The fluorescent reporters commonly
used to visualize proteins can
perturb both protein structure and function. Recently, we found that
4-cyanotryptophan (4CN-Trp), a blue fluorescent amino acid, is suitable
for one-photon imaging applications. Here, we demonstrate its utility
in two-photon fluorescence microscopy by using it to image integrins
on cell surfaces. Specifically, we used solid-phase peptide synthesis
to generate CHAMP peptides labeled with 4-cyanoindole (4CNI) at their
N-termini to image integrins on cell surfaces. CHAMP (computed helical
anti-membrane protein) peptides spontaneously insert into membrane
bilayers to target integrin transmembrane domains and cause integrin
activation. We found that 4CNI labeling did not perturb the ability
of CHAMP peptides to insert into membranes, bind to integrins, or
cause integrin activation. We then used two-photon fluorescence microscopy
to image 4CNI-containing integrins on the surface of platelets. Compared
to a 4CNI-labeled scrambled peptide that uniformly decorated cell
surfaces, 4CNI-labeled CHAMP peptides were present in discrete blue
foci. To confirm that these foci represented CN peptide-containing
integrins, we co-stained platelets with integrin-specific fluorescent
monoclonal antibodies and found that CN peptide and antibody fluorescence
coincided. Because 4CNI can readily be biosynthetically incorporated
into proteins with little if any effect on protein structure and function,
it provides a facile way to directly monitor protein behavior and
protein–protein interactions in cellular environments. In addition,
these results clearly demonstrate that the two-photon excitation cross
section of 4CN-Trp is sufficiently large to make it a useful two-photon
fluorescence reporter for biological applications.