Gaigalas Adolfas K, Choquette Steven, Zhang Yu-Zhong
National Institute of Standards and Technology, Gaithersburg, MD 20899.
Life Technologies, 29851 Willow Creek Rd., Eugene, OR 97402.
J Res Natl Inst Stand Technol. 2013 Jan 14;118:15-28. doi: 10.6028/jres.118.002. eCollection 2013.
Measurements of absorbance and fluorescence emission were carried out on aqueous suspensions of polystyrene (PS) microspheres with a diameter of 2.5 µm using a spectrophotometer with an integrating sphere detector. The apparatus and the principles of measurements were described in our earlier publications. Microspheres with and without green BODIPY(@) dye were measured. Placing the suspension inside an integrating sphere (IS) detector of the spectrophotometer yielded (after a correction for fluorescence emission) the absorbance (called A in the text) due to absorption by BODIPY(@) dye inside the microsphere. An estimate of the absorbance due to scattering alone was obtained by subtracting the corrected BODIPY(@) dye absorbance (A) from the measured absorbance of a suspension placed outside the IS detector (called A1 in the text). The absorption of the BODIPY(@) dye inside the microsphere was analyzed using an imaginary index of refraction parameterized with three Gaussian-Lorentz functions. The Kramer-Kronig relation was used to estimate the contribution of the BODIPY(@) dye to the real part of the microsphere index of refraction. The complex index of refraction, obtained from the analysis of A, was used to analyze the absorbance due to scattering ((A1 - A) in the text). In practice, the analysis of the scattering absorbance, A1-A, and the absorbance, A, was carried out in an iterative manner. It was assumed that A depended primarily on the imaginary part of the microsphere index of refraction with the other parameters playing a secondary role. Therefore A was first analyzed using values of the other parameters obtained from a fit to the absorbance due to scattering, A1-A, with the imaginary part neglected. The imaginary part obtained from the analysis of A was then used to reanalyze A1-A, and obtain better estimates of the other parameters. After a few iterations, consistent estimates were obtained of the scattering and absorption cross sections in the wavelength region 300 nm to 800 nm.
使用带有积分球探测器的分光光度计，对直径为2.5 µm的聚苯乙烯（PS）微球水悬浮液进行吸光度和荧光发射测量。测量仪器和原理在我们早期的出版物中有描述。对含有和不含绿色BODIPY(@)染料的微球进行了测量。将悬浮液置于分光光度计的积分球（IS）探测器内（在对荧光发射进行校正后），可得到由于微球内BODIPY(@)染料吸收而产生的吸光度（文中称为A）。通过从置于IS探测器外部的悬浮液的测量吸光度（文中称为A1）中减去校正后的BODIPY(@)染料吸光度（A），可得到仅由散射引起的吸光度估计值。使用由三个高斯 - 洛伦兹函数参数化的虚折射率来分析微球内BODIPY(@)染料的吸收。利用克莱默 - 克朗尼格关系来估计BODIPY(@)染料对微球折射率实部的贡献。从A的分析中获得的复折射率用于分析由散射引起的吸光度（文中为(A1 - A)）。实际上，对散射吸光度A1 - A和吸光度A的分析是以迭代方式进行的。假设A主要取决于微球折射率的虚部，其他参数起次要作用。因此，首先使用从对由散射引起的吸光度A1 - A进行拟合得到的其他参数值（忽略虚部）来分析A。然后将从A的分析中获得的虚部用于重新分析A1 - A，并获得其他参数的更好估计值。经过几次迭代后，在300 nm至800 nm波长区域内获得了散射和吸收截面的一致估计值。
