A step-by-step protocol and analysis software to measure plasma membrane fluidity in live cells using a fluorescence microscope.
Microscope lasers can cause damage to our samples. We show a common (and mostly ignored) artefact and how to avoid it.
We present a membrane labelling strategy for super-resolution and biophysical imaging without photobleaching.
Vogler BTL, Gentsch GJ, Carravilla P, Helmerich DA, Heiby JC, Reglinski K, Durso W, Klein T, Hildebrandt F, Zahid R, Spengler K, Heller R, Kaether C, Sauer M, Eggeling C, Franke C. NanTex enables computational multiplexing and phenotyping of organelles across super-resolution modalities. bioRxiv (2025) doi: 10.1101/2025.09.16.676574
Aknine N, Holban P, Ragaller F, Carravilla P, Gurdap CO, Cetinkaya A, Sezgin E, Klymchenko AS. Prodan-based solvatochromic probes for polarity imaging of organelles. biorRxiv (2025) doi: 10.1101/2025.10.17.683001
24. Ulrich HF, Klein T, Zhao Z, Cseresnyés Z, Carravilla P, Gerst R, Kasberg A, Scharfenberg FP, Figge MT, Eggeling C, Brendel JC. Selective Activation of Dynamics in Kinetically Frozen Supramolecular Polymer Bottlebrush Assemblies. Small (2025) e05481. doi: 10.1002/smll.202505481
23. Carravilla P, Andronico L, Schlegel J, Urem YB, Sjule E, Ragaller F, Weber F, Gurdap CO, Ascioglu Y, Sych T, Lorent J, Sezgin E. Measuring plasma membrane fluidity using confocal microscopy. Nat Protoc (2025) 20(7):1976-2004. doi: 10.1038/s41596-024-01122-8
22. Dasgupta A, Koerfer A, Kokot B, Urbančič I, Eggeling C, Carravilla P. Effects and avoidance of photoconversion-induced artifacts in confocal and STED microscopy. Nat Methods (2024) 21(7):1171-1174. doi: 10.1038/s41592-024-02297-4
21. Sommerauer C, Gallardo-Dodd CJ, Savva C, Hases L, Birgersson M, Indukuri R, Shen JX, Carravilla P, Geng K, Nørskov Søndergaard J, Ferrer-Aumatell C, Mercier G, Sezgin E, Korach-André M, Petersson C, Hagström H, Lauschke VM, Archer A, Williams C, Kutter C. Estrogen receptor activation remodels TEAD1 gene expression to alleviate hepatic steatosis. Mol Syst Biol (2024) 20(4):374-402. doi: 10.1038/s44320-024-00024-x
20. Seltmann A, Carravilla P, Reglinski K, Eggeling C, Waithe D. Neural network informed photon filtering reduces fluorescence correlation spectroscopy artifacts. Biophys J (2024) 123(6):745-755. doi: 10.1016/j.bpj.2024.02.012
19. Ebrahimi V, Stephan T, Kim J, Carravilla P, Eggeling C, Jakobs S, Han KY. Deep learning enables fast, gentle STED microscopy. Commun Biol (2023) 6(1):674. doi: 10.1038/s42003-023-05054-z
18. Insausti S, Garcia-Porras M, Torralba J, Morillo I, Ramos-Caballero A, de la Arada I, Apellaniz B, Caaveiro JMM, Carravilla P, Eggeling C, Rujas E, Nieva JL. Functional Delineation of a Protein-Membrane Interaction Hotspot Site on the HIV-1 Neutralizing Antibody 10E8. Int J Mol Sci (2022) 23(18):10767. doi: 10.3390/ijms231810767
17. Galiani S, Reglinski K, Carravilla P, Barbotin A, Urbančič I, Ott J, Sehr J, Sezgin E, Schneider F, Waithe D, Hublitz P, Schliebs W, Erdmann R, Eggeling C. Diffusion and interaction dynamics of the cytosolic peroxisomal import receptor PEX5. Biophys Rep (2022) 8;2(2). doi: 10.1016/j.bpr.2022.100055
16. Carravilla P, Dasgupta A, Zhurgenbayeva G, Danylchuk DI, Klymchenko AS, Sezgin E, Eggeling C. Long-term STED imaging of membrane packing and dynamics by exchangeable polarity-sensitive dyes. Biophys Rep (2021) 1(2). doi: 10.1016/j.bpr.2021.100023.
