MRI-R2: Development of an long wavelength nonlinear optical microscope for harmonic and autofluorescence imaging of biological tissues

[0959525Campagnola]“该奖项是根据2009年美国复苏和再投资法案（公法111-5）资助的。”光谱重叠、光漂白、低信噪比和光散射等问题严重限制了荧光在组织成像中的应用。在过去的10-15年中，非线性光学显微镜技术得到了广泛的发展和采用，主要是因为它们能够克服许多这些限制。目前大多数非线性光学显微镜使用的激发波长在700-1000纳米之间，因为这是广泛使用的Ti:Sapphire飞秒激光器的标准调谐范围。虽然这些近红外（NIR）波长比使用可见激发源（例如488,514 nm）的共聚焦激光扫描显微镜（共聚焦激光扫描显微镜）对组织的穿透深度（几百微米vs 10微米）要深得多，但进一步提高这一极限，使与肿瘤和其他病理相关的胶原结构成像达到约1毫米，将具有巨大的生物学和生物医学效益。为了满足这一需求，本提案将邀请来自生物医学工程，物理学，植物生物学，癌症生物学和生物光子学的研究人员建立一个非线性显微镜平台，能够激发从680nm到1300nm的内在和外在荧光团。该仪器将由一个Ti：蓝宝石振荡器/光学参量振荡器（OPO）和一个自制的激光扫描显微镜组成，该显微镜针对更长的波长激发进行了优化。完成后，仪器将提供2和3光子激发荧光，二次谐波和三次谐波的检测。我们预计，使用光学参量振荡器提供的1000-1300 nm调谐范围将提供额外的2-3倍的组织成像深度增加。该仪器的成功构建和实施将使从乳腺癌和卵巢癌的植物和细胞模型到动物模型的广泛动态细胞研究成为可能。这些研究将彻底检查长波的潜力，以进行更深层次的成像和改进的荧光团的激发，自然激发进一步进入近红外。这种结构将为未来的生物学研究奠定实验框架，这些研究可能受益于基于更长波长的非线性光学方法。

0959525Campagnola"This award is funded under the American Recovery and Reinvestment Act of 2009 (Public Law 111-5)."Problems of overlapping spectra, photobleaching, low signal to noise and light scatter pose serious limitations on what can be achieved in practice in imaging tissues by fluorescence. Over the last 10-15 years nonlinear optical microscopy techniques have been developed and adopted widely, largely due to their ability to overcome many of these limitations. Most current nonlinear optical microscopes use excitation wavelengths between 700-1000 nm, as this is the standard tuning range of the widely used Ti:Sapphire femtosecond lasers. While these near infrared (NIR) wavelengths provide significantly deeper penetration (few hundred microns vs. 10 s of microns) into tissue than achievable with confocal laser scanning microscopes using visible excitation sources (e.g. 488, 514 nm), it would be of great biological and biomedical benefit to further improve upon this limit to achieve about 1 mm for imaging the collagen structure associated with tumors and other pathologies. To address this need, this proposal will engage researchers from biomedical engineering, physics, plant biology, cancer biology, and biophotonics to build a nonlinear microscopy platform capable of exciting intrinsic and extrinsic fluorophores from 680nm to 1300 nm. The instrument will be constructed from a Ti:Sapphire oscillator/ optical parametric oscillator (OPO) and a homebuilt laser scanning microscope optimized for longer wavelength excitation. When complete, the instrument will afford detection of 2 and 3-photon excited fluorescence, Second Harmonic Generation and Third Harmonic Generation. We anticipate that using the 1000-1300 nm tuning range provided by the optical parametric oscillator will provide an additional 2-3 fold increase in imaging depth in tissues. The successful construction and implementation of the instrumentation will enable a wide range of dynamic cell studies ranging from plant and cell models to animal models of breast cancer and ovarian cancer. These studies will thoroughly examine the potential of long wavelengths for deeper imaging and improved excitation of fluorophores that naturally excite further into the NIR. This construction will lay down the experimental framework for future biological studies that may benefit from longer wavelength based nonlinear optical approaches.