Koen Clays1 2

1, University of Leuven, Leuven, , Belgium
2, Washington State University, Pullman, Washington, United States

Cellular imaging has become very relevant in such important fields as biophysics, diagnostics and therapeutics. The advances of nonlinear optics for this imaging modality gradually become obvious. Two-photon fluorescence induced by near-infrared light has brought a number of advantages, such as higher resolution and intrinsically confocal imaging, and less scattering and absorption, leading to reduced phototoxicity and longer photostability.

More recently, it has become obvious that an additional imaging modality is possible with the same pulsed laser and microscope equipment. Second-harmonic imaging only requires detection at a different wavelength, but provides additional information, different from one- or two-photon fluorescence. Because these are odd-order processes (either first of third order) they are not sensitive to symmetry. Second-harmonic imaging, being an even-order nonlinear process, is symmetry-sensitive and will provide structural information based on the symmetry requirements for the superstructural features of the probe, while fluorescence will only provide information about the where-about of the probe.

We will report on the design and engineering of new molecular probes for this combined two-photon fluorescence and second-harmonic imaging of cellular structure. These can be molecular probes with combined advanced structural (amphiphilic properties) and optical functions (second- and third- order optical nonlinearities), based on a number of different strategies. We will also report on our approach to screen fluorescent proteins for second-harmonic imaging and our effort to rationally design a new fluorescent protein for this new function of second harmonic imaging.