Two-Photon
Fluorescence Spectroscopy and Imaging of
4‑Dimethylaminonaphthalimide Peptide and Protein Conjugates
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Abstract
We report detailed photophysical
studies on the two-photon fluorescence
processes of the solvatochromic fluorophore 4-DMN as a conjugate of
the calmodulin (CaM) and the associated CaM-binding peptide M13. Strong
two-photon fluorescence enhancement has been observed which is associated
with calcium binding. It is found that the two-photon absorption cross-section
is strongly dependent on the local environment surrounding the 4-DMN
fluorophore in the CaM conjugates, providing sensitivity between sites
of fluorophore attachment. Utilizing time-resolved measurements, the
emission dynamics of 4-DMN under various environmental (solvent) conditions
are analyzed. In addition, anisotropy measurements reveal that the
4-DMN–S38C–CaM system has restricted rotation in the
calcium-bound calmodulin. To establish the utility for cellular imaging,
two-photon fluorescence microscopy studies were also carried out with
the 4-DMN-modified M13 peptide in cells. Together, these studies provide
strong evidence that 4-DMN is a useful probe in two-photon imaging,
with advantageous properties for cellular experiments