Novel Multiphoton Microscopy for Imaging in Cell Biology based on Resonant Nonlinear Optics of Quantum Dots

Our objective is to explore a new approach to microscopy for imaging in cell biology with very high spatial resolution. Light microscopy is an indispensable tool that is driving progress in cell biology, and is still the only practical means of obtaining spatial and temporal resolution within living cells and tissues. However, most cellular constituents have no colour and they are hard to distinguish under a light microscope unless they are stained. Fluorescence microscopy, using antibodies labelled with dyes or fusion of proteins with fluorescent tags has provided a highly sensitive and specific method of visualizing biomolecules. With the introduction of new detection techniques such as confocal, multiphoton, deconvolution, and total internal reflection, fluorescence microscopy is the most rapidly expanding technique employed today, both in the medical and biological sciences, especially when coupled to advances in fluorophore technology. Progresses in the synthesis of semiconductor nanoparticles (called quantum dots - QDs) during the past decades have generated widespread interest in their applications as fluorescent probes in biology and medicine. Modern chemistry now allows fabrication of water-soluble nanocrystals that can be conjugated to biomolecules and still retain very good fluorescent properties, in some cases superior to organic dyes. Besides their fluorescence, QDs also exhibit a strong optical nonlinearity when excited in resonance with their quantized electronic transitions from the valence to the conduction band. The optical signal generated by this interaction, which scales like the third power of the exciting intensity, is known as four-wave mixing and was measured in QDs by a few groups (including us) in recent literature. In this feasibility study we propose to detect the resonant four-wave mixing of water soluble QDs acting as bio-labels, to obtain a fundamentally new multiphoton imaging contrast as opposed to the well established method of fluorescence microscopy. This approach not only allows for a novel application of QDs as bio-markers, but also opens the venue for the engineering of new labels which might generally be not fluorescent and yet exhibit strong coherent nonlinear optical properties such as e.g. metallic nanoparticles. To detect the nonlinear response from QDs we will use a technique already developed by us in the past, which was proven to be very sensitive. To demonstrate the applicability of this detection method to imaging in cell biology we will implement the available set-up with microscope objectives and a scanning unit. The key question will be if enough signal contrast can be obtained with this method. We estimate that this is possible especially when further implementing the set-up for zero-background performance, which is part of our programme of work.This research project, at the interface between laser physics, material science and biology, is an adventurous feasibility study which joins three modern areas of scientific development, namely coherent laser spectroscopy, high-resolution imaging in cell biology and semiconductor nanoparticles. Direct beneficiaries will be collaborators in the School of Biosciences at Cardiff University for the study of biological samples. Moreover, the utilization of colloidal QDs in this project is likely to involve collaborations with the Chemistry Department of Cardiff University. This might generate further research activity toward the development of new types of coherent probes. Reports on the successful outcome of the proposed research will be published in international scientific journals and presented in conferences. Therefore, researchers from both physical sciences and biological disciplines worldwide might benefit from the outcome of this work.