Bratton Benjamin P, Barton Brody, Morgenstein Randy M
Department of Molecular Biology and Lewis-Sigler Institute of Integrative Genomics, Princeton University.
Department of Microbiology and Molecular Genetics, Oklahoma State University.
J Vis Exp. 2019 Oct 29(152). doi: 10.3791/60350.
The shape of a bacterium is important for its physiology. Many aspects of cell physiology such as cell motility, predation, and biofilm production can be affected by cell shape. Bacterial cells are three-dimensional (3D) objects, although they are rarely treated as such. Most microscopy techniques result in two-dimensional (2D) images leading to the loss of data pertaining to the actual 3D cell shape and localization of proteins. Certain shape parameters, such as Gaussian curvature (the product of the two principal curvatures), can only be measured in 3D because 2D images do not measure both principal curvatures. Additionally, not all cells lie flat when mounting and 2D imaging of curved cells may not accurately represent the shapes of these cells. Accurately measuring protein localization in 3D can help determine the spatial regulation and function of proteins. A forward convolution technique has been developed that uses the blurring function of the microscope to reconstruct 3D cell shapes and to accurately localize proteins. Here, a protocol for preparing and mounting samples for live cell imaging of bacteria in 3D both to reconstruct an accurate cell shape and to localize proteins is described. The method is based on simple sample preparation, fluorescent image acquisition, and MATLAB-based image processing. Many high-quality fluorescent microscopes can be simply modified to take these measurements. These cell reconstructions are computationally intensive and access to high-throughput computational resources is recommended, although not necessary. This method has been successfully applied to multiple bacterial species and mutants, fluorescent imaging modalities, and microscope manufacturers.
细菌的形状对其生理功能很重要。细胞生理的许多方面，如细胞运动、捕食和生物膜形成，都可能受到细胞形状的影响。细菌细胞是三维（3D）物体，尽管它们很少被这样看待。大多数显微镜技术会产生二维（2D）图像，导致与实际3D细胞形状和蛋白质定位相关的数据丢失。某些形状参数，如高斯曲率（两个主曲率的乘积），只能在3D中测量，因为2D图像无法测量两个主曲率。此外，并非所有细胞在固定时都会平躺，对弯曲细胞进行2D成像可能无法准确反映这些细胞的形状。在3D中准确测量蛋白质定位有助于确定蛋白质的空间调节和功能。已经开发出一种正向卷积技术，该技术利用显微镜的模糊功能来重建3D细胞形状并准确地定位蛋白质。在此，描述了一种用于制备和固定样品以进行细菌3D活细胞成像的方案，既可以重建准确的细胞形状，又可以定位蛋白质。该方法基于简单的样品制备、荧光图像采集和基于MATLAB的图像处理。许多高质量的荧光显微镜可以简单地进行修改以进行这些测量。这些细胞重建计算量很大，建议使用高通量计算资源，不过并非必需。该方法已成功应用于多种细菌物种和突变体、荧光成像模式以及显微镜制造商。
