Deschout Hendrik, Hagman Joel, Fransson Sophia, Jonasson Jenny, Rudemo Mats, Lorén Niklas, Braeckmans Kevin
Biophotonic Imaging Group, Lab General Biochemistry and Physical Pharmacy, Ghent University, Harelbekestraat 72, B-9000 Gent, Belgium.
Opt Express. 2010 Oct 25;18(22):22886-905. doi: 10.1364/OE.18.022886.
Confocal or multi-photon laser scanning microscopes are convenient tools to perform FRAP diffusion measurements. Despite its popularity, accurate FRAP remains often challenging since current methods are either limited to relatively large bleach regions or can be complicated for non-specialists. In order to bring reliable quantitative FRAP measurements to the broad community of laser scanning microscopy users, here we have revised FRAP theory and present a new pixel based FRAP method relying on the photo bleaching of rectangular regions of any size and aspect ratio. The method allows for fast and straightforward quantitative diffusion measurements due to a closed-form expression for the recovery process utilizing all available spatial and temporal data. After a detailed validation, its versatility is demonstrated by diffusion studies in heterogeneous biopolymer mixtures.
共聚焦或多光子激光扫描显微镜是进行荧光恢复后光漂白（FRAP）扩散测量的便捷工具。尽管其很受欢迎，但准确的FRAP测量仍然常常具有挑战性，因为目前的方法要么限于相对较大的漂白区域，要么对非专业人员来说可能很复杂。为了将可靠的定量FRAP测量带给广大激光扫描显微镜用户群体，我们在此修订了FRAP理论，并提出了一种基于像素的新FRAP方法，该方法依赖于对任何尺寸和纵横比的矩形区域进行光漂白。由于利用所有可用的空间和时间数据对恢复过程有一个封闭形式的表达式，该方法允许进行快速且直接的定量扩散测量。经过详细验证后，通过在异质生物聚合物混合物中的扩散研究证明了其通用性。
