By combining two-photon-excitation and third-harmonic-generation microscopy, researchers at Centre National de la Recherche Scientifique in Talence and Bordeaux, France, have produced images that correlate cell processes with cell structures.Cells respond to their environment with chemical signals, such as ions that move through the cell membrane. Studying this movement may hold the key to disorders such as Alzheimer's disease, explained Regis Barille, one of the researchers on the project.Combining two-photon-excitation and third-harmonic-generation microscopy, a hybrid imaging technique enables researchers to correlate cellular processes and structures. Studying the movement of chemical signals within and between cells may hold the key to disorders such as Alzheimer's disease.The two-photon excitation of fluorescent labels offers information on ion movement without damaging cells. Researchers use two photons of cell-friendly infrared radiation to excite the fluorophore, rather than one at a shorter, cell-damaging wavelength. The catch is that the two photons must strike the tag nearly simultaneously, necessitating a tightly focused, femtosecond excitation source.Third-harmonic generation, on the other hand, images cellular structure. High-power laser radiation generates the third-harmonic frequency at cellular interfaces, the places where two different cell materials meet (for example, at the spot where the membrane meets the cytoplasm). Collecting the third-harmonic radiation creates an image. Like two-photon excitation, third-harmonic generation occurs only in the focal volume of the laser.In a demonstration of the hybrid technique, a Spectra-Physics synchronously pumped optical parametric oscillator generated the 130-fs, 1.5-µm pulses for third-harmonic excitation and the 100-fs, 810-nm pulses for two-photon excitation of the calcium probe Fura-2. The researchers collected the two-photon signal in the backward direction using the excitation objective and the third-harmonic signal in the forward direction using a 0.4-NA condenser. They used Hamamatsu photomultiplier tubes for imaging and two galvanometric scanners to direct the focal point scanning.Focal differencesThe group has performed in vivo imaging of human glial cells with the technique. The 500-nm third-harmonic light does not damage the cells because it is not very powerful and because it is at an absorption minimum of water. Nevertheless, Barille cautioned that it is too early to discuss its implementation."We have not seen damage to our glial cells, but more studies should be done concerning the impact of all femtosecond laser parameters to the viability of cells."One of the biggest challenges for the group was compensating for the focal differences resulting from the use of two illumination wavelengths. "We used two different wavelengths, and the confocal parameter is not the same in both cases," Barille explained. "But the focusing parameters are kept constant and optimized for the third-harmonic signal." The optimization of microscope objectives for IR wavelengths to limit chromatic aberration could improve the technique, he added.In the near term, he said, the researchers hope to correlate the information acquired with both methods. In the future, they will study calcium dynamics, with a goal of eliminating the use of fluorophores altogether.