This article describes a protocol for imaging human neuroblastoma cells using total internal reflection fluorescence (TIRF) microscopy. The method focuses on visualizing synaptic vesicles through the excitation of specific fluorophores.
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 configuration, check the position of the beam that emerges out of the objective on the sample cover. When the beam is positioned in the center of the objective lens, a spot is visible in the center of the turf sample cover, and the cell is imaged in epifluorescence mode.
To reach the critical angle, move the focused spot in the Y direction using the angle adjustment screw on the turf slider. When the beam converges on the sample plane at an angle larger than the critical angle, the spot disappears and a straight, thin, focused line is evident in the middle of the sample cover. To fine tune the turf angle, use a cell sample.
Watch the fluorescence image on the video. At this stage, an epifluorescence-like image is still visible. Gently move the screw until turf condition is achieved. Here, only one optical plane of the cell is in focus, resulting in a flat image with high contrast.
To perform sample imaging, set the single channel time lapse experiment. Appropriate exposure times are between 40 and 80 milliseconds. Acquire images at 1 or 2 Hertz sampling frequency.
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