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

我们的目标是探索一种新的方法显微镜成像细胞生物学具有非常高的空间分辨率。光学显微镜是推动细胞生物学进步的不可或缺的工具，并且仍然是获得活细胞和组织内空间和时间分辨率的唯一实用手段。然而，大多数细胞成分没有颜色，除非染色，否则在光学显微镜下很难区分。荧光显微镜，使用染料标记的抗体或与荧光标记融合的蛋白质，提供了一种高度敏感和特异性的可视化生物分子的方法。随着共聚焦、多光子、反褶积和全内反射等新检测技术的引入，荧光显微镜是当今医学和生物科学中应用最迅速的技术，特别是当荧光团技术取得进展时。在过去的几十年里，半导体纳米粒子（称为量子点- QDs）的合成取得了进展，引起了人们对其作为荧光探针在生物学和医学中的应用的广泛兴趣。现代化学现在允许制造水溶性纳米晶体，这种晶体可以与生物分子结合，并且仍然保持非常好的荧光特性，在某些情况下优于有机染料。除了荧光外，量子点还表现出强烈的光学非线性，当它们从价带到导带的量子化电子跃迁共振激发时。这种相互作用产生的光信号，其尺度类似于激发强度的三次方，被称为四波混频，在最近的文献中，一些小组（包括我们）在量子点中进行了测量。在这项可行性研究中，我们提出检测作为生物标记的水溶性量子点的共振四波混合，以获得一种全新的多光子成像对比度，而不是成熟的荧光显微镜方法。这种方法不仅允许量子点作为生物标记物的新应用，而且还为新标签的工程设计开辟了场所，这些标签通常不是荧光的，但却表现出强相干非线性光学特性，例如金属纳米颗粒。为了检测量子点的非线性响应，我们将使用我们过去已经开发的一种技术，这种技术被证明是非常敏感的。为了证明这种检测方法在细胞生物学成像中的适用性，我们将实现显微镜物镜和扫描单元的可用设置。关键问题是这种方法能否获得足够的信号对比度。我们估计这是可能的，特别是在进一步实施零背景性能的设置时，这是我们工作计划的一部分。该研究项目是激光物理学、材料科学和生物学的交叉点，是一项大胆的可行性研究，它结合了三个现代科学发展领域，即相干激光光谱、细胞生物学中的高分辨率成像和半导体纳米颗粒。直接受益者将是卡迪夫大学生物科学学院研究生物样本的合作者。此外，该项目中胶体量子点的利用可能涉及与卡迪夫大学化学系的合作。这可能会对开发新型相干探针产生进一步的研究活动。关于拟议研究的成功结果的报告将在国际科学期刊上发表，并在会议上提出。因此，来自世界各地的物理科学和生物学科的研究人员可能会从这项工作的结果中受益。

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.