Molecular holography

想象一下，能够以毫秒级的速度在三维空间中同时独特地跟踪细胞中的数百个分子或蛋白质。为了实现这一目标，我建议将分子特异性光散射技术（受激拉曼光谱- SRS）与众所周知的全息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，光以被照射的分子或蛋白质特有的一组波长散射。也就是说，这项技术在散射光中提供了分子的指纹——一种光谱条形码。该技术的进一步改进是使用两个激光器（受激拉曼光谱，SRS），当激光之间的频率差与分子振动相匹配时，产生相干散射光束。全息图，就像我们在货币和信用卡上经常看到的那样，通过将相干散射的激光与原始光源相结合，提供了物体的三维图像。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。我的团队对表皮生长因子受体的成像特别感兴趣，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志物。今年，我们完成了成功的原理验证实验，证明可以创建金纳米颗粒的传统全息图，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，包括波长可调的激光器和固定波长的激光器、针孔、锁定放大器和探测器；以及在达尔豪斯开发的重建图像所需的软件。我的团队现在正准备创造一种有潜力胜过荧光技术的成像方式。分子全息技术将提供几个优势，包括通过一次曝光（全息）捕获更多生物分子的3D分布，而不是从2D图像/切片重建3D轮廓。***与荧光成像不同，我们的技术具有非常真实的无标记潜力，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，无标记分子全息技术可以取代荧光成像技术，成为细胞生物学家的首选方法。我的地方、国家和国际合作者网络将帮助确保这项技术在世界各地的领先机构得到采用

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.**