Long-term label-free assessments of individual bacteria using three-dimensional quantitative phase imaging and hydrogel-based immobilization

Three-dimensional (3D) quantitative phase imaging (QPI) enables long-term label-free tomographic imaging and quantitative analysis of live individual bacteria. However, the Brownian motion or motility of bacteria in a liquid medium produces motion artifacts during 3D measurements and hinders precise cell imaging and analysis. Meanwhile, existing cell immobilization methods produce noisy backgrounds and even alter cellular physiology. Here, we introduce a protocol that utilizes hydrogels for high-quality 3D QPI of live bacteria maintaining bacterial physiology. We demonstrate long-term high-resolution quantitative imaging and analysis of individual bacteria, including measuring the biophysical parameters of bacteria and responses to antibiotic treatments.