Sandoval Ruben M, Wang Exing, Molitoris Bruce A
Indiana University School of Medicine; Indianapolis, IN USA ; The Roudebush VA; Indianapolis, IN USA.
Department of Cellular and Structural Biology; University of Texas Health Science Center; San Antonio, TX USA.
Intravital. 2014 Mar 1;2(1). doi: 10.4161/intv.23674.
Maximizing 2-photon parameters used in acquiring images for quantitative intravital microscopy, especially when high sensitivity is required, remains an open area of investigation. Here we present data on correctly setting the black level of the photomultiplier tube amplifier by adjusting the offset to allow for accurate quantitation of low intensity processes. When the black level is set too high some low intensity pixel values become zero and a nonlinear degradation in sensitivity occurs rendering otherwise quantifiable low intensity values virtually undetectable. Initial studies using a series of increasing offsets for a sequence of concentrations of fluorescent albumin in vitro revealed a loss of sensitivity for higher offsets at lower albumin concentrations. A similar decrease in sensitivity, and therefore the ability to correctly determine the glomerular permeability coefficient of albumin, occurred in vivo at higher offset. Finding the offset that yields accurate and linear data are essential for quantitative analysis when high sensitivity is required.
在获取用于定量活体显微镜检查的图像时，尤其是在需要高灵敏度的情况下，最大化双光子参数仍是一个有待研究的领域。在此，我们展示了通过调整偏移量来正确设置光电倍增管放大器黑电平的数据，以便能够准确量化低强度过程。当黑电平设置过高时，一些低强度像素值会变为零，灵敏度会出现非线性下降，从而使原本可量化的低强度值几乎无法检测到。最初在体外对一系列浓度的荧光白蛋白使用一系列递增偏移量的研究表明，在较低白蛋白浓度下，较高偏移量会导致灵敏度损失。在体内，较高偏移量时也出现了类似的灵敏度下降，进而影响了正确测定白蛋白肾小球通透系数的能力。当需要高灵敏度时，找到能产生准确线性数据的偏移量对于定量分析至关重要。
