Optical imaging at the vicinity of coverslides has developed tremendously in the recent years with the application to cell biology of objective-based Total Internal Reflection Fluorescence Microscopy (TIRFM). Due to reduced fluorescent background, it is the technique of choice to study dynamic phenomena at cell-surface interface and is also perfectly suited to fast and super-resolution imaging. Using membrane labelling and TIRF, it was shown in the lab that primary human cells probe the antigen surface by active microvilli motion that help the cell to decide to spread (Brodovitch 2013). For example, we recently measured the vertical motion of T lymphocyte microvilli to fluctuate of about 70 nm in 1s durations (Brodovitch 2015). Standard commercial setups are now common in biology labs, but we were one of the first in the region PACA to use TIRF and to apply this expertise in immunology.
Traces of surface exploration by a T-lymphocyte on different substrates imaged with TIRF.
We recently acquired a new system using rapid galvanometric mirrors, which permits: (i) fast azimuthal rotation of the beam to correct illumination heterogeneities and reduce background; (ii) rapid change of the incident angle to infer axial information about fluorophore distribution, and ultimately 3D reconstruction of live fluorescent structures.
Brodovitch A, P Bongrand, A Pierres (2013) J. Immunol. 191:2064-2071. Pubmed.
Brodovitch A, Limozin L, Bongrand P, Pierres A (2015) Cell. Mol. Bioeng. 8: 178-186. Pubmed
Our TIRF ILAS-2 system was acquired with funds from the region Provence-Alps-Côte d’Azur.