Distinguishing Free Cytosolic and Vesicle-Confined Nanoparticle Motion by Fluorescence Correlation Spectroscopy.
- 1Institute of Physical Chemistry, Polish Academy of Sciences, Kasprzaka 44/52, 01-224Warsaw, Poland.
- 2Department of Chemistry, University of Zurich, Winterthurerstrasse 190, CH-8057Zurich, Switzerland.
Abstract
Understanding whether internalized nanoparticles remain confined within intracellular vesicles or are released into the cytosol is essential for evaluating their biological activity and potential use as delivery systems. However, this distinction is often difficult to establish by using fluorescence imaging alone, particularly at nanomolar probe concentrations. Here, we propose fluorescence correlation spectroscopy (FCS) combined with length-scale-dependent viscosity analysis and appropriate diffusion models as an approach for assessing nanoparticle uptake and intracellular localization. The method was evaluated in HeLa cells using fluorescent polystyrene nanoparticles, cell-penetrating peptide-coated nanoparticles, dextran of two molecular weights, and receptor-internalized transferrin. Cytosolic probes were identified by autocorrelation curves that fit an anomalous diffusion model and by diffusion coefficients consistent with those predicted by the cytoplasmic length scale-dependent viscosity profile. In contrast, vesicle-associated probes produced characteristic two-step autocorrelation curves that were modeled as the sum of probe diffusion within a confined space, vesicle translation, and directed intracellular transport. Bare nanoparticles, pinocytosed dextrans, and transferrin displayed predominantly vesicle-associated mobility, whereas microinjected dextrans and cell-penetrating-peptide-associated nanoparticles exhibited diffusion in the cytosol. Our results demonstrate that FCS can complement fluorescence imaging by detecting cytosolic probe populations and distinguishing between cytosolic release and vesicular retention.
Related
- siRNA/mRNA payload of lipid nanoparticles quantified using fluorescence microscopy and correlation spectroscopy
- Vision Transformers as a Framework for Imaging Fluorescence Correlation Spectroscopy
- Monitoring intramolecular dynamics across two regions of the mouse prion protein during misfolding and oligomerization using fluorescence correlation spectroscopy
- An efficient hypochlorous acid fluorescence imaging strategy based on toehold-mediated strand displacement for distinguishing cancer cells from normal cells
- Tailoring Fusion Proteins for Interfacial Nanoparticle Functionalization: Toward Fluorescence-Guided Imaging
BibTeX
@article{kwapiszewska2026distinguishing,
title = {{Distinguishing Free Cytosolic and Vesicle-Confined Nanoparticle Motion by Fluorescence Correlation Spectroscopy.}},
author = {Karina Kwapiszewska and Grzegorz Bubak and Krzysztof Bielec and Aneta Magiera and Alicja Zgorzelska and Robert Hołyst},
journal = {The journal of physical chemistry. B},
year = {2026},
month = oct,
doi = {10.1021/acs.jpcb.6c05465},
url = {https://europepmc.org/article/MED/42916719},
note = {CC BY},
}
RIS
TY - JOUR
TI - Distinguishing Free Cytosolic and Vesicle-Confined Nanoparticle Motion by Fluorescence Correlation Spectroscopy.
AU - Karina Kwapiszewska
AU - Grzegorz Bubak
AU - Krzysztof Bielec
AU - Aneta Magiera
AU - Alicja Zgorzelska
AU - Robert Hołyst
PY - 2026
DA - 2026/10/01
JO - The journal of physical chemistry. B
DO - 10.1021/acs.jpcb.6c05465
UR - https://europepmc.org/article/MED/42916719
AB - Understanding whether internalized nanoparticles remain confined within intracellular vesicles or are released into the cytosol is essential for evaluating their biological activity and potential use as delivery systems. However, this distinction is often difficult to establish by using fluorescence imaging alone, particularly at nanomolar probe concentrations. Here, we propose fluorescence correlation spectroscopy (FCS) combined with length-scale-dependent viscosity analysis and appropriate diffusion models as an approach for assessing nanoparticle uptake and intracellular localization. The method was evaluated in HeLa cells using fluorescent polystyrene nanoparticles, cell-penetrating peptide-coated nanoparticles, dextran of two molecular weights, and receptor-internalized transferrin. Cytosolic probes were identified by autocorrelation curves that fit an anomalous diffusion model and by diffusion coefficients consistent with those predicted by the cytoplasmic length scale-dependent viscosity profile. In contrast, vesicle-associated probes produced characteristic two-step autocorrelation curves that were modeled as the sum of probe diffusion within a confined space, vesicle translation, and directed intracellular transport. Bare nanoparticles, pinocytosed dextrans, and transferrin displayed predominantly vesicle-associated mobility, whereas microinjected dextrans and cell-penetrating-peptide-associated nanoparticles exhibited diffusion in the cytosol. Our results demonstrate that FCS can complement fluorescence imaging by detecting cytosolic probe populations and distinguishing between cytosolic release and vesicular retention.
SN - 1520-6106
SN - 1520-5207
ER -