Development of photothermal microscopy for biomedical applications

描述(申请人提供)：厚组织中吸收(即非荧光)发色团的成像对显微镜工作者提出了挑战。最近，一种名为光热显微镜(PM)的新技术引起了人们的关注，该技术将两束激光聚焦到一个样品中。一束(加热束)被调谐到生色团吸收线，而另一束被设置在任何吸收带之外的波长(探针束)。当加热光束被吸收时，能量被沉积到组织中，产生局部密度波动。这种密度变化导致生色团的折射率发生微小的瞬时变化，然后由探测光束进行监测。在早期的应用中，PM已被证明以前所未有的灵敏度跟踪细胞培养中的金纳米颗粒。最近，PM在成像活细胞中的内源性发色团方面也被证明是非常有效的。例如线粒体和红细胞的成像，其3D空间分辨率可与共聚焦显微镜相媲美。尽管首相前景看好，但仍有几个悬而未决的问题。具体地说：在厚厚的组织中进行PM是可能的吗？PM对比的发色团种类到底是什么？到目前为止，只对透射光配置中的薄样品进行了PM。此外，负责PM对比的发色团种类要么未知(在线粒体成像的情况下)，要么推测(在红细胞成像的情况下)。我们建议1)开发一种新型的扫描PM，首次使用单光子或双光子吸收在厚组织中进行光热成像，以及2)使用配备超宽带(UV至THz)激光的光热光谱筛选平台，明确识别和表征现有和新的内源性造影剂。我们将首先集中研究线粒体中的细胞色素、血细胞中的血红素蛋白和通道视紫红质。上述目标的完成将是PM在生物医学成像界获得广泛接受所必不可少的，并将为提供高灵敏度、高分辨率吸收对比和分子特异性的新技术奠定基础。 与公共健康相关：我们建议开发一种光学显微镜技术，提供对组织中吸收(即非荧光)蛋白质或分子的超高灵敏度3D成像。这项技术将有助于体内成像研究的应用和临床上的快速组织诊断。

DESCRIPTION (provided by applicant): The imaging of absorbing (i.e. nonfluorescent) chromophores in thick tissue poses a challenge for microscopists. Recently, a new technique called photothermal microscopy (PM) has been gaining attention, which involves the focusing two laser beams into a sample. One beam (the heating beam) is tuned to a chromophore absorption line while the other is set at wavelength outside any absorption bands (the probe beam). When the heating beam is absorbed, energy is deposited into the tissue, producing a local density fluctuation. This density change results in a small transient change in the refractive index about the chromophore, which is then monitored by the probe beam. In early applications, PM has been shown to track gold nanoparticles in cell cultures with unprecedented sensitivity. More recently, PM has also been shown to be remarkably effective at imaging endogenous chromophores in live cells. Examples include imaging of mitochondria and erythrocytes with 3D spatial resolution comparable to confocal microscopy. As promising as PM is, several open questions still remain. Specifically: is it possible to perform PM in thick tissue, and what exactly are the chromophore species responsible for PM contrast? To date, PM has only been performed with thin samples in a transmitted light configuration. Moreover, the chromophore species responsible for PM contrast are either unknown (in the case of mitochondrial imaging) or speculated (in the case of erythrocyte imaging). We propose to 1) develop a novel scanning PM that can perform photothermal imaging in thick tissue, for the first time, using on one- or two-photon absorption, and 2) unambiguously identify and characterize both existing and new endogenous contrast agents using a photothermal spectroscopy screening platform equipped with an ultra-wide bandwidth (UV to THz) laser. We will initially concentrate on the study of cytochrome in mitochondria, heme protein in blood cells, and channel rhodopsin. A completion of the above aims will be indispensible for PM to gain widespread acceptance in the biomedical imaging community, and will lay the groundwork for a new technology that provides high sensitivity, high resolution absorption contrast with molecular specificity. PUBLIC HEALTH RELEVANCE: We propose to develop an optical microscopy technique that provides ultrahigh sensitivity 3D imaging of absorbing (i.e. non-fluorescent) proteins or molecules in tissue. This technology will be useful for in- vivo imaging research applications and rapid tissue diagnosis in the clinic.