We explore the potential of using the DASPI molecular rotor [trans-4-[4-(Dimethylamino)styryl]-1-methylpyridinium iodide] to probe the rheology of red blood cells. Previous investigations have demonstrated the rotor’s capacity to penetrate cellular membranes and detect temperature-dependent modifications in membrane and cytosol mechanical properties. In this study, we demonstrate that the rotor has no specific affinity for intracellular proteins, leading to the establishment of a DASPI concentration equilibrium between the intracellular and extracellular environments. Using confocal microscopy and a simple geometric model of cells, we estimate that approximately of the fluorescence intensity measured by widefield epifluorescence microscopy originates from the cytosol. We show that the rotor can differentiate healthy donors based on the variations in intracellular hemoglobin concentration and intracellular viscosity. It also detects intra-donor variability due to intrinsic properties of the cells. This study provides direct evidence for the use of the DASPI molecular rotor as a probe for rapid reading of cell rigidity and heterogeneity in blood samples, with potential applications in the study of erythrocyte pathologies.
REFERENCES
1.
Haidekker
, M. A.
, and E. A.
Theodorakis
, “Environment-sensitive behavior of fluorescent molecular rotors
,” J. Biol. Eng.
4
, 11
–14
(2010
). 2.
Haidekker
, M. A.
, M.
Nipper
, A.
Mustafic
, D.
Lichlyter
, M.
Dakanali
, and E. A.
Theodorakis
, Advanced Fluorescence Reporters in Chemistry and Biology I: Fundamentals and Molecular Design, edited by A. P. Demchenko (Springer, Berlin, 2010), pp. 267–308.3.
Haidekker
, M. A.
, T.
Brady
, D.
Lichlyter
, and E.
Theodorakis
, “Effects of solvent polarity and solvent viscosity on the fluorescent properties of molecular rotors and related probes
,” Bioorg. Chem.
33
, 415
–425
(2005
). 4.
Haidekker
, M.
, and E. A.
Theodorakis
, “Molecular rotors—Fluorescent biosensors for viscosity and flow
,” Org. Biomol. Chem.
5
, 1669
–1678
(2007
). 5.
Allen
, B.
, A. C.
Benniston
, A.
Harriman
, S. A.
Rostron
, and C.
Yua
, “The photophysical properties of a julolidene-based molecular rotor
,” Phys. Chem. Chem. Phys.
7
, 3035
–3040
(2005
). 6.
Loutfy
, R. O.
, and A. A.
Bradley
, “Effect of viscosity and temperature on torsional relaxation of molecular rotors
,” J. Phys. Chem.
86
, 4205
–4211
(1982
). 7.
Law
, K.
, “Fluorescence probe for microenvironments: Anomalous viscosity dependence of the fluorescence quantum yield of p-n,n-dialkylaminobenzylidenemalononitrile in 1-alkanols
,” Chem. Phys. Lett.
75
, 545
–549
(1980
). 8.
Förster
, T.
, and G.
Hoffmann
, “Die viskositätsabhängigkeit der fluoreszenzquantenausbeuten einiger farbstoffsysteme
,” Z. Phys. Chem.
75
, 63
–76
(1971
).9.
Howell
, S.
, M.
Dakanali
, E. A.
Theodorakis
, and M. A.
Haidekker
, “Intrinsic and extrinsic temperature-dependency of viscosity-sensitive fluorescent molecular rotors
,” J. Fluoresc.
22
, 457
–465
(2012
). 10.
Iwaki
, T.
, C.
Torigoe
, M.
Noji
, and M.
Nakanishi
, “Antibodies for fluorescent molecular rotors
,” Biochemistry
32
, 7589
–7592
(1993
). 11.
Wang
, K.
, W.
Shi
, J.
Jia
, S.
Chen
, and H.
Ma
, “Characterization of 2-phenylbenzo[g]quinoxaline derivatives as viscosity-sensitive fluorescent probes
,” Talanta
77
, 1795
–1799
(2009
). 12.
Haidekker
, M.
, N.
L’Heureux
, and J.
Frangos
, “Fluid shear stress increases membrane fluidity in endothelial cells: A study with DCVJ fluorescence
,” Am. J. Physiol. Heart Circ. Physiol.
278
, H1401
–H1406
(2000
). 13.
Chen
, T.
, Z.
Chen
, R.
Liu
, and S.
Zheng
, “A NIR fluorescent probe for detection of viscosity and lysosome imaging in live cells
,” Org. Biomol. Chem.
17
, 6398
–6403
(2019
). 14.
Akers
, W. J.
, and M. A.
Haidekker
, “Precision assessment of biofluid viscosity measurements using molecular rotors
,” J. Biomech. Eng.
127
, 450
–454
(2005
). 15.
Haidekker
, M.
, A.
Tsai
, T.
Brady
, H.
Stevens
, J.
Frangos
, E.
Theodorakis
, and M.
Intaglietta
, “A novel approach to blood plasma viscosity measurement using fluorescent molecular rotors
,” Am. J. Physiol. Heart Circ. Physiol.
