Fung J, Hyun W, Dandekar P, Pedersen R A, Weier H U
Department of Obstetrics, Gynecology and Reproductive Sciences, University of California, San Francisco 94143-0720, USA.
J Assist Reprod Genet. 1998 May;15(5):323-30. doi: 10.1023/a:1022508930762.
Our purpose was to evaluate the utility of spectral imaging for multicolor, multichromosome enumeration in human interphase cell nuclei.
Chromosome-specific probes labeled with different fluorochromes or nonfluorescent haptens were obtained commercially or prepared in-house. Metaphase spreads, interphase lymphocytes, or blastomeres cells were hybridized with either 7 or 11 distinctly different probes. Following 46 hr of hybridization, slides were washed and detected using either a filter-based quantitative image processing system (QUIPS) developed in-house or a commercial spectral imaging system.
The filter-based fluorescence microscope system is preferred for simultaneous detection of up to seven chromosome targets because of its high sensitivity and speed. However, this approach may not be applicable to interphase cells when 11 or more targets need to be discriminated. Interferometer-based spectral imaging with a spectral resolution of approximately 10 nm allows labeling of chromosome-specific DNA probes with fluorochromes having greatly overlapping emission spectra. This leads to increases in the number of fluorochromes or fluorochrome combinations available to score unambiguously chromosomes in interphase nuclei.
Spectral imaging provides a significant improvement over conventional filter-based microscope systems for enumeration of multiple chromosomes in interphase nuclei, although further technical development is necessary in its application to embryonic blastomeres. When applied to preconception/preimplantation genetic diagnosis, presently available probes for spectral imaging are expected to detect abnormalities responsible for 70-80% of spontaneous abortions caused by chromosomal trisomies.
我们的目的是评估光谱成像在人类间期细胞核中进行多色、多染色体计数的实用性。
用不同荧光染料或非荧光半抗原标记的染色体特异性探针可通过商业途径获得或自行制备。中期染色体铺展、间期淋巴细胞或卵裂球细胞与7种或11种明显不同的探针进行杂交。杂交46小时后，对玻片进行洗涤，并使用自行开发的基于滤光片的定量图像处理系统（QUIPS）或商业光谱成像系统进行检测。
基于滤光片的荧光显微镜系统由于其高灵敏度和速度，更适合同时检测多达7个染色体靶点。然而，当需要区分11个或更多靶点时，这种方法可能不适用于间期细胞。基于干涉仪的光谱成像，光谱分辨率约为10纳米，允许用发射光谱有很大重叠的荧光染料标记染色体特异性DNA探针。这导致可用于明确计数间期细胞核中染色体的荧光染料或荧光染料组合数量增加。
光谱成像在间期细胞核中多染色体计数方面比传统的基于滤光片的显微镜系统有显著改进，尽管在应用于胚胎卵裂球时还需要进一步的技术发展。当应用于孕前/植入前基因诊断时，目前可用的光谱成像探针有望检测出导致70-80%染色体三体性自然流产的异常情况。
