Molecular holography

想象一下，能够以毫秒的速度同时唯一地跟踪一个细胞中的三维数百个分子或蛋白质。为了实现这一目标，我建议将分子特有的光散射技术(受激拉曼光谱-SRS)与众所周知的全息术的三维成像能力相结合，以创建分子全息图。 在拉曼光谱实验中，光在被照射的分子或蛋白质特有的一组独特的波长上散射。也就是说，这项技术提供了散射光中分子的指纹--一个光谱条形码。这项技术的进一步改进使用了两个激光(受激拉曼光谱，SRS)，当两个激光之间的频差与分子振动匹配时，会产生相干散射束。全息图，就像我们的货币和信用卡上常见的那些，通过将相干散射的激光与原始光源相结合来提供物体的三维图像。分子全息术是这两种技术的结合，以创建分子振动的三维空间分布图。我的团队对成像表皮生长因子受体特别感兴趣，因为它的过度表达与许多癌症有关。然而，只要开发出合适的造影剂(例如，适当功能化的金或银纳米颗粒)，成像技术将广泛适用于任何分子或生物标记物。 今年，我们成功地完成了原理验证实验，证明了可以创建传统的金纳米颗粒全息图，并且可以从附着在纳米颗粒上的分子产生相干光发射(SRS)。将两者结合起来需要集成两个激光器，包括一个波长可调的激光器和另一个波长固定的激光器、一个针孔、锁定放大器和一个探测器；以及在Dalhousie开发的重建图像所需的软件。我的团队现在准备创造一种有可能超越荧光技术的成像模式。分子全息术将提供几个优点，包括通过一次曝光(全息术)捕捉更多生物分子的3D分布，而不是从2D图像/切片重建3D轮廓。 与荧光成像不同，我们的技术具有真正的无标记潜力，从而避免了引入细胞的外来荧光剂的干扰效应。从长远来看，无标记分子全息术可以取代荧光成像，成为细胞生物学家的首选方法。我的本地、全国和国际合作者网络将帮助确保这项技术被世界各地的领先机构采用。

Imagine being able to simultaneously and uniquely track hundreds of molecules or proteins in a cell in three dimensions at millisecond rates. To achieve this goal I propose to combine a molecular-specific light scattering technique (Stimulated Raman spectroscopy - SRS) with the well-known three-dimensional imaging capability of holography, to create molecular holograms. In a Raman spectroscopy experiment, light is scattered at a unique set of wavelengths characteristic of the molecule or protein being illuminated. That is, the technique provides a fingerprint of the molecule in the scattered light - a spectral barcode. A further modification of this technique which uses two lasers (Stimulated Raman spectroscopy, SRS) creates a coherent scattered beam when the frequency difference between the lasers matches a molecular vibration. Holograms, like those commonly observed on our currency and credit cards, provide a three dimensional image of an object by combining coherently scattered laser light with the original light source. Molecular holography is the integration of these two techniques to create a map of the three-dimensional spatial distribution of a molecular vibration. My team is specifically interested in imaging the Epidermal Growth Factor Receptor, as it's overexpression is implicated in a number of cancers. However, the imaging technique would be widely applicable to any molecule or biomarker as long as an appropriate contrast agent is developed (e.g. appropriately functionalized gold or silver nanoparticles). This year, we completed successful proof-of-principle experiments that demonstrate conventional holograms of gold nanoparticles can be created and that coherent light emission (SRS) can be generated from molecules attached to the nanoparticles. Combining the two requires the integration of two lasers comprising a wavelength-tunable one and another with fixed wavelength, a pinhole, lock-in amplifier and a detector; along with the requisite software, developed at Dalhousie, to reconstruct the image. My team is now poised to create an imaging modality that has the potential to outperform fluorescence techniques. Molecular holography would offer several advantages, including capture of the 3D distribution of many more biomolecules with one exposure (holography), rather than reconstruction of a 3D profile from 2D images/slices. Unlike fluorescence imaging our technique has the very real potential to be label-free, thereby avoiding the perturbing effect of a foreign flurophore agent introduced to a cell. In the long term, label-free molecular holography could supplant fluorescence imaging as the cell biologists' modality of choice. My local, national and international network of collaborators will help ensure that the technique is adopted at leading institutions around the world.