Single-molecule light-sheet imaging of suspended T cells

Aleks Ponjavic, James McColl, Alexander R. Carr, Ana Mafalda Santos, Klara Kulenkampff, Anna Lippert, Simon J. Davis, David Klenerman, Steven F. Lee

Research output: Contribution to journalArticlepeer-review

27 Citations (Scopus)


Adaptive immune responses are initiated by triggering of the T cell receptor. Single-molecule imaging based on total internal reflection fluorescence microscopy at coverslip/basal cell interfaces is commonly used to study this process. These experiments have suggested, unexpectedly, that the diffusional behavior and organization of signaling proteins and receptors may be constrained before activation. However, it is unclear to what extent the molecular behavior and cell state is affected by the imaging conditions, i.e., by the presence of a supporting surface. In this study, we implemented single-molecule light-sheet microscopy, which enables single receptors to be directly visualized at any plane in a cell to study protein dynamics and organization in live, resting T cells. The light sheet enabled the acquisition of high-quality single-molecule fluorescence images that were comparable to those of total internal reflection fluorescence microscopy. By comparing the apical and basal surfaces of surface-contacting T cells using single-molecule light-sheet microscopy, we found that most coated-glass surfaces and supported lipid bilayers profoundly affected the diffusion of membrane proteins (T cell receptor and CD45) and that all the surfaces induced calcium influx to various degrees. Our results suggest that, when studying resting T cells, surfaces are best avoided, which we achieve here by suspending cells in agarose.
Original languageEnglish
Pages (from-to)2200-2211
Number of pages12
JournalBiophysical Journal
Issue number9
Early online date8 May 2018
Publication statusPublished - 8 May 2018

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