Periasamy N, Verkman A S
Department of Medicine, University of California, San Francisco 94143-0532.
Anal Biochem. 1992 Feb 14;201(1):107-13. doi: 10.1016/0003-2697(92)90181-6.
Background fluorescence is a major concern in time-resolved microfluorimetry studies of biological samples. A general method for subtraction of an arbitrary background signal in measurements of lifetime and anisotropy decay by multiharmonic Fourier transform spectroscopy is presented. Multifrequency phase and modulation values are measured in parallel by transformation of digitized time-domain waveforms into the frequency domain. For subtraction of background, time-domain waveforms are acquired for emission and reference photomultipliers for sample (e.g., cell containing fluorophore) and blank (e.g., unlabeled cell). Time-domain waveforms obtained in a series of measurements (e.g., sample and blank for parallel and perpendicular orientations of an emission polarizer) are time-justified by least-squares fitting of reference channel waveforms or by phase comparison of the first Fourier harmonics of the reference channel. Background is then subtracted directly in the time domain, and the subtracted waveform is Fourier transformed to the frequency domain for analysis of lifetime or anisotropy decay. This approach yielded excellent background correction over a wide range of background intensities and decay profiles. The method was tested in cuvette fluorimetry with fluorescein and acridine orange and in fluorescence microscopy with living MDCK cells loaded with the pH indicator BCECF. Sample lifetimes and rotational parameters could be recovered accurately with greater than 50% of the signal arising from background. These results establish a direct and practical approach to subtraction of background in complex biological and chemical samples studied by frequency-domain fluorimetry.
背景荧光是生物样品时间分辨微荧光测定研究中的一个主要问题。本文提出了一种通过多谐波傅里叶变换光谱法在寿命和各向异性衰减测量中扣除任意背景信号的通用方法。通过将数字化时域波形变换到频域，并行测量多频相位和调制值。为了扣除背景，采集样品（例如含有荧光团的细胞）和空白（例如未标记的细胞）的发射和参比光电倍增管的时域波形。在一系列测量中获得的时域波形（例如发射偏振器平行和垂直方向的样品和空白）通过参比通道波形的最小二乘拟合或参比通道第一傅里叶谐波的相位比较进行时间校准。然后在时域中直接扣除背景，并将扣除后的波形傅里叶变换到频域以分析寿命或各向异性衰减。该方法在广泛的背景强度和衰减曲线范围内产生了出色的背景校正效果。该方法在使用荧光素和吖啶橙的比色皿荧光测定以及使用加载了pH指示剂BCECF的活MDCK细胞的荧光显微镜检查中进行了测试。样品寿命和旋转参数能够被准确恢复，背景产生的信号超过50%。这些结果建立了一种直接且实用的方法，用于在频域荧光测定研究的复杂生物和化学样品中扣除背景。
