Fauver Mark, Seibel Eric, Rahn J Richard, Meyer Michael, Patten Florence, Neumann Thomas, Nelson Alan
Opt Express. 2005 May 30;13(11):4210-23. doi: 10.1364/opex.13.004210.
A method is presented for imaging single isolated cell nuclei in 3D, employing computed tomographic image reconstruction. The system uses a scanning objective lens to create an extended depth-of-field (DOF) image similar to a projection or shadowgram. A microfabricated inverted v-groove allows a microcapillary tube to be rotated with sub-micron precision, and refractive index matching within 0.02 both inside and outside the tube keeps optical distortion low. Cells or bare cell nuclei are injected into the tube and imaged in 250 angular increments from 0 to 180 degrees to collect 250 extended DOF images. After these images are further aligned, the filtered backprojection algorithm is applied to compute the 3D image. To estimate the cutoff spatial frequency in the projection image, a spatial frequency ratio function is calculated by comparing the extended depth-of-field image of a typical cell nucleus to the fixed focus image. To assess loss of resolution from fixed focus image to extended DOF image to 3D reconstructed image, the 10-90% rise distance is measured for a dyed microsphere. The resolution is found to be 0.9 microm for both extended DOF images and 3D reconstructed images. Surface and translucent volume renderings and cross-sectional slices of the 3D images are shown of a stained nucleus from fibroblast and cancer cell cultures with added color histogram mapping to highlight 3D chromatin structure.
本文介绍了一种利用计算机断层图像重建对单个分离细胞核进行三维成像的方法。该系统使用扫描物镜创建类似于投影或阴影图的扩展景深（DOF）图像。一个微加工的倒V形槽允许微毛细管以亚微米精度旋转，管内外0.02以内的折射率匹配使光学畸变保持在低水平。将细胞或裸露的细胞核注入管中，并以250个角度增量（从0度到180度）成像，以收集250张扩展DOF图像。在这些图像进一步对齐后，应用滤波反投影算法计算三维图像。为了估计投影图像中的截止空间频率，通过将典型细胞核的扩展景深图像与固定焦点图像进行比较来计算空间频率比函数。为了评估从固定焦点图像到扩展DOF图像再到三维重建图像的分辨率损失，对一个染色微球测量10 - 90%上升距离。发现扩展DOF图像和三维重建图像的分辨率均为0.9微米。展示了来自成纤维细胞和癌细胞培养物的染色细胞核的三维图像的表面和半透明体绘制以及横截面切片，并添加了颜色直方图映射以突出三维染色质结构。
