Two-photon absorption spectra of fluorescent isomorphic DNA base analogs.

Alexander Mikhaylov,Aleksander Rebane,Bern Kohler,Jüri Pahapill,Sophie De Reguardati,Patrik R Callis

doi:10.1364/boe.9.000447

Abstract

Fluorescent DNA base analogs and intrinsic fluorophores are gaining importance for multiphoton microscopy and imaging, however, their quantitative nonlinear excitation properties have been poorly documented. Here we present the two-photon absorption (2PA) spectra of 2-aminopurine (2AP), 7-methyl guanosine (7MG), isoxanthopterin (IXP), 6-methyl isoxanthopterin (6MI), as well as L-tryptophan (L-trp) and 3-methylindole (3MI) in aqueous solution and some organic solvents measured in the wavelength range 550 - 810 nm using femtosecond two-photon excited fluorescence (2PEF) and nonlinear transmission (NLT) methods. The peak 2PA cross section values range from 0.1 GM (1 GM = 10-50 cm4 s photon-1) for 2AP to 2.0 GM for IXP and 7MG. Assuming typical excitation conditions for a scanning 2PEF microscope, we estimate a maximum image frame rate of ~175 frames per second (FPS).

Highlights

Two-photon excited fluorescence (2PEF), especially when combined with suitable fluorescent markers and wavelength-agile, high peak intensity ultrafast lasers, is a workhorse for studying biological matter
In a study of leukocyte trafficking in cells, Li et al [8] reported fast 2PEF imaging up to 30 frames per second (FPS) using intrinsic L-trp, significant uncertainty regarding the σ2PA value hindered optimization of the technique
The highest peak σ2PA value is obtained for IXP and 7-methyl guanosine (7MG), σ2PA ~2.0 GM, while the lowest peak value is for 2AP in neat H2O, σ2PA = 0.12 GM (Table 1)

Summary

Introduction

Two-photon excited fluorescence (2PEF), especially when combined with suitable fluorescent markers and wavelength-agile, high peak intensity ultrafast lasers, is a workhorse for studying biological matter. Only limited data is available about the 2PA characteristics of DNA bases and amino acids, which are intrinsic to cells and tissues. This is mainly because the emission efficiency of natural fluorophores is often too low for practical use (quantum yields ~10−4 [4]). In a study of leukocyte trafficking in cells, Li et al [8] reported fast 2PEF imaging up to 30 frames per second (FPS) using intrinsic L-trp, significant uncertainty regarding the σ2PA value hindered optimization of the technique. By applying a simplified model (so-called two-level model) for quantitative description of σ2PA in a dipolar chromophore [14], we estimate the change of permanent electric dipole moment upon the transition from ground- to the lowest excited state

Experimental

Results and discussion

Conclusions