3D Super-Resolution Imaging of Unperturbed Cells

Abstract

Most optical imaging of individual cells has been conducted in 2D on flat glass or culture plastic. Not only is this very abstract from the biological environment and does not imitate physiological conditions, but it may lead to unwanted perturbations of cellular functions and structures due to surface contact. This effect is extremely important in cells whose biological function requires them to interact or sense their local environment, a canonical example of which are T cells, which are responsible for initiating adaptive immune responses to extracellular stimuli.We have developed an experimental protocol capable of using 3D single-molecule super-resolution microscopy with a double-helix point spread function that allows for the imaging of live or fixed cells away from a glass surface. We have empirically determined an ultimate spatial precision of ∼15nm in x and y and ∼30nm in z.We will present both multicolour 3D super-resolution images and single-molecule tracking of human T cells, labelling key proteins involved in their function, including CD45, TCR, Zap70 and CD86.