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Hot-band absorption and anti-Stokes emission properties of an organic fluorescent dye, Alexa Fluor 568, were characterized and compared with those of Rhodamine 101. The comparison of the properties (e.g., quantum efficiency, spectral distribution, thermal properties, and fluorescence lifetime) between the two dyes confirms that both dyes undergo the same process when excited in the red spectral region. Possible undesirable crosstalk effects and applications in dSTORM microscopy were demonstrated and discussed.

Hot-Band Anti-Stokes Fluorescence Properties of Alexa Fluor 568