Building better probes for 2 photon microscopy

为 2 光子显微镜构建更好的探针

• 批准号: 7905078
• 负责人: Mikhail Drobizhev
• 金额: $ 24.1万
• 依托单位: MONTANA STATE UNIVERSITY - BOZEMAN
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-09-30 至 2012-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7905078
• 关键词: Address; Amino Acids; Area; Biological Sciences; Cells; Charge; Chemical Structure; Chemistry; Collaborations; Communities; Complex; Data; Fiber; Genetic Engineering; Hand; Image; Laser Scanning Microscopy; Lasers; Life; Location; Methods; Microscopy; Mutate; Mutation; Pattern; Photons; Physical Chemistry; Physics; Physiologic pulse; Process; Property; Proteins; Resolution; S-1 Antimetabolite agent; Sapphire; Series; Signal Transduction; Spectrum Analysis; Structure; Time; Tissues; Variant; absorption; base; chromophore; dipole moment; electric field; fluorescence imaging; improved; mutant; public health relevance; research study; two-photon

DESCRIPTION (provided by applicant): Fluorescent proteins (FP) are revolutionizing practically all areas of life sciences. Cell targeting with FPs is much more specific than with non-genetically encoded fluorescing probes or markers, and is providing an incredible degree of precision with which process in living tissues can be studied. Starting with the green fluorescing protein, first introduced about 15 years ago, various types of blue-, yellow-, and red-fluorescing proteins have been extensively mutated and studied to create brighter and better probes. Two photon laser scanning microscopy is currently the method of choice for high resolution deep living tissue imaging. Naturally, there is substantial drive to adapt FPs for the two-photon microscopy. However, these efforts have been seriously hampered, so far, by the low cross section of two-photon absorption (2PA), which for currently known FPs is, C2 < 102 GM. (1 Goeppert-Mayer = 10-50 cm4 s-1 photon-1). On the other hand, from numerous studies in other areas of physics and chemistry, it is well known that the 2PA cross sections may be as large as, C2 =103 -104 GM, including fluorescing molecules of the size and complexity comparable to that of FPs. Furthermore, extensive studies of the nonlinear absorption in various organic chromophores (again performed for different reasons) have revealed basic structure-to-property relationships that allow routinely increase the 2PA cross section by orders of magnitude. This proposal is a collaboration between a genetic engineering/fluorescence imaging group and a nonlinear spectroscopy/physical chemistry groups. We are addressing the issue of increasing the efficiency of FPs probes specifically for two-photon fluorescence imaging. Our main challenge comes from the fact (well known in 2PA spectroscopy community) that there is no straightforward relationship between the conventional (one-photon) brightness and the efficiency of two-photon excitation. We are proposing a series of experiments, which will: (Aim 1 and 2) Identify the best two-photon FPs by quantifying the two-photon spectra and cross sections of a broad range of existing fluorescing proteins in a broad spectral range, from 550 to 1500 nm; (Aims 3) Perform specific mutations on the charged amino acids in the surrounding protein cage, such that the 2PA efficiency is maximized by optimizing the strong local electric field at the chromophore location. The extensive preliminary data presented in this proposal strongly supports this hypothesis. We expect to increase the two-photon brightness up to 10-100 times, especially in the red- and near-IR range of excitation wavelengths. Public Health Relevance: This collaborative effort between biologists and physicists will resolve a long-standing obstacle in real- time deep tissue imaging due to insufficient two-photon efficiency of available genetically encoded markers. We are going to dramatically enhance the two-photon efficiency by introducing specific mutations in the protein cage, which will increase the two-photon cross section of the chromophore by up to two orders of magnitude.

描述（由申请人提供）：荧光蛋白（FP）正在彻底改变生命科学的几乎所有领域。使用 FP 进行细胞靶向比使用非基因编码的荧光探针或标记物更具特异性，并且为研究活组织中的过程提供了令人难以置信的精确度。从大约 15 年前首次推出的绿色荧光蛋白开始，各种类型的蓝色、黄色和红色荧光蛋白已被广泛突变和研究，以创造出更亮、更好的探针。双光子激光扫描显微镜是目前高分辨率深层活组织成像的首选方法。自然地，有很大的动力将FP应用于双光子显微镜。然而，到目前为止，这些努力受到双光子吸收（2PA）截面低的严重阻碍，对于目前已知的 FP 来说，C2 < 102 GM。 （1 Goeppert-Mayer = 10-50 cm4 s-1 photon-1）。另一方面，从物理和化学其他领域的大量研究中，众所周知，2PA 横截面可能大至 C2 =103 -104 GM，其中包括与 FP 相当的大小和复杂性的荧光分子。此外，对各种有机发色团的非线性吸收的广泛研究（再次出于不同的原因进行）揭示了基本的结构与性能关系，可以使 2PA 横截面常规增加几个数量级。该提案是基因工程/荧光成像小组和非线性光谱/物理化学小组之间的合作。我们正在解决提高专门用于双光子荧光成像的 FP 探针效率的问题。我们的主要挑战来自这样一个事实（在 2PA 光谱界众所周知）：传统（单光子）亮度与双光子激发效率之间没有直接关系。我们提出了一系列实验，这些实验将：（目标 1 和 2）通过量化 550 至 1500 nm 宽光谱范围内现有荧光蛋白的双光子光谱和横截面来识别最佳双光子 FP； （目标 3）对周围蛋白笼中的带电氨基酸进行特定突变，通过优化发色团位置的强局部电场来最大化 2PA 效率。该提案中提供的大量初步数据有力地支持了这一假设。我们期望将双光子亮度提高至 10-100 倍，特别是在激发波长的红光和近红外范围内。公共卫生相关性：生物学家和物理学家之间的合作将解决由于可用基因编码标记的双光子效率不足而导致的实时深层组织成像中长期存在的障碍。我们将通过在蛋白质笼中引入特定突变来显着提高双光子效率，这将使发色团的双光子横截面增加多达两个数量级。