15. Rujas E, Leaman DP, Insausti S, Carravilla P, García-Porras M, Largo E, Morillo I, Sánchez-Eugenia R, Zhang L, Cui H, Iloro I, Elortza F, Julien JP, Eggeling C, Zwick MB, Caaveiro JMM, Nieva JL. Focal accumulation of aromaticity at the CDRH3 loop mitigates 4E10 polyreactivity without altering its HIV neutralization profile. iScience (2021) 24(9):102987. doi: 10.1016/j.isci.2021.102987
14. Largo E, Queralt-Martín M, Carravilla P, Nieva JL, Alcaraz A. Single-molecule conformational dynamics of viroporin ion channels regulated by lipid-protein interactions. Bioelectrochemistry (2021) 137:107641. doi: 10.1016/j.bioelechem.2020.107641
13. Hidalgo A, Garcia-Mouton C, Autilio C, Carravilla P, Orellana G, Islam MN, Bhattacharya J, Bhattacharya S, Cruz A, Pérez-Gil J. Pulmonary surfactant and drug delivery: Vehiculization, release and targeting of surfactant/tacrolimus formulations. J Control Release (2021) 329:205-222. doi: 10.1016/j.jconrel.2020.11.042
12. Rujas E, Insausti S, Leaman DP, Carravilla P, González-Resines S, Monceaux V, Sánchez-Eugenia R, García-Porras M, Iloro I, Zhang L, Elortza F, Julien JP, Saéz-Cirión A, Zwick MB, Eggeling C, Ojida A, Domene C, Caaveiro JMM, Nieva JL. Affinity for the Interface Underpins Potency of Antibodies Operating In Membrane Environments. Cell Rep (2020) 32(7):108037. doi: 10.1016/j.celrep.2020.108037
11. Torralba J, de la Arada I, Carravilla P, Insausti S, Rujas E, Largo E, Eggeling C, Arrondo JLR, Apellániz B, Nieva JL. Cholesterol Constrains the Antigenic Configuration of the Membrane-Proximal Neutralizing HIV-1 Epitope. ACS Infect Dis (2020) 6(8):2155-2168. doi: 10.1021/acsinfecdis.0c00243
10. Carravilla P, Nieva JL, Eggeling C. Fluorescence Microscopy of the HIV-1 Envelope. Viruses (2020) 12(3):348. doi: 10.3390/v12030348
9. Carravilla P, Darré L, Oar-Arteta IR, Vesga AG, Rujas E, de Las Heras-Martínez G, Domene C, Nieva JL, Requejo-Isidro J. The Bilayer Collective Properties Govern the Interaction of an HIV-1 Antibody with the Viral Membrane. Biophys J (2020) 118(1):44-56. doi: 10.1016/j.bpj.2019.11.005
8. Carravilla P, Chojnacki J, Rujas E, Insausti S, Largo E, Waithe D, Apellaniz B, Sicard T, Julien JP, Eggeling C, Nieva JL. Molecular recognition of the native HIV-1 MPER revealed by STED microscopy of single virions. Nat Commun (2019) 10(1):78. doi: 10.1038/s41467-018-07962-9
7. Carravilla P, Nieva JL. HIV Antivirals: Targeting the Functional Organization of the Lipid Envelope. Future Virol (2018) 13(2):129-140. doi: 10.2217/fvl-2017-0114
6. Carravilla P, Cruz A, Martin-Ugarte I, Oar-Arteta IR, Torralba J, Apellaniz B, Pérez-Gil J, Requejo-Isidro J, Huarte N, Nieva JL. Effects of HIV-1 gp41-Derived Virucidal Peptides on Virus-like Lipid Membranes. Biophys J (2017) 113(6):1301-1310. doi: 10.1016/j.bpj.2017.06.061
5. Chojnacki J, Waithe D, Carravilla P, Huarte N, Galiani S, Enderlein J, Eggeling C. Envelope glycoprotein mobility on HIV-1 particles depends on the virus maturation state. Nat Commun (2017) 8(1):545. doi: 10.1038/s41467-017-00515-6
4. Huarte N, Carravilla P, Cruz A, Lorizate M, Nieto-Garai JA, Kräusslich HG, Pérez-Gil J, Requejo-Isidro J, Nieva JL. Functional organization of the HIV lipid envelope. Sci Rep (2016) 6:34190. doi: 10.1038/srep34190
3. Carravilla P, Nieva JL, Goñi FM, Requejo-Isidro J, Huarte N. Two-photon Laurdan studies of the ternary lipid mixture DOPC:SM:cholesterol reveal a single liquid phase at sphingomyelin:cholesterol ratios lower than 1. Langmuir (2015) 31(9):2808-17. doi: 10.1021/la504251u
2. Apellániz B, Rujas E, Carravilla P, Requejo-Isidro J, Huarte N, Domene C, Nieva JL. Cholesterol-dependent membrane fusion induced by the gp41 membrane-proximal external region-transmembrane domain connection suggests a mechanism for broad HIV-1 neutralization. J Virol (2014) 88(22):13367-77. doi: 10.1128/JVI.02151-14
1. Serrano S, Araujo A, Apellániz B, Bryson S, Carravilla P, de la Arada I, Huarte N, Rujas E, Pai EF, Arrondo JLR, Domene C, Jiménez MA, Nieva JL. Structure and immunogenicity of a peptide vaccine, including the complete HIV-1 gp41 2F5 epitope: implications for antibody recognition mechanism and immunogen design. J Biol Chem (2014) 289(10):6565-6580. doi: 10.1074/jbc.M113.527747