SINGLE CELL FLUORESCENT PROBES

这个子项目是许多研究子项目中的一个 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得主要资金， 因此可以在其他CRISP条目中表示。所列机构为 中心，不一定是研究者的机构。 通常希望同时观察生化混合物中的几种荧光标记的组分，即使用多路复用。 当使用相同的激光源激发所有标签时，用于此的设备是最简单的，并且这种布置还具有更容易解释数据的优点，因为所有标签接收相同的输入功率。 然而，用一个激发源进行多路复用是困难的。 这是因为靠近激发源发射的染料倾向于最有效地吸收光，因为它们在该波长处具有最大吸收，而那些发射进一步进入红色的染料具有红移吸收光谱并且在激发波长处收获较少的光子。 已使用排列成使FRET最大化的染料的组合来缓解该问题。 不幸的是，这只是一个部分的解决方案，因为在FRET系统中的能量转移的效率是由重叠积分。 如果供体染料的发射与受体染料的吸收的重叠小，则能量转移将小。 这项工作的中心假设是，当供体和受体系统通过不允许它们变成平面的共轭连接基连接时，则可以通过键发生从供体到受体的快速能量转移。 氢键能量转移的机理与FRET的F？rster基础不同，并且没有已知的供体片段的发射与受体部分的吸收重叠的要求。因此，适当设计的通过键能量转移盒可以通过供体部分或多个供体部分在方便的波长（例如488 nm：来自Ar激光器的激发）下吸收光子，通过缀合的连接体将能量快速转移到在远更长的波长下发射的受体片段。在该方案中，对施主吸收和受主发射波长之间的差没有限制。 因此，可以设计在短波长处强烈吸收并在几个波长处（由受体的化学性质决定）以非常相似的强度明亮地发射的染料，这些波长相隔许多波数，即具有优异的分辨率。 将共轭体系中的多于一个供体与受体偶联有利于吸收更多的光，从而增加发射强度。总之，通过键能量转移盒具有增加从由在单一波长下操作的激光源激发的若干探针获得的分辨率和荧光强度两者的潜力。 蛋白质通常不能通过被动扩散进入细胞，而是需要主动转运。 虽然一些蛋白质也可以通过显微注射、包埋在脂质体、病毒载体和电穿孔中转运到细胞中，但这些方法费力、耗时，并且通常效率低。 最近开发的一种方法涉及一种称为 战车 （Active Motif，卡尔斯巴德，CA）克服了这些问题。Chariot与蛋白质非共价复合成肽，并促进其运输到细胞中。 Chariot肽是无细胞毒性的，并且不依赖于转运蛋白或特异性受体而穿过质膜，从而避免了溶酶体降解途径。 Chariot肽具有高转运效率（65-95%），并且已经显示出快速共转运大荧光蛋白。 一旦内化，荧光蛋白-Chariot肽复合物迅速解离，从而允许荧光标记的蛋白质前进到其细胞内靶标，同时Chariot肽迅速降解。 使用Pep 1肽（或类似的载体系统）将蛋白质/通过键盒缀合物转移到活细胞中开辟了新的研究前景。 然而，使用Chariot系统导入细胞的蛋白质在胞质溶胶中是游离的并不明显;它们可以被封装在胞内囊泡中。我们研究的目标之一是通过在该中心进行的单分子研究来阐明这一点。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. It is often desirable to simultaneously observe several fluorescently tagged components in a biochemical mixture, i.e. using multiplexing. The apparatus for this is simplest when all the tags are excited using the same laser source, and this arrangement also has the advantage that data are more easily interpreted because all the labels receive the same input power. Multiplexing with one excitation source is, however, difficult. This is because dyes that emit close to the excitation source tend to absorb the light most efficiently since they have the greatest absorption at that wavelength, while those dyes that emit further into the red have red-shifted absorption spectra and harvest less photons at the excitation wavelength. Combinations of dyes arranged to maximize FRET have been used to alleviate this problem. Unfortunately, this is only a partial solution because the efficiency of the energy transfer in FRET systems is governed by the overlap integral. If the overlap of the emission of the donor dye with the absorption of the acceptor dye is small, then the energy transfer will be small. The central hypothesis of this work is that when a donor and acceptor systems are connected via a conjugated linker that does not allow them to become planar then rapid energy transfer from the donor to the acceptor may occur through bonds. Through-bond energy transfer is mechanistically different to the F¿rster basis for FRET, and there is no known requirement for overlap of the emission of the donor fragment with the absorption of the acceptor part. Thus, appropriately designed through-bond energy transfer cassettes could absorb photons via a donor part, or parts, at a convenient wavelength (eg 488 nm: excitation from an Ar-laser), transfer the energy rapidly through the conjugated linker to the acceptor fragment that emits at a far longer wavelength. There is no constraint on the difference between the donor absorption and the acceptor emission wavelengths in this scheme. It therefore is possible to design dyes that absorb strongly at a short wavelength and emit brightly with very similar intensities at several wavelengths (governed by the chemical nature of the acceptor) that are many wavenumbers apart, ie with excellent resolution. Coupling more than one donor in a conjugated system with an acceptor facilitates absorption of more light thereby increasing the intensity of the emission. In summary, through bond energy transfer cassettes have the potential to increase both the resolution and fluorescence intensities obtained from several probes excited by a laser source operating at a single wavelength. Proteins generally cannot enter cells by passive diffusion, but require active transport. While some proteins can also be transported into cells by microinjection, entrapped in liposomes, viral vectors, and electroporation, such methods are laborious, time consuming, and often have low efficiencies. A recently developed method involving a peptide called Chariot (Active Motif, Carlsbad, CA) overcomes these problems. Chariot non-covalently complexes with proteins to peptides and facilitates their transport into cells. The Chariot peptide is non-cytotoxic, and crosses plasma membranes independent of transporters or specific receptors, thus avoiding the lysosomal degradative pathway. The Chariot peptide has high transport efficiency (65-95%) and has already been shown to rapidly co-transport large fluorescent proteins. Once internalized, the fluorescent protein-Chariot peptide complex rapidly dissociates, thereby allowing the fluorescent-tagged protein to proceed to its intracellular target while concomitantly the Chariot peptide is rapidly degraded. Use of the Pep1 peptide (or similar carrier systems) to transfer protein/through-bond cassette conjugates into living cells opens new vistas of research. However, it is not evident that proteins imported into cells using the Chariot system are free in the cytosol; they could be encapsulated in intracellular vesicles. One of the objectives of our research is to elucidate this with single molecule studies performed at the center.