Violet laser diodes as light sources for cytometry.
Violet laser diodes as light sources for cytometry.
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DOI：
10.1002/1097-0320(20010601)44:2
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发表时间：
2001-06
期刊：
影响因子：
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通讯作者：
H. Shapiro;N. Perlmutter
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文献类型：
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作者：
H. Shapiro;N. Perlmutter
背景技术紫光激光二极管最近已经商业化。这些器件在 395-415 nm 范围内发射 5-25 mW 的功率，可用于将二极管与准直光学器件和稳压电源集成到外壳中的系统中，该外壳装有热电冷却器，这是保持稳定输出所必需的。此类系统目前售价数千美元，但预计价格将大幅下降。材料和方法 实验室建造的流式细胞仪中使用 4 mW、397 nm 紫色二极管系统来激发透化和完整细胞中 DAPI 和 Hoechst 染料的荧光。还测量了前向和正交光散射。结果DAPI染色得到精度良好的DNA含量直方图（G(0)/G(1)变异系数1.7%）；使用 Hoechst 33342 的精度较低。Hoechst 34580 的激发最大值接近 400 nm，产生最高的荧光强度，但在溶液中短时间内似乎会分解。散射信号表现出相对广泛的分布。结论 对于使用 DAPI 和 Hoechst 染料进行 DNA 荧光测量，紫罗兰激光二极管是相对便宜、紧凑、高效且安静的光源；它们还可以激发其他几种荧光探针。
BACKGROUND Violet laser diodes have recently become commercially available. These devices emit 5-25 mW in the range of 395-415 nm, and are available in systems that incorporate the diodes with collimating optics and regulated power supplies in housing incorporating thermoelectric coolers, which are necessary to maintain stable output. Such systems now cost several thousand dollars, but are expected to drop substantially in price. Materials and Methods A 4-mW, 397-nm violet diode system was used in a laboratory-built flow cytometer to excite fluorescence of DAPI and Hoechst dyes in permeabilized and intact cells. Forward and orthogonal light scattering were also measured. RESULTS DNA content histograms with good precision (G(0)/G(1) coefficient of variation 1.7%) were obtained with DAPI staining; precision was lower using Hoechst 33342. Hoechst 34580, with an excitation maximum nearer 400 nm, yielded the highest fluorescence intensity, but appeared to decompose after a short time in solution. Scatter signals exhibited relatively broad distributions. CONCLUSIONS Violet laser diodes are relatively inexpensive, compact, efficient, and quiet light sources for DNA fluorescence measurement using DAPI and Hoechst dyes; they can also excite several other fluorescent probes.