Abstract
This chapter focuses on the role of total internal reflection fluorescence (TIRF) microscopy in facilitating single molecule imaging in living cells. TIRF can be achieved using one of a number of illumination schemes, but most researchers use either prism- or objective-type configurations. In prism-type TIRF, the excitation laser does not enter the internal optical path of the microscope, but rather is directed at the sample from above, through a prism that is optically coupled to the glass slide via a layer of glycerol. Due to their extreme sensitivity and high readout speed, intensified charge-coupled device-based cameras are excellent for single molecule imaging. High-end intensified charge-coupled devices (ICCDs) have single photon sensitivity under ideal conditions and can acquire 640 × 480 images directly to digital storage media at rates of over 100 frames per second. Traditional, cooled CCDs are a feasible alternative in some applications and are desirable for their high image quality and the strong linearity of their response.
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