Periodic Reporting for period 1 - CELL-FORCE (Validation of a novel device for real-time, long-term measurement of cellular forces)
Periodo di rendicontazione: 2024-01-01 al 2025-06-30
The conventional thinking in cell biology, which often assumes that cells communicate mostly via bio-chemical signalling, has recently been challenged with several examples where mechanical rather than chemical cues play an important role in development, physiology and disease. Biological cells continually exert forces on their environment, which can vary substantially in magnitude, spatial distribution and temporal evolution. These forces are key to many processes including cell growth, tissue formation, wound healing and the invasion of cancer cells into healthy tissue. Understanding how cellular forces affect the micro-environment hinges on our ability to image them with sufficient local and temporal resolution (e.g. continuously over several days with subcellular spatial resolution), adequate field of view (e.g. to study cell sheets) and relevant sensitivity (typical forces are in the pico to nano Newton range). Despite significant advances made in this area of functional bioimaging over the last years, existing methods still struggle to meet the requirements, thus precluding new commercial opportunities in cell biomechanics.
The CELL-FORCE project worked to demonstrate Elastic Resonator Interference Stress Microscopy (ERISM) as a new microscopy method that allows direct, robust and non-destructive imaging of forces associated with various mechanical cell-substrate interactions, and validate its commercial feasibility. The greatly increased sensitivity offered by ERISM over other methods was used to allow accurate measurements of vertical forces and of cells exerting only weak force. Moreover, with a low light intensity requirement and no need to detach cells after a measurement, using ERISM makes it possible to take long-term measurements of multiple cells without photodamage and facilitates downstream applications such as immunostaining.
The CELL-FORCE project worked to demonstrate Elastic Resonator Interference Stress Microscopy (ERISM) as a new microscopy method that allows direct, robust and non-destructive imaging of forces associated with various mechanical cell-substrate interactions, and validate its commercial feasibility. The greatly increased sensitivity offered by ERISM over other methods was used to allow accurate measurements of vertical forces and of cells exerting only weak force. Moreover, with a low light intensity requirement and no need to detach cells after a measurement, using ERISM makes it possible to take long-term measurements of multiple cells without photodamage and facilitates downstream applications such as immunostaining.
The main goal of CELL-FORCE was to take the Technology Readiness Level (TRL) of the ERISM slides and the protocol for the measurement of cellular forces from TRL 2/3 to 4. To achieve this, the project followed two scientific / technical streams of work, namely (1) optimization and validation of measurement slides and (2) Improving and miniaturising the optical design of the read-out system. A main achievement in these activities was to replace the stack forming the ERISM slide, which originally consisted of an elastomer layer and top gold film, by a single layer of a high refractive index elastomer. In this way, interference is generated through Fresnel reflection at the elastomer-cell interface rather than via reflection at an elastomer-metal interface. While this design was found to show a decreased interference contrast, which required adapting the measurement conditions and to some extent data analysis pipeline, it also greatly reduced the complexity of the slide architecture. As a further important achievement, the ability to record fluorescence images of the sample in parallel to ERISM measurements was fully integrated, both in hardware and software, addressing an important need of many potential end-users of the technology.
The main technical achievements described above represent a significant advance beyond the state of the art. For instance, an immediate impact of these achievements was that it became possible to monitor the mechancial impact of kidney malfunction in an advanced cell culture model, monitoring in parallel and for the exact same cells, the change in protein expression pattern through immunostaining and potentially in the future through genetically encoded fluorescent reporters. While this and other achievements during the project represent important steps, it also became clear for example that further research will be required in order to lower the cost of hardware for ERISM measurements so that it can be used more widely.