MIT engineers have used fluorescent labels that toggle on and off to examine the interactions of molecules within a cell and their impact on cellular behaviour.
Living cells constantly receive various molecular signals that affect their behaviour, and the ability to measure these signals and understand how cells respond through downstream molecular signalling networks is crucial for scientific understanding. This includes insights into cellular ageing and disease processes. However, comprehensive studies in this area are currently hindered by the limitations of existing cell imaging techniques, which can only simultaneously analyse a few different types of molecules within a cell.
MIT researchers have developed an alternative method that allows them to observe up to seven different molecules at a time, potentially even more. The new approach uses green or red fluorescent molecules that blink at different rates, allowing for tracking target protein levels in a cell over time through image capture and computational analysis of the fluorescent signals.
The team identified and engineered several green and red switchable fluorophores, each blinking at a different rate, to label cellular molecules like enzymes and signalling proteins. By imaging the cell over extended periods and applying a computational algorithm akin to how the human ear discerns sound frequencies, they employed linear unmixing to isolate each fluorophore’s signal. This technique, similar to a Fourier transform in auditory analysis, allowed them to observe the location and timing of each fluorescently labelled molecule within the cell throughout the imaging. Remarkably, this advanced imaging can be performed with a standard light microscope, requiring no special equipment.
In this study, researchers demonstrated a technique to label molecules in mammalian cells’ division cycle, tracking changes in enzymes like cyclin-dependent kinases. They applied this method to various kinases, cell structures, and organelles and successfully used it in zebrafish larvae brains. This approach is versatile and practical for observing cell responses to numerous stimuli, including nutrients and hormones. It can aid in studying growth, ageing, and cancer, highlighting its potential in diverse biological research areas.
The team is expanding their switchable fluorophores to study more cellular signals and adapting their system for use in mouse models.