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. ? ? ?
