The fluorescing capability of general fluorophores is limited, both in terms of the rate and number of emitted photons. These limitations, in combination with the limited detection efficiency of the microscope and the spatial size of the optical-probe volume, have direct implications for the rate at which images in confocal fluorescence microscopy can be acquired, as well as for the number of images that can be acquired from a certain specimen.
Consider a confocal fluorescence microscope working at typical high-resolution conditions as specified in Table 3.1. The fluorophore used in this example is fluorescein. The detection pinhole is, by assumption, set equal to the projected Airy disk. In this case, the lateral resolution, or width of the confocal probing voxel, is 90% of the wide-field case, whereas the axial resolution is 120% compared to the confocal case with an infinitely small detection pinhole (see Sec. 2.3).
The boundary conditions set by the fluorophore are shown in Table 3.2. They result from the dye concentration in the sample, the fluorescence lifetime, and the number of times the fluorophore can be excited before it irreversibly photobleaches (the Q-number; see Sec. 2.4) and is lost for fluorescence measurements. In practice, the average total of fluorescent photons emitted per fluorophore varies between 102â106. In some cases, removing oxygen or adding protective agents, can increase the Q-number and therefore the number of images that can be acquired from the specimen.
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