Genetically Encoded Phase Contrast Agents for Digital Holographic Microscopy

Abstract

Quantitative phase imaging and digital holographic microscopy have shown great promise for visualizing the motion, structure and physiology of microorganisms and mammalian cells in three dimensions. However, these imaging techniques currently lack molecular contrast agents analogous to the fluorescent dyes and proteins that have revolutionized fluorescence microscopy. Here we introduce the first genetically encodable phase contrast agents based on gas vesicles, a unique class of air-filled protein nanostructures derived from buoyant microbes. The relatively low index of refraction of the air-filled core of gas vesicles results in optical phase advancement relative to aqueous media, making them a "positive" phase contrast agent easily distinguished from organelles, dyes, or microminerals. We demonstrate this capability by identifying and tracking the motion of gas vesicles and gas vesicle-expressing bacteria using digital holographic microscopy, and by imaging the uptake of engineered gas vesicles by mammalian cells. These results give phase imaging a biomolecular contrast agent, greatly expanding the capabilities of this powerful technology for three-dimensional biological imaging.

Measuring gas vesicle dimensions by electron microscopy

“…Gas Vesicles (GVs) are hollow, gas-filled protein nanostructures natively expressed in certain types of cyanobacteria, heterotrophic bacteria, and Archaea as a buoyancy aid (Walsby 1994). Recently, it was discovered that the unique physical properties of GVs enable them to serve as genetically encodable contrast agents for ultrasound and other imaging methods, allowing deep tissue imaging of cellular function (Shapiro, Goodwill, et al 2014;Shapiro, Ramirez, et al 2014;Bourdeau et al 2018;Lu et al 2018;Farhadi et al 2019Farhadi et al , 2020Lakshmanan et al 2020). In addition, GVs are being applied to acoustic manipulation and therapeutic uses of engineered cells (Bar-Zion et al 2019;Wu et al 2019).…”

Dehydration of plant cells shoves nuclei rotation allowing for 3D phase-contrast tomography

“…In the absence of an adequate refractive index difference between the structure of interest and the surrounding medium, even high spatial resolution is not sufficient to guarantee neither a correct quantitative phase-contrast mapping nor tomographic imaging. In order to improve the phase contrast, the use of dyes or the intracellular injection of glycerol, genetically encoded agents, or other strategies for DH microscopy have been proposed 30 – 32 . Here we deliberately induced dehydration in epidermal onion cells and exploit it to achieve a more convenient imaging condition in 3D.…”

Microscopic Object Classification through Passive Motion Observations with Holographic Microscopy

“…Protein-coated air-filled vesicles were purified from the buoyant alga Anabaena flos-aquae as previously described [ 19 , 20 ]. The 100 nm-scale vesicles were clustered by first biotinylating with 10 5 molar excess of EZ-Link-Sulfo-NHS-LC-biotin (ThermoFisher Scientific, Waltham, MA, USA) in PBS for 4 h. After dialyzing twice vs. PBS, the biotinylated vesicles were clustered by incubation with streptavidin (Geno Technology, St. Louis, MO, USA) for 30 min at room temperature at a streptavidin-to-vesicle ratio of 100:1.…”

“…Cyanobacteria are unique in being able to regulate their buoyancy by means of intracellular air-filled vesicles, resulting in cells with positive buoyancy [ 18 ]. These vesicles may be purified from the cyanobacteria and used for labeling other cells as they provide contrast for quantitative phase and ultrasonic imaging and MRI [ 19 , 20 ]. Their density, which is that of air surrounded by a thin protein shell, has an average reported value of 0.119 mg/µL [ 21 ].…”