Lattice Light Sheet Microscopy to Visualize Receptor-Ligand Interactions in Live Cells
Transcrição
Lattice light-sheet microscopy, LLSM, enables the visualization of cell-surface receptor-ligand interactions in live cells with high spatiotemporal resolution.
Take a coverslip containing transduced ligand-expressing cells emitting red fluorescence. Attach the coverslip cell-side-up onto a greased sample holder. Fix the holder on the piezo of a microscope stage to allow precise sample positioning and scanning during imaging. Adjust the software settings to focus on a single ligand-expressing cell.
Pipette the receptor-expressing cell suspension onto the coverslip. The receptors are tagged with green fluorophore-labeled antibodies. Image the interacting cell pairs.
The microscope's excitation laser emits a circular, linearly-polarized laser beam that passes through cylindrical lenses to create a light sheet. The light sheet is projected onto a modulator that removes excess diffraction and transforms it into an ultrathin lattice pattern.
The lattice light sheet illuminates an ultrathin section of the interacting cell pair, and the fluorescence emitted by the interacting cells is captured perpendicular to the incident beam. Image the interacting cell pair in multiple Z-planes, and capture two-dimensional images in each focal plane with a high-speed camera.
Using suitable software, combine these images to form a z-stack — a high-resolution four-dimensional image demonstrating the spatiotemporal dynamics of the cell-surface receptor-ligand interaction.
