Method of calibration of a fluorescence microscope for quantitative studies
Method of calibration of a fluorescence microscope for quantitative studies
复制标题
DOI：
10.1111/j.1365-2818.2011.03514.x
复制
发表时间：
2011-10-01
影响因子：
2
通讯作者：
Bernas, Tytus
中科院分区：
文献类型：
--
作者：
Kedziora, Katarzyna M.;Prehn, Johen H. M.;Bernas, Tytus
共焦显微镜是基于测量的荧光强度起源于一个有限的体积在成像标本。强度以绝对（尽管是任意的）单位量化，产生数字3D显微照片。因此，可以获得关于细胞和组织中生物分子的局部浓度的定量信息。这种方法需要估计光测量的精度（受噪声限制）并将数字强度单位转换为感兴趣分子的浓度（或数量）的绝对值。为了满足第一个先决条件，我们提出了一种测量信号和噪声的技术。该方法涉及任何固定的显微镜标本的图像的时间序列的注册。分析是一个多步骤的过程，它分离像素强度变化的单调，周期性和随机分量。这种方法允许同时确定噪声的暗和光子分量。因此，对于每个信号电平获得置信区间（总噪声估计）。该算法也可以应用于检测显微镜的机械不稳定性和照明光源的不稳定性。所提出的技术与一个简单的强度标准相结合，以提供相对强度单位到其绝对对应物（定量成像的第二个先决条件）的转换。此外，光漂白动力学的标准是用来估计光的功率传递到显微镜标本。因此，所提出的方法在一个步骤中提供了绝对强度校准、显微镜系统的精度和灵敏度的估计。
Confocal microscopy is based on measurement of intensity of fluorescence originating from a limited volume in the imaged specimen. The intensity is quantized in absolute (albeit arbitrary) units, producing a digital 3D micrograph. Thus, one may obtain quantitative information on local concentration of biomolecules in cells and tissues. This approach requires estimation of precision of light measurement (limited by noise) and conversion of the digital intensity units to absolute values of concentration (or number) of molecules of interest. To meet the first prerequisite we propose a technique for measurement of signal and noise. This method involves registration of a time series of images of any stationary microscope specimen. The analysis is a multistep process, which separates monotonic, periodic and random components of pixel intensity change. This approach permits simultaneous determination of dark and photonic components of noise. Consequently, confidence interval (total noise estimation) is obtained for every level of signal. The algorithm can also be applied to detect mechanical instability of a microscope and instability of illumination source. The presented technique is combined with a simple intensity standard to provide conversion of relative intensity units into their absolute counterparts (the second prerequisite of quantitative imaging). Moreover, photo bleaching kinetics of the standard is used to estimate the power of light delivered to a microscope specimen. Thus, the proposed method provides in one step an absolute intensity calibration, estimate of precision and sensitivity of a microscope system.