Total Internal Reflection Fluorescence (TIRF) for Single-Molecule Imaging
A. Differential interference contrast (DIC) image of a young hippocampal neuron developed in a primary culture for two days. The growth cone (in the black frame) is enlarged in the fluorescence image, B, in which individual ErbB4 receptors were visualized using TIRF.
Total internal reflection fluorescence (TIRF) microscopy takes advantage of the refractive behavior of light traversing a glass and water interface to lessen background fluorescence. When shining the light in an angle that is larger than 61°, an evanescent wave is formed in the lower refractive index medium, where the intensity drops exponentially with the distance from the interface. Therefore, fluorescent molecules more than approximately 200 nm from the glass will not be excited, leading to a dramatic decrease in background fluorescence. For this reason, TIRF microscopy is the preferred single-molecule fluorescence imaging approach for studying plasma membrane-associated processes. At Pacific Northwest National Laboratory (PNNL) a TIRF module is implemented on our wide-field single-molecule fluorescence microscope, enabling us to acquire fluorescence images from the basal membrane of living cells with high spatial and temporal resolution.
Overlay of DIC and TIRF images of hippocampal neurons developed in primary culture for five days. Endogenously expressed ErbB4 receptors are tagged with fluorescent antibodies (orange).
