Figure 1
The ME-MPM design. (a) Optical setup for multiple-excitation multiphoton microscopy (ME-MPM) measurements. Beams from Mai Tai broad-band (BB) and Mai Tai high power (HP) sources were directed to either an upright confocal microscope (FV300) or an inverted confocal microscope (FV1000). Note that the Nicol prisms split the beam into two paths directed to either microscope for both lasers. The setting of each polarizer can be easily adjusted by rotating the preceding half-wave plate to direct more or less light toward each system, allowing for optimal excitation of each fluorophore during dual color imaging. The control of half-wave plates i, ii, and iii is described in the text and in Additional file 1. (b) Two-photon absorption (TPA) spectra for EGFP and DsRedII fluorescent proteins in Goeppert-Mayer units. (c) TPA spectra for the hydrazine derivatives of FITC (green), and Alexa Fluor 594 (red) in Goeppert-Mayer units. We do not present the spectra in terms of TPA brightness, σ2 × φ, where φ is the fluorescence quantum yield, because the quantum yields of these dyes are similar in the same environment and they are quite sensitive to pH and other conditions [22]. Imaging experiments presented here were performed at 920 nm for green-emitting fluorophores and at 1015 to 1040 nm for red-emitting fluorophores.