflow remained under cryogenic conditions. To our knowledge, nobody had previously prepared and imaged such a specimen with this level of precision.
Markéta Dalecká:
I transitioned from competing systems to Tescan, and I have to say the user interface is excellent. It is simpler, more intuitive, and highly configurable. Every operator can customise the layout according to their workflow. The integrated Help system is also extremely useful.
Dominik Pinkas:
Another major advantage is Tescan's willingness to collaborate directly with users. Unlike large multinational corporations, we have direct communication with the software developers. Automatic functions - such as autofocus - used to present challenges, but after extensive collaboration and testing of development versions, automation has now reached a level where it can be relied upon during routine operation.
Dominik Pinkas:
The biggest challenge is the extreme beam sensitivity of biological material. Every electron used for imaging inevitably damages the specimen. The electron beam breaks chemical bonds, generates local heating, and activates residual gas molecules inside the microscope chamber. These molecules become deposited onto the sample surface, creating contamination exactly where the beam is focused.
Our ultimate goal is therefore to minimise radiation damage while still obtaining meaningful structural information.
Markéta Dalecká:
That is precisely why we avoid exposing the sample whenever possible. The beam remains blanked during navigation and alignment. We only illuminate the specimen briefly to verify our position before immediately blanking it again. Likewise, focusing is performed away from the region of interest because high-energy electrons can permanently damage the sample, especially at high magnifications where the irradiated area becomes clearly visible.
Markéta Dalecká:
Electron microscopy is always a matter of trade-offs. Every advantage comes at a cost. Conventional resin embedding inevitably alters the native ultrastructure through dehydration, but it provides excellent image contrast because heavy metals can be chemically incorporated into the specimen.
Dominik Pinkas:
Cryogenic preparation is considerably faster because the sample is vitrified almost immediately, allowing subsequent processing steps to follow directly. Rapid freezing under high pressure prevents ice crystal formation, producing vitreous ice with essentially the same density as liquid water.
However, cryogenic specimens exhibit much lower intrinsic contrast and are extremely susceptible to thermal damage.
Dominik Pinkas:
Cryogenic transfer is unquestionably the most critical part of the entire workflow. During summer, when laboratory humidity reaches around 58%, cryogenic specimens immediately accumulate frost. Winter conditions are much more favourable because lower humidity greatly reduces ice contamination.
Whenever a cryogenic specimen is exposed to ambient air near the liquid nitrogen workstation, contamination becomes a serious concern.
Fortunately, we are currently in the process of acquiring a fluorescence microscope that will be integrated directly into the electron microscope chamber. This will eliminate the need for this high-risk transfer step altogether.
Markéta Dalecká:
The greatest benefit will be dramatically simplified localisation of regions of interest. Fluorescence-guided navigation, already very well integrated within the Tescan software, will allow us to precisely identify and verify target structures directly inside the microscope.
I strongly believe that core facilities should work closely together. This integrated solution will seamlessly combine fluorescence and electron microscopy into a single workflow without requiring risky specimen transfers between laboratories or even different floors of the building.
Thank you for the interview, and we wish you continued success in your research.
Written by Jana Šilarová
Head of Marketing Department