Interference Traction Force Microscopy (iTFM) for Bioimaging of Cellular Forces

The human body consists of over 100,000 billion individual cells, with each cell being on some level an independent entity. Coordination of this hugely complex system relies on an intricate network of signalling, many aspects of which are still poorly understood. While the involvement of chemical signals (e.g. hormones) in this challenging coordination is widely accepted, there is now rapidly increasing interest in how cells interact with each other and with their environment on the mechanical level, for example by pushing and pulling or by sensing the stiffness or mechanical stress in their local environment. This is motivated by strong evidence that mechanical signalling (known as mechanotransduction) plays a critical role in a number of important processes, ranging from controlling how stem cells differentiate in the growing human embryo to affecting the progression of important diseases. However, while mechanotransduction is therefore obviously both fascinating and important for improving human health, our ability to study mechanics on a cellular level remains limited due to a lack of suitable methods to 'image' the forces that cells or components of cells apply. (Several microscopy methods to investigate cell force have been developed but their application requires special expertise that is often not available in a biology lab and even if this expertise is available it remains very challenging to follow the mechanical behaviour of cells over the extended time periods over which many relevant processes in cell biology occur. In addition, existing methods are not sensitive enough to study the activity of weaker cells.)

In this project, light is used as an optical ruler to accurately and robustly image the force pattern applied by cells. Using an effect known as optical interference, the short wavelength of visible light will facilitate an increase in the sensitivity of force imaging by 5 to 10-fold over existing techniques. In addition, light-waves will provide an internal reference for the new method which means that it will be able to track force patterns over long periods of time (up to several weeks) without a need for disruptive reference measurements which many of the existing methods currently require.

There are many areas of cell biology where the new technique may prove to be highly useful. Due to the short-term nature of this project, it will initially be applied to one specific example: a study of the forces involved in the adhesion of cells normally found in the kidney where they are responsible for blood filtration and thus for removal of waste products from the body. These cells are under constant mechanical pressure and their failure is associated with kidney malfunction. Studying the involved forces therefore promises to improve our functional understanding of the kidney and may in the long-run lead to methods for early detection and treatment of kidney failure, a condition often associated with obesity and diabetes.

Cell forces play a critical role in many cellular processes and control the development of multi-cellular organisms. For a number of important diseases, loss or change in the mechanical function of cells has been identified as a causal factor. However, the number of cell culture models and processes for which the role of cell forces has been studied remains limited compared to the vast amount of data available from biochemical assays. This is largely due to a lack of bioimaging tools that offer sufficient throughput, mechanical sensitivity and lateral resolution and that provide the non-invasive, stable and reference-free measurements required for long-term studies. For example, an investigation of cell adhesion forces associated with podocyte damage will require quantitative imaging of cell forces for over three weeks. We propose to address this shortcoming by developing a new cell force imaging technology - interference Traction Force Microscopy (iTFM) - that will utilize in-plane interference in elastic optical gratings to map the traction forces exerted by cells. Due to its interference-based measurement principle, iTFM is expected to achieve 5 to 10-fold higher force sensitivity than the best existing methods (which rely on localization microscopy). In addition, the self-referencing nature of iTFM will obviate the zero-force reference measurements required for most existing methods and will thus provide a step-change in throughput and in the ability to perform long-term studies. We envisage that iTFM will enable a wide range of previously impossible or impractical experiments, ranging from sub-cellular to cell sheet level and spanning diverse cell types and time scales. Here we will validate and introduce iTFM by applying it to the measurement of forces exerted by podocytes. In particular, we will carry out long-term investigations of the forces involved during podocyte differentiation and podocyte damage.

The functional bioimaging technology developed here is relevant for bioscience researchers across different fields of biology and medicine, including in academia, in the pharmaceutical industry (drug screening) and in the healthcare sector (e.g., for pathology, blood work). To maximize the impact of our research, we will follow a combined strategy of (i) academic translation (via broad dissemination, including in high-impact journals, and via academic collaboration with leading cell biology labs world-wide) and (ii) of pursing routes for commercialization of the developed technology (via IP protection and licensing or via a spin-out company).

For most of the omics approaches pursued in research today, commercial assays and screening methods are available. Interestingly, this is not the case for mechanotransduction and specifically not for traction force microscopy. The academic cell biomechanics community has grown rapidly over recent years and has on its own now reached a level where we expect commercialization of a bioimaging tool for traction force mapping to be economically viable. This vision is amplified by the potential to apply mechanical assays in other sectors (pharmaceutical, healthcare). However, existing technology is not particularly amenable to commercialization, mainly because its use requires extensive training and throughput and hence productivity are limited. In addition, for many existing approaches there would not be a clearly defined product. The interference based, self-referenced, highly sensitive yet robust bioimaging approach suggested here, however, has much stronger potential for successful commercialization: iTFM chips are based on storable elastomer materials and could be confected and sold with user-specified stiffness. The hardware required for iTFM readout can be developed as an add-on for existing live cell imaging systems or as a high throughput stand-alone bioimaging station.

To maximize economic and societal impact, we will seek protection of the IP developed in the project. In particular, we expect to protect the iTFM principle itself and several aspects of the process for producing iTFM chips. We will actively approach possible partners for licensing of this IP, through using our existing network and by disseminating our work at meetings with industry participation. Besides licensing, we will also explore commercialization through a university spin-out, which would lead to direct creation of value and jobs.

The interdisciplinary nature of this project and the interdisciplinary environment in St Andrews provide excellent training and will be highly stimulating and inspiring for all involved. This project will have direct impact through the training given to a PDRA. In addition, PhD students working in the investigators' groups will be closely involved in the research in order to maximize their exposure to interdisciplinary research and problem-oriented thinking.