Cell Imaging Using Total Internal Reflection Fluorescence Microscopy

Begin with a total internal reflection fluorescence (TIRF) microscope setup with a covered imaging chamber containing a glass coverslip with adhered human neuroblastoma cells in a buffer.

These cells express fluorophores specific to synaptic vesicle membranes.

In epifluorescence mode, select a cell for imaging.

In TIRF mode, through the objective, direct a laser at the sample, appearing as a spot on the cover.

Adjust the angle to exceed the critical angle at the interface between the high-refractive-index glass coverslip and the lower-refractive-index sample, causing total internal reflection (TIR), seen as a thin line.

TIR generates an evanescent wave that excites fluorophores on the nearest membrane.

The fluorescence emitted is collected by the objective and captured by a camera.

Fine-tune for a high-contrast image of the cell membrane near the coverslip.

Optimize acquisition settings to minimize photobleaching and set sampling frequency.

Begin TIRF imaging to visualize fused synaptic vesicles.

In epifluorescence mode, focus on the coverslip, and choose transfected cells placed in the chamber center. Under software control, switch to turf illumination in live mode. To set the turf configu