Quantitative phase deformability cytometry (QP-DC) for precise and clinically relevant multiparametric immune cell profiling
Kyoohyun Kim,
Eoghan O’Connell,
Christine Schauer,
Janina Schoen,
Jiwoo Shim,
Florian Mayerle,
Philipp Radler,
Philipp Lebhardt,
Martin Kräter, et al.
bioRxiv 2025.10.27.684875v1
(2025)
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Imaging flow cytometry enables the detailed analysis of cell morphology and internal structures through high-throughput cell imaging, and quantitative phase imaging (QPI)-based microfluidic approaches have extended this by providing label-free measures such as dry mass and refractive index (RI). Building on these developments, we present quantitative phase deformability cytometry (QP-DC), which integrates QPI with deformability cytometry to simultaneously measure morphology, mechanics, and intrinsic biophysical parameters such as mass density and dry mass. Numerical refocusing ensures in-focus images independent of axial position, improving precision in contour detection and feature extraction. Using microspheres and whole blood, we validated QP-DC and then applied it to neutrophils under lipopolysaccharide (LPS) stimulation and from patients with systemic lupus erythematosus (SLE). QP-DC revealed LPS-induced reductions in neutrophil mass density and identified heterogeneous subpopulations in SLE. These results demonstrate the capability of QP-DC for precise biophysical and mechanical characterization, offering significant potential for research and clinical diagnostics.
Biphasic inflammation control by fibroblasts enables spinal cord regeneration in zebrafish
Nora John,
Thomas Fleming,
Julia Kolb,
Olga Lyraki,
Sebastián Vásquez-Sepúlveda,
Asha Parmer,
Kyoohyun Kim,
Maria Tarczewska,
Kanwarpal Singh, et al.
Fibrosis and persistent inflammation are interconnected processes that inhibit axon regeneration in the mammalian central nervous system (CNS). In zebrafish, by contrast, fibroblast-derived extracellular matrix deposition and inflammation are tightly regulated to facilitate regeneration. However, the regulatory cross-talk between fibroblasts and the innate immune system in the regenerating CNS remains poorly understood. Here, we show that zebrafish fibroblasts possess a dual role in inducing and resolving inflammation, which are both essential for regeneration. We identify a transient, injury-specific cthrc1a+ fibroblast state with an inflammation-associated, less differentiated, and non-fibrotic profile. Induction of this fibroblast state precedes and contributes to the initiation of the inflammatory response. At the peak of neutrophil influx, cthrc1a+ fibroblasts coordinate the resolution of inflammation. Disruption of these inflammation dynamics alters the mechano-structural properties of the lesion environment and inhibits axon regeneration. This establishes the biphasic inflammation control by dedifferentiated fibroblasts as a pivotal mechanism for CNS regeneration.
Although micron-sized microgels have become important building blocks in regenerative materials, offering decisive interactions with living matter, their chemical composition mostly significantly varies when their network morphology is tuned. Since cell behavior is simultaneously affected by the physical, chemical, and structural properties of the gel network, microgels with variable morphology but chemical equivalence are of interest. This work describes a new method to produce thermoresponsive microgels with defined mechanical properties, surface morphologies, and volume phase transition temperatures. A wide variety of microgels is synthesized by crosslinking monomers or star polymers at different temperatures using thermally assisted microfluidics. The diversification of microgels with different network structures and morphologies but of chemical equivalence offers a new platform of microgel building blocks with the ability to undergo phase transition at physiological temperatures. The method holds high potential to create soft and dynamic materials while maintaining the chemical composition for a wide variety of applications in biomedicine.
A label-free method for measuring the composition of multicomponent biomolecular condensates
Patrick M. McCall,
Kyoohyun Kim,
Anna Shevchenko,
Martine Ruer-Gruß,
Jan Peychl,
Jochen Guck,
Andrej Shevchenko,
Anthony A. Hyman,
Jan Brugués
Nature Chemistry
17
1891-1902
(2025)
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Many subcellular compartments are biomolecular condensates made of multiple components, often including several distinct proteins and nucleic acids. However, current tools to measure condensate composition are limited and cannot capture this complexity quantitatively because they either require fluorescent labels, which can perturb composition, or can distinguish only one or two components. Here we describe a label-free method based on quantitative phase imaging and analysis of tie-lines and refractive index to measure the composition of reconstituted condensates with multiple components. We first validate the method empirically in binary mixtures, revealing sequence-encoded density variation and complex ageing dynamics for condensates composed of full-length proteins. We then use analysis of tie-lines and refractive index to simultaneously resolve the concentrations of five macromolecular solutes in multicomponent condensates containing RNA and constructs of multiple RNA-binding proteins. Our measurements reveal an unexpected decoupling of density and composition, highlighting the need to determine molecular stoichiometry in multicomponent condensates. We foresee this approach enabling the study of compositional regulation of condensate properties and function.
