Multiparametric high-resolution imaging of barley embryos by multiphoton microscopy and magnetic resonance micro-imaging.

Nonlinear optical microscopy and magnetic resonance imaging (MRI) address different properties of the sample and operate on different geometrical scales. MRI maps density and mobility of molecules tracking specific molecular signatures. Multiphoton imaging profits from the nonlinear absorption of light in the focus of a femtosecond laser source stimulating the autofluorescence of biomolecules. As this effect relies on a high light intensity, the accessible field of view is limited, but the resolution is very high (a few hundred nanometers). Here, we aim to link the different accessible scales and properties addressed in the different techniques to obtain a synoptic view. As model specimen we studied embryos of barley. Multiphoton stimulated autofluorescence images and images of second harmonic generation are achieved even down to low magnification (10x), low numerical aperture (N.A. 0.25) conditions. The overview images allowed morphological assignments and fluorescence lifetime imaging provides further information to identify accumulation of endogenous fluorophores. The second, complementary contribution from high-resolution MR images provides a 3D model and shows the embedding of the embryo in the grain. Images of the proton density were acquired using a standard 3D spin-echo imaging pulse sequence. Details directly comparable to the low magnification optical data are visible. Eventually, passing from the MR images of the whole grain via low magnification to high resolution autofluorescence data bridges the scale barrier, and might provide the possibility to trace transport and accumulation of, e.g., nutrients from large structure of the plant to the (sub-) cellular level.