MSCA Fellow postdoc at the Membrane Enzymology group, University of Groningen; tweeting about fluorescence microscopy, smFRET, membrane proteins, bacteria & GPCRs
https://scholar.google.nl/citations?user=lC_EQr4AAAAJ&hl=en
How is one dye #NileRed (NR) the sensor of both polarity and microviscosity?
Sarkar et al. correlate spectral shifts and fluorophore orientations of single molecules of NR in Lo/Ld supported lipid bilayers
PhysChemB: https://doi.org/10.1021/acs.jpcb.4c07578
π bioRxiv: https://doi.org/10.1101/2024.06.21.600028
Single-molecule microscopy is full of surprises:
see how anisotropy can be used to follow the conformational dynamics! π€― #24-state-model
"Examination of conformational dynamics of AdiC transporter with fluorescence-polarization microscopy"
π₯π¬
π¨π¬#preprint :
Ξ»-STED π¬improves spectral sensitivity with polarity-sensitive probes π and enables correlative measurements of membrane order and anomalous lipid diffusion
πhttps://www.biorxiv.org/content/10.1101/2025.02.06.636942v1
Congrats to the authors! ππ
@faldalf
Molecular plasma membrane organization and dynamics play an important role in cellular signalling. Advances in our understanding of the nanoscale architecture of the plasma membrane heavily rely on the development of non-invasive experimental methods, particularly of advanced fluorescence microscopy and spectroscopy techniques with high spatio-temporal resolution and sensitivity to local molecular properties. However, it remains difficult to combine several of them for a multimodal characterisation that would reduce the possibility of misinterpretations. Here, we integrated a spectral detector into a super-resolution stimulated emission depletion (STED) microscope, achieving three goals. First, we show that compared to the standard ratiometric detection using fixed bandpass filters, the spectrally resolved acquisition together with spectral fitting or phasor analysis improves the accuracy of experiments determining membrane lipid order with polarity-sensitive probes multifold. Secondly, we demonstrate that this acquisition scheme allows the use of such probes in combination with other dyes with overlapping spectra, enabling co-localisation of the membrane order maps with other cellular structures of interest, e.g. fluorescently labelled proteins. Finally, we correlate the obtained membrane lipid order with the anomalous trapped diffusion properties of a fluorescent sphingomyelin lipid analogue in the plasma membrane of living cells, as determined by STED fluorescence correlation spectroscopy, and highlight that some of the most apparent trapping sites locate at the boundaries of local ordered environments discernible by the introduced spectral STED microscopy. With additional measurements in model membranes and Monte-Carlo simulations we conclude that for sub-100 nm ordered environments uneven probe partitioning cannot by itself explain the trapping diffusion of SM in cells. ### Competing Interest Statement The authors have declared no competing interest.
"Direct effect of membrane environment on the activation of mGluR2 revealed by single-molecule FRET" in #Structure
π https://doi.org/10.1016/j.str.2025.01.011
𧡠#GPCR #mGluRπ§Ό#detergents #cholesterol
π¬ #smFRET
π»π§ͺ #MD #mutants
Congrats to C. Banerjee, B. Liauw, and R. Vafabakhsh!
Can antibiotics kill intracellular bacteria in vivo?
Researchers from Northeast Agricultural University, Harbin, China, developed safe and biocompatible nanopetides that penetrate host cells and kill intracellular bacteria by disrupting bacterial cell membranes and inducing excessive accumulation of ROS
Yongjie Zhu et al. (2025) in Science Advances: https://www.science.org/doi/10.1126/sciadv.ads3844
#review #microscopy #methods
"Advanced fluorescence lifetime-enhanced multiplexed nanoscopy of cells"
by Samrat Basak and RomanTsukanov
FLIM // super-resolution // SMLM // DNA-PAINT // multiplexed imaging // MIET
https://www.degruyter.com/document/doi/10.1515/mim-2024-0029/html
In this review paper, we summarize the significant advancements in the field of fluorescence lifetime imaging microscopy (FLIM), particularly wide-field FLIM with single-molecule sensitivity, achieved using the time-correlated single-photon counting-based position-sensitive LINCam system. Fluorescence lifetime adds valuable information beyond conventional intensity-based imaging, enabling diverse applications across research fields. Here, we focus on three primary bioimaging applications: (I) single-molecule FLIM in the far-red spectral region, (II) fast and multiplexed super-resolution imaging of cells, and (III) three-dimensional super-resolution imaging with high axial localization precision. Recent advances in position-sensitive detector technologies offer exciting opportunities for high-throughput super-resolution imaging with enhanced localization precision.
