Wavelength and alignment tests for confocal spectral imaging systems
Wavelength and alignment tests for confocal spectral imaging systems
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共焦光谱成像系统的波长和对准测试
DOI：
10.1002/jemt.20249
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发表时间：
2005
影响因子：
2.5
通讯作者：
J. Lerner
中科院分区：
文献类型：
--
作者：
R. Zucker;J. Lerner
目前市场上的共聚焦光谱成像（CSI）显微镜系统通过进行光谱表征来描绘生物标本中的多种荧光蛋白、标记或染料。然而，我们发现一些CSI在特定系统内以及相关和不相关仪器之间呈现出不一致的参考光谱曲线。我们还发现不稳定的证据，如果不诊断，可能导致不一致的数据。这种可变性证实了对诊断工具的需求，以提供一种标准化的、客观的方法来表征不稳定性、不对准的证据，以及执行校准和验证功能。我们的方案使用一种廉价的多离子放电灯（MIDL），它含有Hg+， Ar+和无机荧光团，可以发出清晰，稳定的光谱特征，代替样品。MIDL表征验证了CSI系统的准确性和一致性，并验证了生物样品的获取。我们总共检查了10个CSI系统，所有这些系统都显示出光谱不一致，使我们能够识别出故障的子系统。10种仪器中只有一种达到了最佳性能预期。我们发现，使用发出绝对标准“参考光谱”的主光源使我们能够诊断仪器错误，并在标准化条件下测量精度和再现性。利用这些信息，CSI作业人员可以确定CSI系统是否处于最佳工作状态，并与其他CSI系统的性能进行客观比较。很明显，如果类似制造和模型的CSI系统被标准化，以从标准光源显示相同的光谱轮廓，那么研究人员可以确信，现实生活中的实验结果可以在任何类似的系统上重复。Microsc。科学通报，2005,(6):357 - 357。出版于2005年Wiley‐Liss， Inc。
Confocal spectral imaging (CSI) microscope systems now on the market delineate multiple fluorescent proteins, labels, or dyes within biological specimens by performing spectral characterizations. However, we find that some CSI present inconsistent spectral profiles of reference spectra within a particular system as well as between related and unrelated instruments. We also find evidence of instability that, if not diagnosed, could lead to inconsistent data. This variability confirms the need for diagnostic tools to provide a standardized, objective means of characterizing instability, evidence of misalignment, as well as performing calibration and validation functions. Our protocol uses an inexpensive multi‐ion discharge lamp (MIDL) that contains Hg+, Ar+, and inorganic fluorophores that emit distinct, stable spectral features, in place of a sample. An MIDL characterization verifies the accuracy and consistency of a CSI system and validates acquisitions of biological samples. We examined a total of 10 CSI systems, all of which displayed spectral inconsistencies, enabling us to identify malfunctioning subsystems. Only one of the 10 instruments met its optimal performance expectations. We have found that using a primary light source that emits an absolute standard “reference spectrum” enabled us to diagnose instrument errors and measure accuracy and reproducibility under normalized conditions. Using this information, a CSI operator can determine whether a CSI system is working optimally and make objective comparisons with the performance of other CSI systems. It is evident that if CSI systems of a similar make and model were standardized to reveal the same spectral profile from a standard light source, then researchers could be confident that real‐life experimental findings would be repeatable on any similar system. Microsc. Res. Tech. 68:307–319, 2005. Published 2005 Wiley‐Liss, Inc.