Measuring Molecular Mass Densities at Subcellular Resolution Using Optical Diffraction Tomography
Kyoohyun Kim,
Abin Biswas,
Jochen Guck,
Simone Reber
The Nuclear Membrane. Methods in Molecular Biology
119-141
(2025)
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Biological systems intricately regulate their density and volume throughout their life cycles and in response to physiological changes. Mass density, as a fundamental physical quantity, plays significant roles in biological processes such as differentiation, cell growth, protein synthesis, and condensate formation. Loss of density homeostasis on the other hand can have severe consequences including cellular senescence and disease states. Recent developments in biophotonics now enable high-resolution density quantification, providing new insights into the biophysical properties of cells and subcellular structures. One such technique is optical diffraction tomography (ODT), which offers label-free, high-resolution measurements of mass density distribution based on refractive index (RI) measurements. In this chapter, we present a comprehensive guide to implementing ODT for quantitative characterization of mass density distribution in biological systems, including in vivo (adherent cell culture) and in vitro (Xenopus egg extract) samples. We begin by detailing the optical setups, emphasizing key considerations for optimizing tomography acquisition. Subsequently, we introduce preparation protocols tailored to biological samples in various types of sample carriers and offer guidance on standard image acquisition and data analysis procedures. Finally, we address the challenges posed by the linear relationship between RI and mass density in complex substances, offering strategies for overcoming these limitations.
Conserved nucleocytoplasmic density homeostasis drives cellular organization across euraryotes
Abin Biswas,
Omar Muñoz,
Kyoohyun Kim,
Carsten Hoege,
Benjamin M. Lorton,
Rainer Nikolay,
Matthew L. Kraushar,
David Shechter,
Jochen Guck, et al.
Nature Communications
16
7597
(2025)
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The confinement of macromolecules has profound implications for cellular biochemistry. It generates environments with specific physical properties affecting diffusion, macromolecular crowding, and reaction rates. Yet, it remains unknown how intracellular density distributions emerge and affect cellular physiology. Here, we show that the nucleus is less dense than the cytoplasm and that living systems establish a conserved density ratio between these compartments due to a pressure balance across the nuclear envelope. Nuclear transport establishes a specific nuclear proteome that exerts a colloid osmotic pressure, which, assisted by chromatin pressure, increases nuclear volume. During C. elegans development, the nuclear-to-cytoplasmic density ratio is robustly maintained even when nuclear-to-cytoplasmic volume ratios change. We show that loss of density homeostasis correlates with altered cell functions like senescence and propose density distributions as key markers in pathophysiology. In summary, this study reveals a homeostatic coupling of macromolecular densities that drives cellular organization and function.
Thermally Assisted Microfluidics to Produce Chemically Equivalent Microgels with Tunable Network Morphologies
Dirk Rommel,
Bernhard Häßel,
Philip Pietryszek,
Matthias Mork,
Oliver Jung,
Meike Emondts,
Nikita Norkin,
Iris Christine Doolaar,
Yonka Kittel, et al.
Angewandte Chemie, International Edition in English
64
(1)
(2025)
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Although micron-sized microgels have become important building blocks in regenerative materials, offering decisive interactions with living matter, their chemical composition mostly significantly varies when their network morphology is tuned. Since cell behavior is simultaneously affected by the physical, chemical, and structural properties of the gel network, microgels with variable morphology but chemical equivalence are of interest. This work describes a new method to produce thermoresponsive microgels with defined mechanical properties, surface morphologies, and volume phase transition temperatures. A wide variety of microgels is synthesized by crosslinking monomers or star polymers at different temperatures using thermally assisted microfluidics. The diversification of microgels with different network structures and morphologies but of chemical equivalence offers a new platform of microgel building blocks with the ability to undergo phase transition at physiological temperatures. The method holds high potential to create soft and dynamic materials while maintaining the chemical composition for a wide variety of applications in biomedicine.
Contact
Core Facility Microscopy Kyoohyun Kim
Max-Planck-Zentrum für Physik und Medizin Kussmaulallee 2 91054 Erlangen, Germany
