Travel Grant for Adaptive Optics in CARS Microscopy

光学显微镜为科学家和临床医生提供了大约400年来以亚细胞分辨率成像活组织的能力。这种能力导致了生命科学和物理科学的一些科学突破。人们希望以更高的分辨率对样品成像，以便能够分辨出最精细的结构，从而在普通光学领域取得了重大进展。事实上，就在130多年前，阿贝通过引入衍射理论和研究光学像差，在改进的光学显微镜方面的工作推动了光学的研究。然而，对于所有形式的显微镜来说，关键是能够在样品中存在的不同结构之间进行对比。在过去50年的大部分时间里，这是通过在样品中添加荧光标签来实现的，最终使用了转基因样品，使它们自然地表达了荧光标签。即使在这种情况下，样品也不处于其自然状态，因此该方法不适用于许多样品，并且使得显微镜应用于活组织变得困难，并且不可能在许多临床应用中应用。相干反斯托克斯拉曼散射(CARS)显微镜是一种可以使用特定分子的光谱指纹作为造影剂来进行光学显微镜的方法。通过将两个激光光源调谐到正确的波长，可以激发特定的分子光谱特征，从而实现高度特定的显微镜。这个过程是非线性的，因此成像体积被限制在三维空间，这使得显微镜能够自然地产生高分辨率的光学切片，这些切片可以被重建以产生三维图像。然而，当人们希望更深入地成像到样品中时，激光必须穿过样品，并且被像差，从而在远远超过100微米的深度处导致信号和空间分辨率的损失。这个问题类似于地面天文学家通过大气层检查太空时所面临的问题。通过使用有源光学元件(自适应光学)，他们已经证明了大气像差可以消除，或者至少可以最小化。以前，我们对多光子和共聚焦显微镜采用了相同的技术，并取得了相当大的成功；然而，这两种成像方式都需要以某种形式向样品中添加荧光化合物，从而扰乱组织的自然状态。该项目的目的是探索自适应光学(AO)在CARS显微镜中的应用，通过两周的时间将自适应光学应用于显微镜和CARS成像方面的世界领先专业知识联系起来。通过将光子学研究所的AO系统安装到哈佛CARS显微镜上，我们将能够迅速确定AO是否如人们强烈怀疑的那样在CARS显微镜中发挥作用。因此，这一建议提供了一种快速、低成本的方法来评估用于深度CARS显微镜的AO。

Optical microscopy has provided scientists, and clinicians, with the ability to image living tissue with sub-cellular resolution for around four hundred years. This ability has lead to a number of scientific breakthroughs both in the life and physical sciences. Significant advances in general optics have frequently been achieved by the desire to image samples with increasing resolution so that the finest structures can be resolved. Indeed just over 130 years ago Abbe advanced the study of optics with his work on improved optical microscopy with the introduction of diffraction theory and the study of optical aberrations. However, crucial to all forms of microscopy is the ability to have contrast between the different structures present within the sample. For most of the last fifty years this has been provided by the addition of fluorescent labels to the sample culminating in the use of genetically modified samples so that they naturally express a fluorescent label. Even in this case the sample is not in its natural state and thus the method is not practical for a number of samples, and makes the application of microscopy to living tissue difficult, and impossible for many clinical applications.Coherent Anti-Stokes Raman Scattering (CARS) microscopy is a method by which optical microscopy can be undertaken using the spectral fingerprint of a specific molecule as the contrast agent. By tuning two laser sources to the correct wavelengths specific molecular spectral features can be excited enabling highly specific microscopy. The process is non-linear and hence the imaging volume is restricted in three dimensions offering the ability of the microscope to naturally generate high resolution optical slices which can be reconstructed to produce three dimensional images.However, as one wishes to image more deeply into a sample the laser light has to pass through the sample and is aberrated leading to a loss of signal and spatial resolution at depths much beyond 100microns. This problem is similar to that faced by ground based astronomers examining space through the atmosphere. Through the use of active optical elements (adaptive optics) they have demonstrated that the atmospheric aberrations can be removed, or at least minimised. Previously we have adopted the same technology to multiphoton and confocal microscopy with considerable success; however, both of these imaging modalities require the addition of fluorescent compounds to the sample in some form thus perturbing the natural state of the tissue. The aim of this project is to explore the use of adaptive optics (AO) in CARS microscopy by linking, for two weeks, world leading expertise in both the application of adaptive optics to microscopy and CARS imaging. By fitting the Institute of Photonics AO system to the Harvard CARS microscope we will rapidly be able to determine if AO has a role to play in CARS microscopy as is strongly suspected. This proposal thus offers a rapid, low cost route to the evaluation of AO for in-depth CARS microscopy.