Acquiring images and simultaneously recording relevant sensor parameters from the sample is a powerful way to understand how materials alter under changing conditions such as temperature, humidity, tensile forces, shear stress, corrosive environments, or aging. This often requires an integrated system capable of recording and imaging dynamic processes that occur during an experiment.
Many materials change color, shape, and size with changing conditions. For example, ferroelectric materials realign, and thermotropic liquid crystals undergo phase transitions and color changes as temperature changes; polymeric films can tear when under tensile strain, and many materials change color as they oxidize.
Optical microscopes can visualize these changes, but microscopists increasingly need to capture and correlate image data with stage sensor parameters to quantify a transformation or gain additional information about visual differences. Events such as a color shift can be analyzed, the extent of a tear can be measured, and the size and shape of particles can be quantified.
Assembling a dynamic data and image capture system
The basics of an imaging setup are straightforward. Systems are comprised of the microscope, relevant sample stage (to accurately control temperature, environmental conditions, or tensile/shear forces), CCD or CMOS camera of appropriate performance, and suitable software to acquire and analyze the images and data collected.
Recent advances, such as scientific CMOS (sCMOS) cameras, focus on offering high sensitivity and speed, which is ideal for live cell fluorescence imaging. However, they are not necessarily the best option for applications where dynamic samples evolve on the microscale, especially with changing environmental conditions such as temperature and humidity.
Consideration should also be given to microscope lenses and their correction for imaging through optical windows. For higher numerical aperture (NA) lenses, typically for 20x magnification and above, lenses with correction rings are preferred and allow matching the optical settings to the window thickness. This reduces spherical aberration and improves image quality.
Selecting a camera with the appropriate performance for microscopy can suffer from the same biases as consumer cameras. More is often considered better – particularly in sensor size and pixel count – without due regard to specific needs. For example, balancing the improved spatial resolution of smaller pixels with the resulting limit on the dynamic range of the sensor.
Camera resolution is a key parameter to consider in an imaging system, but it is only one of several parameters. Pixel size and pitch of the camera should be matched to the resolution of the microscope, which is governed by the NA of the optical system. Due to the optical limitations of the microscope, increasing the number of pixels or decreasing their size does not necessarily provide higher resolution.
The overall dynamic range, i.e., the range of brightness levels between the darkest and brightest areas, dark noise, and temporal noise are also key considerations. Dynamic range and signal-to-noise can be affected by pixel size, and this is especially important in applications where observations of small changes in color are often a key measurement parameter.
Frame rate determines the smoothness of operation in live-view imaging. While higher frame rates produce smoother transitions, matching frame rate to the demands of the intended application is important. For example, dimensionally larger images typically result in slower frame rates, while high frame rates can quickly produce extremely large and unwieldly image data sets.
Imaging in practice
With an optimized instrument setup in place, there are many applications where dynamic imaging can add insight. Two recent examples are outlined below:
1. Freeze drying pharmaceuticals
Products that are prone to degradation must be stabilized by immobilizing or reducing water content. Freeze drying (lyophilization) removes most of the water in a sample, providing a dry, active, shelf-stable, and readily soluble product. However, freeze drying is a complex process, so pharma and biotech companies can decrease costs by optimizing protocols to speed up timelines and increase product yields.
Freeze-drying microscopy (FDM) combines light microscopy techniques with a thermal stage, and has become a widely-used method to determine how a drug product will react to different thermal and pressure conditions.
Researchers at the UK’s National Institute for Biological Standards and Control (NIBSC), led by Paul Matejtschuk, PhD, are using the latest FDM technology to investigate the development of formulation and freeze-drying processes on protein therapeutics. The group used FDM to predict the ideal freeze-drying conditions for liposome-cryoprotectant mixtures, using a cryostage mounted on an optical microscope, connected to a control unit and liquid nitrogen pump. Images were taken every 20 seconds for the duration of the experiment and physical changes to the liposomal formulations could be observed (Figure 1).
Results showed that the presence of protein adds stability to neutral and charged formulations, with the same amount of Ovalbumin (OVA) retained after freeze drying. The minimal leakage of the OVA suggests that pre-cooling the shelf and rapid freezing could prevent egress resulting from the formation of large crystals. Liposomal size also changed upon rehydration, with cationic liposomes showing the greatest increase. The study demonstrated the ability to freeze-dry liposomal formulations in microplates and vials for the rapid screening, preservation, and optimization of liposomal formulations.
2. Analyzing the mechanical properties of meat alternatives
Reading Scientific Services Ltd (RSSL) was tasked by a meat alternative brand to compare properties of chicken with chicken alternatives. Scientists at RSSL used tensile testing to relate these properties to sensory profiles, including taste, smell, and texture to discover which properties most impacted the eating experience.
Extension experiments were performed using a mechanical testing device placed under an optical zoom microscope. Key parameters calculated were extensivity (how far the sample stretches before failing), and failure mode—i.e., clean break or fibrillar staggered breakage.
Chicken breast and three plant-based chicken products were tested. Extension was performed at a speed of 200µm/s. The extension tests continued until the samples visibly failed and the force dropped to zero.
The images showed that under tensile testing, all samples failed in a similar way with fibers breaking at the point of failure. It was interesting to note that only the Soy-Protein-S2 had similar fine fibers to chicken [Figure 2], while the other plant-based samples did not.
Understanding the physical and microstructural properties of food is vital in product development. This experiment and data could help product formulators to develop meat-free alternatives based on different ingredients, that mimic the eating experience of real chicken and potentially other meats.
Dynamic imaging offers microscopists a powerful technique to enhance their insight into the materials they are studying. Important changes can be visualized and correlated with information from multiple sensors, and even quantified. System selection and optimization is a multi-faceted process, where traditional optical parameters must be considered together with detailed specifications of cameras and software. With an optimized system, dynamic imaging is a powerful approach that can add value to many fields of research.
1. Hussain MT, Forbes N, Perrie Y, Malik KP, Duru C, Matejtschuk P. (2020) Freeze-drying cycle optimization for the rapid preservation of protein-loaded liposomal formulations. International Journal of Pharmaceutics 573 (2020)118722. https://doi.org/10.1016/j.ijpharm.2019.118722