MRI Instrumentation Development: Construction of a Real-Time Fluorescence and Phosphorescence Lifetime Imaging 3D Microscope Based on a Novel Analog Acquisition Method

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。该仪器开发项目基于一种新颖的实时衰减时间测量技术，创造了一种新型的三维荧光和磷光寿命成像显微镜。衰变时间或寿命是指分子在吸收光子后保持电子激发态的时间。激发态寿命测量对差异染料分布和样品散射和吸收差异不敏感，这些参数仅影响荧光强度测量，可用于测量离子浓度，标记物之间的相互作用或通过NADH成像的能量代谢。某些化合物的磷光寿命可用于测量氧浓度。在荧光寿命成像显微镜（FLIM）中，图像像素值反映荧光团的寿命而不是强度。在这里描述的项目中，开发了一种新型的寿命确定方法，并将其应用于FLIM系统中，该系统将寿命图像直接实时显示在屏幕上，从而大大加快了寿命图像的获取速度。显微镜的构建将在生物物理成像光电子学发展资源（DRBIO）中进行。将建造两台仪器，一台为磷光时间尺度（微秒）设计，将在需要使用氧敏感荧光粉进行3d分辨率氧成像的项目中进行测试。第二种仪器将用于荧光时间尺度（纳秒）的开发，并测试一个项目，该项目将利用更快的寿命成像来更好地阐明分子转录启动子在体内的调节和相互作用。该仪器的独特功能——比现有的FLIM系统进行3d分辨终身成像的速度快10-1000倍——将确保许多未来的合作项目受益于该系统。该技术不仅可以作为一种成像方式，而且可以应用于许多分析应用，例如通过光纤检测氧浓度。这些仪器的设计和制造将为培养光学、非线性光学、高速电子学、生物物理学和光物理学方面的研究生和其他研究人员提供一个极好的机会。除了最初参与试验台实验的合作实验室外，世界各地的研究人员还将通过合作生物成像中心DRBIO获得该显微镜。一旦开发和测试，该仪器设计将通过在商业激光扫描成像系统上实施而商业化。

This award is funded under the American Recovery and Reinvestment Act of 2009 (Public Law 111-5).This instrument development project creates a new type of 3D fluorescence and phosphorescence lifetime imaging microscope based on a novel decay time measurement technique that operates in real-time. The decay time or lifetime is the time a molecule remains in an electronically excited state after absorbing a photon. Excited state lifetime measurements are insensitive to differential dye distributions and sample scattering and absorption differences, parameters that plague fluorescence intensity-only measurements, and can be used to measure ion concentrations, interactions between labeled or energy metabolism via NADH imaging. Phosphorescence lifetimes of certain compounds can be used for measuring oxygen concentrations. In fluorescence lifetime imaging microscopy (FLIM), the image pixel values reflect the fluorophore's lifetime rather than the intensity. In the project described here a new type of lifetime determination method is developed and applied in a FLIM system in which the lifetime image is displayed directly on the screen in real time enabling dramatically faster acquisition of lifetime images. The microscope construction will be carried out in the Developmental Resource for Biophysical Imaging Optoelectronics (DRBIO). Two instruments will be built, one designed for phosphorescence timescales (microsecond) which will be tested in projects requiring 3D-resolved oxygen imaging using oxygen-sensitive phosphors. A second instrument will be developed for fluorescence timescales (nanosecond) and tested a project that will utilize faster lifetime imaging for better elucidation of the regulation and interactions of molecular transcription initiators in vivo. The unique capabilities of the instrument - 3D-resolved lifetime imaging carried out 10-1000 times faster than possible on the currently available FLIM systems - will ensure that a number of future collaborative projects that will benefit from the system. The novel technique developed is not only applicable as an imaging modality, but can be applied in a number of analytical applications such as oxygen concentration sensing through an optical fiber. Design and fabrication of the instruments will provide an excellent opportunity for training graduate students and other researchers in optics, nonlinear optics, high speed electronics, biophysics and photophysics. In addition to the initial collaborating laboratories involved in the test bed experiments, the microscope will be available to researchers from around the world through DRBIO, a collaborative bioimaging center. Once developed and tested, the instrument design will be made commercially available by implementation on commercial laser scanning imaging systems.