Cryogenic electron tomography provides nanometer-scale views of subcellular structures but often lacks essential physiological context, such as metabolic state or ion concentrations. To bridge this gap, we incorporated fluorescent biosensors into a cryogenic correlative light and electron microscopy (cryoCLEM) workflow, enabling direct measurement of physiological conditions alongside structural data. We focused on biosensors for calcium, pH, and osmotic stress, validating and calibrating their responses under cryogenic conditions. This was achieved through an in vitro workflow using high-pressure freezing of purified biosensors in defined chemical environments, followed by cryogenic ratiometric fluorescence imaging. We further applied these biosensors in plunge-frozen samples ranging from bacteria to human cells. This approach links localized environmental changes to 3D structural and organizational dynamics of proteins. Looking ahead, expanding the biosensor toolkit and combining cryoCLEM with subtomogram averaging will enable identification of condition-specific structural states and provide key insight into structure-function relationships.
Azaldegui et al. (Sun,) studied this question.