Fluorescence recovery after photobleaching on the confocal laser-scanning microscope: generalized model without restriction on the size of the photobleached disk
Fluorescence recovery after photobleaching on the confocal laser-scanning microscope: generalized model without restriction on the size of the photobleached disk
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DOI：
10.1117/1.3569620
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发表时间：
2011-04-01
影响因子：
3.5
通讯作者：
Ameloot, Marcel
中科院分区：
文献类型：
--
作者：
Smisdom, Nick;Braeckmans, Kevin;Ameloot, Marcel
在共焦激光扫描显微镜 (CLSM) 上进行的光漂白后荧光恢复 (FRAP) 对于与漂白光束分辨率相比较大的光漂白圆盘表现良好。对于接近这一分辨率的较小圆盘，当前提供封闭式解决方案的 FRAP 模型不允许准确提取扩散系数。我们提出的新的广义圆盘模型通过考虑漂白分辨率和总共焦成像分辨率来解决这个缺点。在线性光漂白的假设下获得封闭形式的解。此外，同时分析以不同磁盘尺寸收集的 FRAP 数据可以内在确定仪器分辨率参数，从而避免了外部校准的需要。引入了一种估计 FRAP 数据方差的新方法，以便通过非线性最小二乘法在这种全局分析方法中进行适当的加权。在同质样品上的两个独立 CLSM 上进行实验，提供大范围扩散系数的验证。 (C) 2011 年光电仪器工程师协会 (SPIE)。 [DOI：10.1117/1.3569620]
Fluorescence recovery after photobleaching (FRAP) carried out on a confocal laser-scanning microscope (CLSM) performs well for photobleached disks that are large compared to the resolution of the bleaching beam. For smaller disks approaching this resolution, current FRAP models providing a closed-form solution do not allow one to extract the diffusion coefficient accurately. The new generalized disk model we present addresses this shortcoming by bringing into account the bleaching resolution and the total confocal imaging resolution. A closed-form solution is obtained under the assumption of linear photobleaching. Furthermore, simultaneous analysis of FRAP data collected at various disk sizes allows for the intrinsic determination of the instrumental resolution parameters, thereby obviating the need for an extrinsic calibration. A new method to estimate the variance of FRAP data is introduced to allow for proper weighting in this global analysis approach by nonlinear least squares. Experiments are performed on two independent CLSMs on homogeneous samples providing validation over a large range of diffusion coefficients. (C) 2011 Society of Photo-Optical Instrumentation Engineers (SPIE). [DOI: 10.1117/1.3569620]