Development of photothermal microscopy for biomedical applications

描述（由申请人提供）：厚组织中吸收（即非荧光）发色团的成像对显微镜学家提出了挑战。最近，一种名为光热显微镜（PM）的新技术引起了人们的关注，该技术涉及将两束激光聚焦到样品上。一束（加热束）被调谐到发色团吸收线，而另一束被设置在任何吸收带之外的波长（探测束）。当加热光束被吸收时，能量沉积到组织中，产生局部密度波动。这种密度变化会导致发色团的折射率发生微小的瞬态变化，然后由探针光束监测。在早期应用中，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也被证明在活细胞内源性发色团成像方面非常有效。例如，线粒体和红细胞的三维空间分辨率成像可与共聚焦显微镜相媲美。尽管总理很有前途，但仍存在几个悬而未决的问题。具体来说：是否有可能在厚组织中进行PM，以及究竟是哪些发色团负责PM对比？迄今为止，PM仅在透射光配置下使用薄样品进行。此外，负责PM对比的发色团种类要么是未知的（在线粒体成像的情况下），要么是推测的（在红细胞成像的情况下）。我们建议1)开发一种新型扫描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，2)使用配备超宽带（UV到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。上述目标的完成对于PM在生物医学成像界获得广泛接受是必不可少的，并且将为提供高灵敏度，高分辨率吸收对比度和分子特异性的新技术奠定基础。

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.