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

Abstract

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.

Quantifying the Dynamics of Bacterial Biofilm Formation on the Surface of Soft Contact Lens Materials Using Digital Holographic Tomography to Advance Biofilm Research

“…It should also be pointed out that the average RI of the bacteria cells and the range of its changes related to the physiological processes depends on the medium in which they are found or on which they are immobilized [ 28 ]. This should therefore be considered when comparing the RI data across the bacteria being tested.…”

Quantitative phase imaging based on holography: trends and new perspectives

“…The results imply that the OA treatment does not affect the dry mass density of the cytoplasm and LDs. Also, QPI was used for long‑term assessments of bacteria 374 , distinguishing bacteria from sulfur mineral grains 375 , and analysis of the size of planktons 376 . Recently, the RI reconstruction can identify the cell nucleus within the label-free tomograms by using a novel computational segmentation method based on statistical inference 377 .…”

Nanomaterial‐assisted oncolytic bacteria in solid tumor diagnosis and therapeutics

“…This decrease can be attributed to the dilution resulting from bacterial cell growth and division 196 . Bacterial motility may induce artifacts or blur images, affecting their localization and morphology accuracy 197 . Moreover, various imaging modalities encounter challenges when utilizing NAOB.…”

“… 196 Bacterial motility may induce artifacts or blur images, affecting their localization and morphology accuracy. 197 Moreover, various imaging modalities encounter challenges when utilizing NAOB. Optical imaging is limited by its penetration depth (1 cm) and usually receives small molecule interferences such as hemoglobin, water, and cytochromes.…”