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

Lead Research Organisation: Cardiff University
Department Name: School of Physics and Astronomy

Abstract

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.
 
Description In this feasibility study we detected the resonant four-wave mixing of water soluble CdSe/ZnS 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. We detected the nonlinear response, specifically four-wave mixing, using a very sensitive technique developed by us in the past. To demonstrate the applicability of this detection method to imaging in cell biology we have implemented the available set-up with microscope objectives and a sample scanning unit with nanometric resolution. We have demonstrated that this method is sensitive enough to detect only few quantum dots within the focal volume, specifically we find a noise floor equivalent to 10 QDs for 1 second integration. Using co-polarized excitation and a two-pulse experiment, large background from the non-resonant response of the medium and of the detection diodes was observed. We eliminated this problem in two ways. Firstly, we used cross-polarization between pulse 1 and detection, for which the background due to wave mixing in the detectors is vanishing. Secondly, we extended the setup to a three-beam configuration with cross-polarization. In this setup, we achieved both zero non-resonant background, and additionally a lifetime imaging. We demonstrated a spatial resolution significantly better than the diffraction limit (150nm lateral and 590nm axial). We investigated the photobleaching of the QDs, which was found to be negligible in the polymer reference samples, but visible for QDs in fixed cells, antibody labelled to stain the Golgi. Finally, we have imaged the Golgi in a 3D stack (see attachment). We plan to continue this research by investigating different quantum dot materials (PbS, PbSe, InAs) in the more infrared range of the spectrum, and by using gold nanoparticles to locally enhance the non-linear response.
Exploitation Route The findings establich a new microscopy technique which is been taken forward for particle tracking and correlative electron-light microscopy
Sectors Healthcare,Pharmaceuticals and Medical Biotechnology

 
Description The novel optical microscopy technique which can be used for correlative light-electron microscopy
First Year Of Impact 2008
Sector Healthcare,Pharmaceuticals and Medical Biotechnology
Impact Types Societal,Economic

 
Title SURFACE PLASMON FOUR-WAVE MIXING MICROSCOPY 
Description Laser pulses are applied to surface plasmon resonant articles such as gold nanoparticles within a microscopy sample to generate a four-wave mixing signal that is detected as the output of the microscopy process. 
IP Reference US8817261 
Protection Patent granted
Year Protection Granted 2014
Licensed No
Impact This technique is used for correlative electron - light microscopy
 
Title SURFACE PLASMON FOUR-WAVE MIXING MICROSCOPY 
Description Laser pulses are applied to surface plasmon resonant articles such as gold nanoparticles within a microscopy sample to generate a four-wave mixing signal that is detected as the output of the microscopy process. 
IP Reference WO2010070337 
Protection Patent granted
Year Protection Granted 2010
Licensed No
Impact This technique is used for correlative electron - light microscopy