Switchable Solvatochromic Probes for Live‐Cell Super‐resolution Imaging of Plasma Membrane Organization

Danylchuk, Dmytro I.; Moon, Seonah; Xu, Ke; Klymchenko, Andrey S.

doi:10.1002/anie.201907690

Cited by 135 publications

(208 citation statements)

References 46 publications

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“…Solvatochromic dyes can report polarity in different cellular compartments, including cell plasma membranes 11,12 the endoplasmic reticulum, 13,14 mitochondria, 15 the Golgi apparatus, 16 as well as lipid droplets. 14,17,18 At the level of lipid membranes, they allow direct monitoring of lipid order in live cells 12,14,[19][20][21] and can distinguish liquid ordered (Lo) from disordered (Ld) phases, 11,22 which were hypothesized to play a role in the formation of lipid microdomains (rafts) in cell membranes. 23,24 In lipid compartments of cell organelles, solvatochromic dyes can detect changes in polarity upon cell starvation and oxidative stress.…”

Section: Introductionmentioning

confidence: 99%

“…25,26 One should distinguish probes that stain exclusively cell plasma membrane and those that penetrate inside the cells (cell permeant). The former are typically di-4-ANEPPDHQ, 27 which is a styryl pyridinium derivative, and NR12S 12 and its recently improved analogues, 21 based on Nile Red. On the other hand, cell permeant probes are neutral dyes that can rapidly cross cell plasma membranes.…”

Section: Introductionmentioning

confidence: 99%

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Polarity Mapping of Cells and Embryos by Improved Fluorescent Solvatochromic Pyrene Probe

et al. 2020

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Solvatochromic dyes enable sensing and imaging of biomolecular organization in living systems by monitoring local polarity (lipophilicity), but most such dyes suffer from limited brightness, photostability, lack of a convenient spectral range and limited sensitivity to polarity. Moreover, the presence of an electron acceptor group, typically a carbonyl, in their push-pull structure raises concerns about their potential chemical reactivity within the biological environment. In order to achieve robust bioimaging, we synthesized a push-pull pyrene probe bearing a ketone acceptor group (PK) and compared it with a recently developed aldehyde analogue (PA). We found that in live cells the aldehyde analogue PA transforms slowly (in ~100 min) into blue-emissive species, assigned to in situ formation of an imine analogue, whereas the PK probe is stable in the presence of primary amines and inside cells. Like the parent PA, the new probe shows strong solvatochromism and an emission color response to lipid order in membranes (ordered vs. disordered liquid phases), while its blue-shifted absorption is more optimal for excitation with 400-nm light sources. In live cells, the PK probe enables high-contrast polarity mapping of organelles using two-color ratiometric detection, suggesting that polarity increases in the following order: lipid droplets < plasma membranes < endoplasmic reticulum. In the zebrafish embryo, polarity imaging with the PK probe reveals a new dimension in visualizing the organization of tissueslipophilicity distribution, where biomembranes, lipid droplets, cells, yolk, extracellular space and newly formed organs are revealed by specific emission wavelengths of the probe. The newly developed probe and the proposed approach of polarity mapping open new opportunities for bioimaging at the cellular and animal level.

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Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Polarity Mapping of Cells and Embryos by Improved Fluorescent Solvatochromic Pyrene Probe

et al. 2020

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“…26 However, the progress has been hampered by the lack of reliable experimental methods and by the multiple controversies related to the lipid raft hypothesis. 27 Environment-sensitive probes, such as LAURDAN, 28 di-4-ANEPPDHQ, 29 Nile Red (NR) derivatives, 30,31 could be used to address the lipid order in biomembranes through shifts in the emission color. 32 However, these probes report the average lipid order of the bulk of cell membrane and are not suitable for probing specifically the receptor microenvironment.…”

Section: Introductionmentioning

confidence: 99%

Nile Red-Based GPCR Ligands as Ultrasensitive Probes of the Local Lipid Microenvironment of the Receptor

et al. 2021

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The local lipid microenvironment of transmembrane receptors is an essential factor in GPCR signaling. However, tools are currently missing for studying endogenously expressed GPCRs in primary cells and tissues. Here we introduce fluorescent environment-sensitive GPCR ligands for probing the microenvironment of the receptor in living cells using fluorescence microscopy under no-wash conditions. We designed and synthesized antagonist ligands of the oxytocin receptor (OTR) by conjugating a high-affinity non-peptidic OTR ligand PF-3274167 to the environment-sensitive fluorescent dye Nile Red. The length of the polar PEG spacer between the pharmacophore and the fluorophore was adjusted to lower the non-specific interactions of the probe while preserving a strong fluorogenic response. We demonstrated that the new probes embed into the lipid bilayer in the vicinity of the receptor and convey the information about the local polarity and the lipid order via the wavelength-shifting emission of the Nile Red fluorophore.

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“…The ventral (bottom) plasma membrane was lowly labeled owing to its inaccessibility to the probe-containing medium, similar to our observations for other PAINT probes. 26,29 Meanwhile, spatially binning the accumulated transient singlemolecule displacement d into 100×100 nm 2 bins enabled their individual fitting 20 to obtain the local diffusivity D, hence color-coded SMdM super-resolution maps (Figure 2c, Figure S1, and Figure S2).…”

Section: Timementioning

confidence: 99%

Probing nanoscale diffusional heterogeneities in cellular membranes through multidimensional single-molecule and super-resolution microscopy

Yan

Chen²,

Xu³

2020

Preprint

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Diffusion properties notably determine the behavior of biomembranes. Here we report the concurrent nanoscale fine-mapping of membrane topography, diffusivity, and packing order in live mammalian cells through a synergy of single-molecule and super-resolution methods. By identifying a bright, lipophilic fluorescence turn-on probe that enables sustained single-molecule imaging of cellular membranes under stroboscopic excitation, we accumulate the positions and transient displacements of >10^6 probe molecules to achieve super-resolution topography and diffusivity mapping. We thus determine a trend that the membrane diffusivity drops with increased lipid packing order when comparing the endoplasmic reticulum (ER) membrane, plasma membrane, and nanodomains induced by cholera toxin B. Utilizing our nanoscale mapping capability, we further unveil reduced diffusivity in the ER membrane at ER-plasma membrane contact sites. By next integrating spectrally resolved single-molecule imaging, we show this localized diffusion slowdown is not due to altered lipid packing order, but may instead be attributed to local protein crowding. Our integrated multidimensional single-molecule approach thus unveils and differentiates between nanoscale diffusional heterogeneities of different origins in live-cell membranes.

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Switchable Solvatochromic Probes for Live‐Cell Super‐resolution Imaging of Plasma Membrane Organization

Abstract: This is the author manuscript accepted for publication and has undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record.

Cited by 135 publications

References 46 publications

Polarity Mapping of Cells and Embryos by Improved Fluorescent Solvatochromic Pyrene Probe

Polarity Mapping of Cells and Embryos by Improved Fluorescent Solvatochromic Pyrene Probe

Nile Red-Based GPCR Ligands as Ultrasensitive Probes of the Local Lipid Microenvironment of the Receptor

Probing nanoscale diffusional heterogeneities in cellular membranes through multidimensional single-molecule and super-resolution microscopy

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