Figure 1.

Probe delivery and characteristics. Left, Probe delivery. The first step toward quantification of translational mobility is the introduction of the fluorescently tagged molecules to the cell of interest. Fluorescent probes that are conjugated with the molecule of interest or genetically encoded are delivered to the interior of the cell most commonly by transfection, microinjection, electroporation, viral infection, or some combination. Here, a dissociated hippocampal neuron was transfected using liposome-mediated gene transfer for high expression of GFP (the image is pseudocolored according to fluorescence intensity). The delivery method strongly depends on the optical technique and tagging method being used. Top right, The Jablonski diagram compares the difference between the electronic states of a fluorescent molecule excited by 1PE or 2PE. For 1PE, a higher-energy photon (shown in blue) is used to generate fluorescence, while for 2PE, two lower-energy photons (shown in magenta) are combined to excite the fluorophore from the ground state to an excited state. The fluorescence emission is used in each optical technique for quantifying the mobility of molecules through a specified volume. Bottom right, A comparison between the excitation (illumination) volume with 1PE or 2PE is shown. To achieve a confocal volume with 1PE (gray mesh), a pinhole placed in the detection path is introduced to spatially restrict the signal, while 2PE has an intrinsic spatial confinement by the square of the intensity profile of the laser (magenta ellipsoid).