Abstract
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.
Highlights
The emission spectra of fluorescent dyes can be elucidated via radiative/non-radiative transitions between the electronic states and internal conversions
We report the anti-Stokes fluorescence properties of Alexa Fluor 568 (AF568) when it is excited in the red-edge region of its absorption spectrum
The output power of each source was calibrated and measured after the microscope objective (Nikon CFI Plan Apo Lambda 60x oil). Both Rhodamine 101 (Rh101) and AF568 dyes were prepared in a 10−4 M solution of methanol and PBS (1:1)
Summary
The emission spectra of fluorescent dyes can be elucidated via radiative/non-radiative transitions between the electronic states and internal conversions. The process begins with a single photon absorption when the molecule is excited to its higher S1 or S2 electronic states from the S0 ground state. During the anti-Stokes fluorescence, the emitted photon has higher energy (lower wavelength) than the excitation photon. During two-photon absorption process, a virtual state is assumed to exist between S0 and S1 to excite the molecule.
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