Concurrent Scientific Session (Imaging): Super-Resolution Alternatives
Light-sheet microscopy with isotropic, subcellular 3D resolution
Light-sheet fluorescence microscopy (LSFM) has revolutionized volumetric imaging of large samples. LSFM illuminates only a single optical slice and thereby avoids excitation outside of the focal plane, which otherwise causes image blur and excessive sample bleaching. However, LSFM typically does not improve spatial resolution compared to confocal microscopy and in the context of large samples, it only provides modest, cellular-scale resolution.
Using propagation invariant beams instead of Gaussian optics, Lattice light-sheet microscopy (LLSM) is able to increase the axial resolution in LSFM by generating thin, extended sheets of light. However, common to all propagation invariant beams, the confinement of excitation is reduced, meaning more out-of-focus fluorescence is excited outside of the focal plane. This is undesirable, as it degrades axial resolution and increases photo-bleaching. Hence, LLSM is most commonly operated in a mode that provides a compromise between axial resolution and excitation confinement.
I will present our work on LSFM to yield isotropic, subcellular 3D resolution (i.e. equal spatial resolution in every direction) over large field of views while minimizing out-of-focus fluorescence excitation. Using two-photon excitation, we create high-aspect ratio light-sheets that match the diffraction limited lateral resolution over large sample volumes. We employ this technology to quantitatively image cancer cell morphodynamics and signalling in collagen matrices and Zebrafish xenografts. The use of photo-switchable fluorophores and specially engineered light-sheets allows to confine a population of active fluorophores to a thin layer whose thickness is theoretically not limited by diffraction. A mathematical theorem for the creation of suitable light-sheets for photo-activation and off-switching is discussed and preliminary experimental results are presented.