Extended depth of field imaging for high speed cell analysis
Extended depth of field imaging for high speed cell analysis
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DOI：
10.1002/cyto.a.20370
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发表时间：
2007-04-01
期刊：
影响因子：
3.7
通讯作者：
Basiji, David A.
中科院分区：
文献类型：
--
作者：
Ortyn, William E.;Perry, David J.;Basiji, David A.
背景：荧光显微镜是一种非常有用的工具，可以分析细胞上、细胞内或细胞间荧光标记分子的强度、位置和运动。然而，该技术的缺点是图像采集速度慢，景深有限。共聚焦显微镜通过“光学切片和重建”解决了景深问题，但只能通过进一步降低图像采集率来在多个焦平面上重复扫描细胞。在本文中，我们描述了一种使用改进的ImageStream (R)系统执行高速扩展景深（EDF）成像的技术，该技术在不到一分钟的时间内收集来自数千个细胞的高分辨率多模式图像，焦点保持在16 μ m焦距范围内。方法：采用包含波前编码（TM）元件的EDF ImageStream原型系统，从荧光标记的微珠中捕获图像。利用光度和形态学特征定量分析图像，以评估特征值与焦点位置的一致性。使用悬浮荧光原位杂交技术（fishs (TM)）检测Jurkat细胞Y染色体，比较标准和基于波前编码的EDF成像方法用于自动染色体计数。结果：头部图像的定性视觉检查显示，带有EDF的原型ImageStream系统在16 μ m的焦距范围内保持焦距质量。定量分析表明，与标准成像相比，扩展景深采集模式对焦敏感特征值的变化减少了约10倍。使用fishs协议探测Jurkat细胞图像的自动染色体计数使用EDF成像显着更准确。结论：EDF技术的使用可以显著增强细胞成像的定量，特别是在FISH等应用中，必须在细胞内的宽焦范围内检测到小的离散信号。(c) 2007年国际分析细胞学学会。
Background: Fluoresence microscopy is an extremely useful tool to analyze the intensity, location and movement of fluorescently tagged molecules on, within or between cells. However, the technique suffers from slow image acquisition rates and limited depth of field. Confocal microscopy addresses the depth of field issue via "optical sectioning and reconstruction", but only by further reducing the image acquisition rate to repeatedly scan the cell at multiple focal planes. In this paper we describe a technique to perform high speed, extended depth of field (EDF) imaging using a modified ImageStream (R) system whereby high resolution, multimode imagery from thousands of cells is collected in less than a minute with focus maintained over a 16 mu m focal range.Methods: A prototype EDF ImageStream system incorporating a Wavefront Coded (TM) element was used to capture imagery from fluorescently labeled beads. Bead imagery was quantitatively analyzed using photometric and morphological features to assess consistency of feature values with respect to focus position. Jurkat cells probed for chromosome Y using a fluorescence in situ hybridization in suspension protocol (FISHIS (TM)) were used to compare standard and Wavefront Coded-based EDF imaging approaches for automated chromosome enumeration.Results: Qualitative visual inspection of bead imagery reveals that the prototype ImageStream system with EDF maintains focus quality over a 16 mu m focus range. Quantitative analysis shows the extended depth field collection mode has approximately ten-fold less variation in focus-sensitive feature values when compared with standard imaging. Automated chromosome enumeration from imagery of Jurkat cells probed using the FISHIS protocol is significantly more accurate using EDF imaging.Conclusions: The use of EDF techniques may significantly enhance the quantitation of cell imagery, particularly in applications such as FISH, where small discrete signals must be detected over a wide focal range within the cell. (c) 2007 international Society for Analytical Cytology.