High-resolution imaging of bacterial spatial organization with vertical cell imaging by nanostructured immobilization (VerCINI)

Light microscopy is indispensable for analysis of bacterial spatial organization, yet the sizes and shapes of bacterial cells pose unique challenges to imaging. Bacterial cells are not much larger than the diffraction limit of visible light, and many species have cylindrical shapes and so lie flat on microscope coverslips, yielding low-resolution images when observing their short axes. In this protocol, we describe a pair of recently developed methods named VerCINI (vertical cell imaging by nanostructured immobilization) and mu VerCINI (microfluidic VerCINI) that greatly increase spatial resolution and image quality for microscopy of the short axes of bacteria. The concept behind both methods is that cells are imaged while confined vertically inside cell traps made from a nanofabricated mold. The mold is a patterned silicon wafer produced in a cleanroom facility using electron-beam lithography and deep reactive ion etching, which takes similar to 3 h for fabrication and similar to 12 h for surface passivation. After obtaining a mold, the entire process of making cell traps, imaging cells and processing images can take similar to 2-12 h, depending on the experiment. VerCINI and mu VerCINI are ideal for imaging any process along the short axes of bacterial cells, as they provide high-resolution images without any special requirements for fluorophores or imaging modalities, and can readily be combined with other imaging methods (e.g., STORM). VerCINI can easily be incorporated into existing projects by researchers with expertise in bacteriology and microscopy. Nanofabrication can be either done in-house, requiring specialist facilities, or outsourced based on this protocol.

Automated summary

Light microscopy is crucial for analyzing bacterial spatial organization, but the sizes and shapes of bacterial cells present unique challenges for imaging. This article introduces a pair of newly developed methods, VerCINI and mu VerCINI, which greatly improve spatial resolution and image quality for microscopy of the short axes of bacteria.