Lasers for Science, Central Laser Facility, Science and Technology Facilities Council, Rutherford Appleton Laboratory, Harwell Science and Innovation Campus, Didcot OX110QX, UK.
Eur Biophys J. 2011 Feb;40(2):131-41. doi: 10.1007/s00249-010-0631-x. Epub 2010 Oct 15.
The ability to detect raft structures in membranes continues to present a problem, especially in the membranes of live cells. Rafts, generally considered to be small (< 200 nm) sphingolipid-rich regions, are commonly modelled using lipid vesicle systems where the ability of fluorophore-labelled lipids to preferentially locate into domains (basically large rafts) is investigated. Instead, in this study the motional properties of different fluorophores were determined using two-photon excitation and time-correlated single-photon counting coupled with diffraction-limited imaging with polarizing optics in scanning mode to obtain nanosecond rotational correlation time images. To develop the method, well-characterized domain-containing models consisting of giant unilamellar vesicles comprising mixtures of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine, sphingomyelin and cholesterol were used with the fluorophores diphenylhexatriene, 1-palmitoyl-2-{6-[(7-nitro-2-1,3-benzoxadiazol-4-yl)amino]hexanoyl}-sn-glycero-3-phosphocholine and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(7-nitro-2-1,3-benzoxadiazol-4-yl). Accordingly, images of rotational correlation times of the probes revealed domain structures for all three probes consistent with other studies using different approaches. Rotational correlation time images of living cell membranes were also observed. The method has the advantage that not only does it enable domains to be visualised or imaged in a unique manner but that it can also potentially provide useful information on the lipid dynamics within the structures.
检测膜中的筏结构仍然是一个问题，尤其是在活细胞的膜中。筏通常被认为是小的（<200nm）富含鞘脂的区域，通常使用脂质囊泡系统来建模，其中荧光标记的脂质优先定位到域（基本上是大筏）的能力得到了研究。相反，在这项研究中，使用双光子激发和时间相关的单光子计数，结合具有偏光的扫描模式的衍射受限成像，确定了不同荧光团的运动性质。为了开发该方法，使用了具有特征的含有域的模型，这些模型由包含 1-棕榈酰基-2-油酰基-sn-甘油-3-磷酸胆碱、神经鞘磷脂和胆固醇的混合体的巨大单层囊泡组成，并用荧光团二苯己烯、1-棕榈酰基-2-{-6-[(7-硝基-2-1,3-苯并恶二唑-4-基)氨基]己酰基}-sn-甘油-3-磷酸胆碱和 1,2-二油酰基-sn-甘油-3-磷酸乙醇胺-N-（7-硝基-2-1,3-苯并恶二唑-4-基）进行了测试。因此，探针的旋转相关时间图像揭示了所有三种探针的域结构，与使用不同方法的其他研究一致。还观察到活细胞膜的旋转相关时间图像。该方法的优点不仅在于它能够以独特的方式可视化或成像域，而且还可以潜在地提供有关结构内脂质动力学的有用信息。
