Determination of two-photon excitation and emission spectra of fluorescent molecules in single living cells

Abstract

Modelocked Ti:Sapphire lasers are widely used in two-photon microscopes (TPM), partly due to their tunability over a broad range of wavelengths (between 700 nm and 1000 nm). Many biophysical applications, including quantitative Forster Resonance Energy Transfer (FRET) and photoswitching of fluorescent proteins between dark and bright states, require wavelength tuning without optical realignment, which is not easily done in tunable Ti:Sapphire lasers. In addition, for studies of dynamics in biological systems the time required for tuning the excitation should be commensurate with the shortest of the time scales of the processes investigated. A set-up in which a modelocked Ti:Sapphire oscillator providing broad-bandwidth (i.e., short) pulses with fixed center wavelength is coupled to a pulse shaper incorporating a spatial light modulator placed at the Fourier plane of a zero-dispersion two-grating setup, represents a faster alternative to the tunable laser. A pulse shaping system and a TPM with spectral resolution allowed us to acquire two-photon excitation and emission spectra of fluorescent molecules in single living cells. Such spectra may be exploited for mapping intracellular pH and for quantitative studies of protein localization and interactions in vivo.