Determination of particle number and brightness using a laser scanning confocal microscope operating in the analog mode
Determination of particle number and brightness using a laser scanning confocal microscope operating in the analog mode
复制标题
DOI：
10.1002/jemt.20526
复制
发表时间：
2008-01-01
影响因子：
2.5
通讯作者：
Gratton, Enrico
中科院分区：
文献类型：
--
作者：
Dalal, Rooshin B.;Digman, Michelle A.;Gratton, Enrico
我们描述了一种方法来获得的亮度和分子的数量在每个像素的图像堆栈与激光扫描显微镜获得。该方法是基于由于分子在像素中的扩散的强度波动。对于在模拟模式下操作的检测器，方差必须与强度成比例。一旦这个常数被校准，我们使用方差和强度之间的比率来推导粒子亮度。然后，从强度与亮度的比率，我们获得像素中的平均粒子数。我们表明，该方法与分子在溶液中，并与波动相关光谱得到的结果是可比的。我们比较了在模拟和光子计数模式下工作的探测器所获得的结果。虽然在光子计数模式下工作的探测器的动态范围是上级的，模拟探测器的性能是可以接受的，在一般的实验条件下。由于大多数商业激光扫描显微镜在模拟模式下操作，亮度和粒子数的计算可以应用于这些仪器获得的数据，只要方差与强度成正比。我们证明，恢复亮度的mEGFP，浓度无关，是相似的，无论是在溶液中测量，或在两种不同的细胞类型。此外，我们区分移动的和非移动的组件，并介绍了一种方法来校正强度的缓慢变化。
We describe a method to obtain the brightness and number of molecules at each pixel of an image stack obtained with a laser scanning microscope. The method is based on intensity fluctuations due to the diffusion of molecules in a pixel. For a detector operating in the analog mode, the variance must be proportional to the intensity. Once this constant has been calibrated, we use the ratio between the variance and the intensity to derive the particle brightness. Then, from the ratio of the intensity to the brightness we obtain the average number of particles in the pixel. We show that the method works with molecules in solution and that the results are comparable to those obtained with fluctuation correlation spectroscopy. We compare the results obtained with the detector operating in the analog and photon counting mode. Although the dynamic range of the detector operating in the photon counting mode is superior, the performance of the analog detector is acceptable under common experimental conditions. Since most commercial laser scanning microscopes operate in the analog mode, the calculation of brightness and number of particles can be applied to data obtained with these instruments, provided that the variance is proportional to the intensity. We demonstrate that the recovered brightness of mEGFP, independent of concentration, is similar whether measured in solution or in two different cell types. Furthermore, we distinguish between mobile and immobile components, and introduce a method to correct for slow variations in intensity.