Building better probes for 2 photon microscopy

描述（由申请人提供）：荧光蛋白（FP）正在彻底改变生命科学的所有领域。与非遗传编码荧光探针或标记物相比，FPs的细胞靶向性更强，并且提供了令人难以置信的精确度，可以研究活组织中的过程。从大约15年前首次引入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经被广泛地突变和研究，以创造更亮、更好的探针。双光子激光扫描显微镜是目前高分辨率深部活体组织成像的首选方法。自然地，有大量的驱动，以适应FPs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）低截面的严重阻碍，目前已知的FPs为C2 < 102 GM. （1 Goeppert-Mayer = 10-50 cm4 s-1光子-1）。另一方面，从其他物理和化学领域的大量研究中，我们都知道2PA的横截面可能大到，C2 =103 -104 GM，包括荧光分子的大小和复杂性可与FPs相比较。此外，对各种有机发色团的非线性吸收的广泛研究（同样出于不同的原因）已经揭示了基本的结构-性质关系，这些关系允许常规地将2PA横截面增加几个数量级。该提案是由基因工程/荧光成像小组和非线性光谱/物理化学小组合作完成的。我们正在解决的问题，提高效率的FPs探针，特别是双光子荧光成像。我们的主要挑战来自这样一个事实（在2PA光谱界众所周知），即传统的（单光子）亮度和双光子激发效率之间没有直接的关系。我们提出了一系列的实验，这将：（目标1和2）通过在550到1500 nm的宽光谱范围内定量现有荧光蛋白的双光子光谱和横截面来确定最佳的双光子FPs；（Aims 3）对周围蛋白笼中的带电氨基酸进行特异性突变，通过优化发色团位置的强局部电场，使2PA效率最大化。本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到10-100倍，特别是在激发波长的红色和近红外范围内。公共卫生相关性：生物学家和物理学家之间的这项合作努力将解决长期以来由于现有遗传编码标记的双光子效率不足而导致的实时深层组织成像障碍。我们将通过在蛋白质笼中引入特定的突变来显著提高双光子效率，这将使发色团的双光子横截面增加两个数量级。

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.