In the last two decades, cryomicroscopy methods have become widespread and effective tools used by life scientists, pharmaceutical researchers, and more, to examine biological structures close to their native state.1 The capabilities of cryomicroscopy to visualize the structure of biological molecules, such as proteins and protein complexes, make a valuable addition to existing methods such as X-ray crystallography and nuclear magnetic resonance (NMR). Determining the structure of proteins and protein complexes, which are of particular interest as drug targets and are the subject of investigation to gain a deeper understanding of disease mechanisms, is an important element of drug discovery.
In this article, we will take a look at the use of cryomicroscopy techniques, including cryo-correlative light and electron microscopy (cryo-CLEM), freeze-drying microscopy (FDM), and cryopreservation in pharma research, and how temperature-controlled microscopy stages are enabling researchers to advance drug discovery and development studies at cryogenic temperatures.
Cryo-CLEM
Electron microscopy (EM) enables molecules to be studied in a variety of different functional states, using tiny amounts of material, at near atomic resolution. Cryo-EM uses extremely low, cryogenic temperatures to overcome the challenge of measuring biological specimens with high water content using electron beams in vacuum conditions.
Before cryo-EM became commercially available in the 1980s, biological specimens were prepared for electron microscopy by methods such as chemical fixation or staining, but these procedures suffer from preservation artifacts that compromise the image resolution. Rapid freezing is often used to maintain the sample in a frozen state that resembles the natural physiological environment, which is particularly important in pharmaceutical research, where results achieved during the preclinical phase must be replicable in clinical studies.
Cryo-CLEM brings the advantages of low temperature fluorescence together with cryo-EM, to increase sensitivity for the detection of biological, chemical, and genetic processes inside living cells. Cryo-CLEM enables direct fluorescent labelling and targeting of molecules or molecular assemblies (such as intracellular membranes, DNA or cyto-structural elements) in cryo-immobilized samples, pinpointing regions for subsequent high-resolution imaging using EM.
In order to make a biological sample compatible with the vacuum conditions found in EM and preserve the structural detail, samples are embedded in vitrified “glass like” ice and need to be kept below -140°C. Any contact with moisture contained in the air has to be avoided since ice crystals would form immediately and contaminate the sample. Under cryo-conditions, the fluorescence signals' structural detail is preserved and photobleaching is significantly reduced.
Advances in cryo-CLEM technology include innovative cryo-fluorescence stages, such as the Linkam CMS196, which enables the automated acquisition of high-resolution fluorescence maps of a whole EM grid. This is then used to navigate the sample and correlate with EM in the case of cryo-CLEM, or with other techniques such as X-ray microscopy.
Using fluorescence microscopy, transmission electron microscopy (TEM), and cryo-soft X-ray tomography (cryo-SXT), a group of researchers and clinicians in Barcelona, Spain, could observe the effectiveness of the anti-cancer drug cisplatin at extremely low concentrations, to establish the lowest possible dose needed to produce an effect in order to minimize toxicity.2 The group imaged cryogenically frozen cell samples on a fluorescence microscope, keeping them vitrified at liquid nitrogen temperatures using a CMS196 cryo-fluorescence stage. The samples were then analysed using cryo-SXT, which enabled 3D investigation on a nanometric scale.
Thanks to the cryogenic imaging techniques available, results showed that tricine – one of two adjuvants studied – facilitated effective therapeutic use of cisplatin at lower doses than previously used, possibly paving the way for the development of chemotherapy treatments with reduced side effects for patients.
Freeze-drying Microscopy
Figure 1: Instrument set-up at the NIBSC lab. Linkam FDCS196 Freeze-Drying Cryo Stage, T94 Controller and Liquid Nitrogen Pump, Vacuum Pump, and Olympus BX51 Optical Microscope. Image shows an older version of the FDM system.
Many drugs are produced as lyophilized – or freeze-dried – formulations, in order to increase stability and extend shelf-life. Drug developers must create an optimized freeze-drying process for new pharmaceutical compounds, which can be a complex and expensive endeavour. It is important to understand the temperature and pressure requirements of each of the three main freeze-drying steps in order to streamline the process and develop more efficient freeze-drying cycles. Using freeze drying microscopy (FDM), researchers can directly visualize each step and establish how the drug product will behave under different thermal conditions.