β¨ Multifunctional Si-rhodamine dyes for
𧬠live cell labeling and
𧲠affinity purification of proteins and organelles!
π Preprint by Kumar et al.:
Optimizing multifunctional fluorescent ligands for intracellular labeling
π https://www.biorxiv.org/content/10.1101/2022.07.02.498544v2
π Congrats to all authors! @So_lets_kiLab π₯³
Enzyme-based self-labeling tags enable covalent attachment of synthetic molecules to proteins inside living cells. A frontier of this field is designing multifunctional ligands that contain both fluorophores and affinity tags or pharmacological agents and can still efficiently enter cells. Self-labeling tag ligands with short linkers can enter cells readily but often show less activity due to steric issues; ligands with long linkers can be more potent but show lower cell permeability. Here, we overcome this tug-of-war between efficacy and cell-permeability by devising a rational strategy for making cell permeable multifunctional ligands for labeling HaloTag fusions. We found that the lactoneβzwitterion equilibrium constant ( K LβZ) of rhodamines inversely correlates with their distribution coefficients (log D 7.4), suggesting that ligands based on dyes exhibiting low K LβZ and high log D 7.4 values, such as Si-rhodamines, would efficiently enter cells. We designed cell-permeable multifunctional HaloTag ligands with a biotin moiety to purify mitochondria or a JQ1 appendage to translocate BRD4 from euchromatin to the nucleolus or heterochromatin. We discovered that translocation of BRD4 to constitutive heterochromatin in cells expressing HaloTagβHP1a fusion proteins can lead to apparent increases in transcriptional activity. These new reagents enable affinity capture and translocation of intracellular proteins in living cells and the use of Si-rhodamines and other low K LβZ/high log D 7.4 dye scaffolds will facilitate the design of new multifunctional chemical tools for biology. ### Competing Interest Statement Patents and patent applications covering azetidine-containing rhodamine dyes (with inventors Jonathan B. Grimm, Pratik Kumar, and Luke D. Lavis) are assigned to HHMI.
Dual-color πβ€οΈ sub-millisecond single-molecule tracking via MINFLUX π©π¬
reveals concurrent diffusion of nAchR receptors π and fluorescent cholesterol
#preprint
https://www.researchsquare.com/article/rs-5619606/v1
Congrats to the authors! ππ
Nicotinic acetylcholine receptors (nAChRs) are ubiquitous neurotransmitter receptors predominantly located at the cell-surface of neurons and muscle cells. Their dynamics affect synaptogenesis at neurodevelopmental stages and the efficacy of synaptic transmission in the adult synapse. Here we ...
π¨ New insights into GPCR dynamics!
A simulation-driven mutagenesis study of A2A adenosine receptor shows how water molecules act as key facilitators of conformational change & receptor activity π§π¬
By tweaking water-mediated contact networks, basal & ligand-induced activity can be predictably tuned
π Nature Chemistry: https://www.nature.com/articles/s41557-024-01719-2
Solvent-mediated interaction networks control a wide range of protein functions, but their design has been neglected owing to a lack of accurate computational tools. Now it is shown that allosteric signalling membrane proteins can be engineered through such networks, revealing a broader space of designable protein interactions and functions.
π¨ Amazing work:
"Highly multiplexed spatial transcriptomics in bacteria" by Sarfatis et al. in Science
1000-fold volumetric expansion of E. coli cells paves the way to image-based single-cell transcriptomic π¦ π¬
#MERFISH
https://www.science.org/doi/10.1126/science.adr0932
bioRxiv:
https://www.biorxiv.org/content/10.1101/2024.06.27.601034v1