Abstract
The two-photon excitation (TPE) microscopy has become an important tool of noninvasive imaging due to the better penetration and relative harmlessness of the longer wavelength. However, the high photon flux in two-photon excitation can potentially lead to higher-order photobleaching within the focal volume. This paper measured the relationship between the photobleaching rate and the excitation power for Chemical dyes and green fluorescence proteins (GFPs) in vivo at biological imaging level. As expected, the photobleaching rate increased near-linearly with the excitation power for one-photon excitation. However, the two-photon photobleaching rate increased with high-order power (≥3.5) of excitation power, indicating the presence of high-order photon interaction in two-photon excitation microscopy. As a consequence, the use of multi-photon excitation microscopy to study may be limited by increased photobleaching.
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