By Karen M. Gooding, Fred E. Regnier
Revised and accelerated to mirror the newest suggestions in HPLC from the previous decade. offers functional innovations for the assessment and research of proteins, peptides, and polynucleotides.
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The probability of two-photon absorption with CW illumination is extremely low . 2)). Solid-state lasers such as Cr:LiSAF, Nd:YLF, Nd:glass, and Cr:fosterite lasers, as well as dye- and ﬁber-based lasers have been employed for two-photon microscopy (for review see ). The most prominent light source, however, is the Ti:sapphire laser. Because of its high average power (up to several Watt), its broad tuning range (700–1 100 nm), short pulse duration (∼100 fs), repetition rates matching the nanosecond ﬂuorescence lifetimes of many ﬂuorophores, as well as reliable and robust operation, the Ti:sapphire laser has become the excitation source of choice for biomedical imaging.
We give an overview of ﬂuorescence labeling techniques and how they are combined with 2PLSM for functional imaging and photomanipulation in living tissue. Finally, we discuss limitations and provide some future perspectives. 1 Two-Photon Fluorescence Excitation Two-photon absorption is a quantum event, in which two photons are virtually simultaneously absorbed within a narrow temporal window (of typically less than 10−15 s), each photon providing half the energy for the molecular transition to an excited state (Fig.