Fluorescent Probes for Multiplexed Intracellular Imaging

描述（由申请人提供）：相对于用于观察细胞荧光的精密仪器，用于细胞内成像的染料尚不发达。高级探测器有几个重要的特点。它们应该是小的、无细胞毒性的、水溶性的实体，不会干扰它们所结合的分子的功能。它们的光谱特性应该是这样的，它们在与所用激发源对应的波长上吸收非常强，然后以高量子产量发射。如果要同时标记和观察几个生物分子（即多路复用），则需要在一个方便的激发波长上强烈吸收，但发出具有不同斯托克位移的强烈荧光信号，从而获得高分辨率的染料组。应进行修饰，以促进在激发波长/功率下的吸收，避免细胞生物分子的自身荧光和细胞损伤。发射波长应该发生在相对较长的波长（例如800纳米），其中细胞基本上是透明的，使检测更容易。对于单分子检测方法，探针应抵抗光分解和涉及非荧光激发态的“闪烁”。最后，还有其他特性可以被设计成荧光探针。这些特性包括过渡偶极的各向同性，以消除基于FRET的距离测量中由取向因素引起的不确定性，以及通过门控测量观察的有利寿命特性，以增加信噪比。

DESCRIPTION (provided by applicant): Dyes for intracellular imaging are underdeveloped relative to the sophisticated instrumentation available for observing fluorescence in cells. Superior probes would have several important characteristics. They should be small, noncytotoxic, water-soluble entities that do not interfere with the function of the molecules to which they are conjugated. Their spectroscopic properties should be such that they absorb very strongly at wavelengths that correspond to the excitation source used, then emit with high quantum yields. If several biomolecules are to be labeled and observed simultaneously (ie multiplexing), then sets of dyes are required that absorb strongly at one convenient excitation wavelength, but emit sharp intense fluorescence signals with different Stoke's shifts giving high resolutions. Modifications should be available to facilitate absorption at excitation wavelengths/powers that avoid autofluorescence from cellular biomolecules and cell damage. The emission wavelengths should occur at relatively long wavelengths (eg 800 nm) where cells are essentially transparent enabling easier detection. For single molecule detection methods, the probes should resist photodecomposition and "blinking" involving non-fluorescent excited states. Finally, there are other properties that might be engineered into fluorescent probes. These include isotropy of transition dipoles to remove uncertainties caused by orientation factors in FRET- based distance measurements, and favorable lifetime characteristics for observation via gated measurements to increase signal-to-noise. In preliminary studies, a set of novel probes was devised for multiplexing in DNA sequencing. The conceptual basis for these is also applicable to intracellular imaging. They contain donor parts that absorb strongly at one excitation wavelength, twisted, but otherwise conjugated, linker systems that facilitate rapid "through-bond energy transfer", and acceptor parts that emit at wavelengths far from the excitation source. Thus they can be excited with high cross-sections by a single laser, then fluoresce strongly at different, resolvable wavelengths. This proposal is to create similar sets of "through-bond energy transfer cassettes" that are optimized for intracellular imaging, ie that conform to most or all of the criteria described above. They will be prepared (Burgess), optimized for superior spectroscopic properties (Burgess/Hochstrasser), and attached to a set of four model proteins (Schroeder/Burgess). The labeled-proteins will be transported into cells and observed using multicomponent, equilibrium fluorescence measurements to detect protein-protein interactions via FRET donor (Schroeder). The data collected will be analyzed via novel computer algorithms (Liu), then compared with that obtained by Hochstrasser et al for the same intracellular events observed at the single molecule level. This work proposed will provide superior intracellular probes for equilibrium and single molecule detection methods.