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几乎所有领域。用FP靶向细胞比用非遗传编码的荧光探针或标记物更具特异性，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突变和研究，以创造更明亮，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，有大量的驱动器，以适应FP的双光子显微镜。然而，迄今为止，这些努力受到双光子吸收（2 PA）的低横截面的严重阻碍，对于目前已知的FP，其为C2 < 102 GM。（1 Goeppert-Mayer = 10-50 cm 4 s-1光子-1）。另一方面，从物理学和化学的其它领域中的大量研究中，众所周知，2 PA横截面可以大到C2 =103 - 104 GM，包括大小和复杂性与FP相当的荧光分子。此外，对各种有机发色团中的非线性吸收的广泛研究（再次出于不同的原因进行）已经揭示了基本的结构与性质的关系，其允许常规地以数量级增加2 PA横截面。该提案是基因工程/荧光成像组和非线性光谱/物理化学组之间的合作。我们正在解决的问题，提高效率的FP探针，特别是双光子荧光成像。我们的主要挑战来自于这样一个事实（在2 PA光谱学界众所周知），即传统（单光子）亮度和双光子激发效率之间没有直接的关系。我们提出了一系列实验，这些实验将：（目标1和2）通过在从550至1500 nm的宽光谱范围内量化宽范围的现有荧光蛋白质的双光子光谱和横截面来识别最佳双光子FP;（目的3）对周围蛋白质笼中的带电氨基酸进行特异性突变，使得通过优化发色团位置处的强局部电场来最大化2 PA效率。本提案中提出的大量初步数据有力地支持了这一假设。我们希望将双光子亮度提高10-100倍，特别是在红色和近红外激发波长范围内。公共卫生相关性：生物学家和物理学家之间的这种合作努力将解决由于可用的遗传编码标记的双光子效率不足而导致的真实的时间深层组织成像中的长期障碍。我们将通过在蛋白质笼中引入特定突变来显著提高双光子效率，这将使发色团的双光子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.