Label‐Free Quantitative Analysis of Coacervates via 3D Phase Imaging

Abstract

Coacervation is considered as a ubiquitous mechanism for assembling biomolecular materials outside cells and organizing membraneless organelles inside cells. Despite the importance of mapping binodals to understand the driving forces and thermodynamics of coacervate, quantifying protein concentration within a droplet is significantly challenging owing to its dynamic and viscous nature. A direct imaging‐based method is presented to quantify coacervate using real‐time 3D quantitative phase imaging. The proposed method utilizes the measurements of the refractive index tomograms of individual coacervates and retrieves the protein concentration and volume of individual protein droplets exploiting light‐scattering analysis. The retrievals of accurate protein concentrations are demonstrated in droplets, whereas conventional fluorescence‐based techniques present underestimations. With its simple, direct, real‐time, and quantitative analysis capability, the present method can be utilized in various protein analyses and quantifications for the study of coacervation both in vitro and in vivo.

Hydrophobicity of arginine leads to reentrant liquid-liquid phase separation behaviors of arginine-rich proteins

“…To determine the protamine and hyaluronic acid concentrations in the droplets, a quantitative amino acid analysis was performed with a SYKAM System S4300 Amino Acid Analyzer (SYKAM, Gewerbering, Germany), according to the previously reported method 64 . Specifically, protamine and hyaluronic acid were mixed at a ratio of 3:7 and then centrifuged at 920 × g for 1 h to obtain the dilute phase and sedimented droplet phase.…”

3D imaging lipidometry in single cell by in-flow holographic tomography

“…In the 2D case, a deep learning approach has been employed to identify LDs inside the QPMs 44 . Instead, in the 3D case, to segment LDs in microalgal cells 34 and in foam cells 32 , the RI threshold has been selected according to the FM image of the same cell obtained from the channel mounted on the static TPM system. However, this is not possible in TPM flow-cytometry.…”

“…The first demonstration in visualizing and measuring LDs in live cells by DH was reported in the last decade 28 . Recently, the tomographic phasecontrast microscopy (TPM) was applied for LD 3D imaging within mammalian cells 29−31 and microalgae 32 , for 4D tracking of the LDs dynamics in live hepatocytes 33 , and for recording time-lapses of living foam cells 34 . Nevertheless, the up-to-date available label-free techniques allow to investigate LDs only in static, adherent cells, strongly limiting both throughput and reliability of the information regarding the LD spatial organization, the latter being significantly affected by the cell culture mode.…”

Quantitative Phase Imaging: Recent Advances and Expanding Potential in Biomedicine

“…284 This process of intracellular phase separation was examined by 3D QPI with identification confirmed by fluorescence. 285 Future applications of QPI tomography includes combinations with other QPI data analysis methods to reveal the essential biological mechanism(s) behind these structures. Another promising application of QPI tomography is the measurement of mass within multicellular specimens, such as whole animals 277 (see in vivo section, below), or 3D organoids that are often used as in vitro models of development or disease.…”

“…A promising application of tomographic QPI to measure subcellular structures is the interrogation of biomolecular condensates, which are membrane-less organelles or organelle subdomains that have been implicated in a wide range of cell behaviors including bone metastasis and autophagy . This process of intracellular phase separation was examined by 3D QPI with identification confirmed by fluorescence . Future applications of QPI tomography includes combinations with other QPI data analysis methods to reveal the essential biological mechanism(s) behind these structures.…”