FDM involves a purpose-built light microscope combined with a dedicated thermal stage, which accurately controls the sample temperature and pressure, and allows thermal measurements to be made in real-time. One critical parameter of freeze-drying is the collapse temperature (Tc) – the temperature at which the product loses its structural integrity and results in processing defects – and FDM allows drug developers to closely monitor samples and adjust the freeze-drying protocol quickly and efficiently.
One research group at the National Institute for Biological Standards and Control (NIBSC), UK, is working to understand the complexities of freeze-drying pharmaceuticals using advanced FDM techniques. The group, led by Dr. Paul Matejtschuk, is focusing on optimizing the formulations of freeze-dried liposomal drugs, which pose developmental challenges due to their physical and chemical instability. By using a specialised cryo-stage (FDCS196, Linkam Scientific Instruments) mounted on an optical microscope (Figure 1), Matejtschuk and his team can predict the ideal freeze-drying conditions for liposome-cryoprotectant mixtures, by estimating the freezing, collapse, and melt temperatures.3
Figure 2: Latest version of the FDM system.
Experiments such as this are vital in the continued effort to develop rapid, transferable, and scalable freeze-drying methods to stabilize pharmaceutical compounds like liposomes.
Cryopreservation
Storing biological specimens for research relies on effective preservation techniques, to retain the cell’s physical and biological integrity. Chilling or freezing samples can lead to the possible build-up of ice crystals, leading to terminal cell damage. Cryoprotectants are important substances that prevent cell damage during freezing by lowering the melting point of water. Many organisms such as polar insects, fish, and amphibians produce their own cryoprotectants, or antifreeze compounds, that scientists are studying in order to develop new cryoprotectants for preserving cells for research.
For example, researchers at the University of Warwick, UK, led by Dr. Matthew Gibson, are investigating antifreeze (glyco) proteins (AFP) with the aim to develop novel synthetic AFP mimic compounds. The lab uses a cryobiology stage (BCS196, Linkam Scientific Instruments) to measure ice crystal growth in cells, relying on the stage’s temperature control capabilities while observing AFPs. Gibson investigated the use of gold nanoparticles as probes for measuring ice recrystallisation inhibition activity, using the cryobiology stage to alter the temperature and develop a high-throughput method to screen materials for structural features that behave like AFPs.4
Discoveries such as this hold the potential for the development of novel cryoprotectants that can prevent ice growth in cryopreserved cells, maintaining their integrity and therefore their potential use in biomedical and pharmaceutical research.
Future Pharma Research
The techniques described in this article highlight a selection of the many cryomicroscopy methods available that are helping to advance pharmaceutical research. Cryo-CLEM brings together the power of cryo-EM and cryo-fluorescence and as a relatively new technique, its success relies on developments in specialized cryo-stages that facilitate the cryo-CLEM workflow. Such stages are capable of maintaining vitrified samples at liquid nitrogen temperatures, keeping them contamination-free as they are moved from the fluorescence microscope for cryo-EM imaging. Other dedicated cryo-stages are available that are compatible with a wide range of microscopy techniques, such as FDM, which provide precise control over the temperature of samples during imaging, down to -196°C. These innovations provide pharma researchers with the tools needed for a vast number of applications, from evaluating new therapies and production processes, to preserving biological samples for future research.
References
- Booy, F. and Orlova, E.V. Cryomicroscopy, in: Chemical Biology: Applications and Techniques (eds Larijani, B., Rosser, C.A., and Woscholski, R.) 2007.
- Gil, S., Solano, E., Martinez-Trucharte, F., et al. Multiparametric analysis of the
- effectiveness of cisplatin on cutaneous squamous carcinoma cells using two different types of adjuvants. PLoS ONE. 2020; 15(3): e0230022.
- Hussain M.T., Forbes N., Perrie Y., Malik K.P., Duru C. and Matejtschuk P. Freeze-drying cycle optimization for the rapid preservation of protein-loaded liposomal formulations. International Journal of Pharmaceutics 573, 2020; 118722.
- Mitchell, D. E., Congdon, T., Rodger, A., and Gibson, M. I. Gold Nanoparticle Aggregation as a Probe of Antifreeze (Glyco) Protein-Inspired Ice Recrystallization Inhibition and Identification of New IRI Active Macromolecules. Scientific Reports, 2015; 5: 15716.