282
, H1609
–H1614
(2002
).16.
Kuimova
, M. K.
, G.
Yahioglu
, J. A.
Levitt
, and K.
Suhling
, “Molecular rotor measures viscosity of live cells via fluorescence lifetime imaging
,” J. Am. Chem. Soc.
130
, 6672
–6673
(2008
). 17.
Kuimova
, M. K.
, S. W.
Botchway
, A. W.
Parker
, M.
Balaz
, H. A.
Collins
, H. L.
Anderson
, K.
Suhling
, and P. R.
Ogilby
, “Imaging intracellular viscosity of a single cell during photoinduced cell death
,” Nat. Chem.
1
, 69
–73
(2009
). 18.
Woodcock
, E. M.
, P.
Girvan
, J.
Eckert
, I.
Lopez-Duarte
, M.
Kubànkovà
, J.
van Loon
, N. J.
Brooks
, and M. K.
Kuimova
, “Measuring intracellular viscosity in conditions of hypergravity
,” Biophys. J.
116
, 1984
–1993
(2019
). 19.
Thompson
, J.
, T.
Herling
, M.
Kubankova
, A.
Vysniauskas
, T.
Knowles
, and M. K.
Kuimova
, “Molecular rotors provide insights into microscopic structural changes during protein aggregation
,” J. Phys. Chem. B
119
, 10170
–10179
(2015
). 20.
Kubankova
, M.
, I.
Lopez-Duarte
, J. A.
Bull
, D. M.
Vadukul
, L.
Serpell
, M.
de Saint Victor
, E.
Stride
, and M. K.
Kuimova
, “Probing supramolecular protein assembly using covalently attached fluorescent molecular rotors
,” Biomaterials
139
, 195
–201
(2017
).21.
Goh
, W. L.
, M.
Lee
, T.
Joseph
, S.
Quah
, C.
Brown
, C.
Verma
, S.
Brenner
, F.
Ghadessy
, and Y. N.
Teo
, “Molecular rotors as conditionally fluorescent labels for rapid detection of biomolecular interactions
,” J. Am. Chem. Soc.
136
, 6159
–6162
(2014
). 22.
Yu
, W. T.
, T. W.
Wu
, C. L.
Huang
, I. C.
Chen
, and K. T.
Tan
, “Protein sensing in living cells by molecular rotor-based fluorescence-switchable chemical probes
,” Chem. Sci.
7
, 301
–307
(2016
). 23.
Gatzogiannis
, E.
, Z.
Chen
, L.
Wei
, R.
Wombacher
, Y.
Kao
, G.
Yefremov
, V. W.
Cornish
, and W.
Min
, “Mapping protein-specific micro-environments in live cells by fluorescence lifetime imaging of a hybrid genetic-chemical molecular rotor tag
,” Chem. Commun.
48
, 8694
–8696
(2012
). 24.
Hawe
, A.
, V.
Filipe
, and W.
Jiskoot
, “Fluorescent molecular rotors as dyes to characterize polysorbate-containing IgG formulations
,” Pharm. Res.
27
, 314
–326
(2010
). 25.
Sawada
, S.
, T.
Iio
, Y.
Hayashi
, and S.
Takahashi
, “Fluorescent rotors and their applications to the study of G-F transformation of actin
,” Anal. Biochem.
204
, 110
–117
(1992
). 26.
Kung
, C. E.
, and J. K.
Reed
, “Fluorescent molecular rotors: A new class of probes for tubulin structure and assembly
,” Biochemistry
28
, 6678
–6686
(1989
).m 27.
Loutfy
, R. O.
, and D. M.
Teegarden
, “Effect of polymer chain tacticity on the fluorescence of molecular rotors
,” Macromolecules
16
, 452
–456
(1983
). 28.
Zhu
, D.
, M. A.
Haidekker
, J.-S.
Lee
, Y.-Y.
Won
, and J.-M.
Lee
, “Application of molecular rotors to the determination of the molecular weight dependence of viscosity in polymer melts
,” Macromolecules
40
, 7730
–7732
(2007
). 29.
Kung
, C. E.
, and J. K.
Reed
, “Microviscosity measurements of phospholipid bilayers using fluorescent dyes that undergo torsional relaxation
,” Biochemistry
25
, 6114
–6121
(1986
). 30.
Viriot
, M. L.
, M. C.
Carre
, C.
Geoffroy-Chapotot
, A.
Brembilla
, S.
Muller
, and J. F.
Stoltz
, “Molecular rotors as fluorescent probes for biological studies
,” Clin. Hemorheol. Microcirc.
19
, 151
–160
(1998
).31.
Ma
, C.
, W.
Sun
, L.
Xu
, Y.
Qian
, J.
Dai
, G.
Zhong
, Y.
Hou
, J.
Liu
, and B.
Shen
, “A minireview of viscosity-sensitive fluorescent probes: Design and biological applications
,” J. Mater. Chem. B
8
, 9642
–9651
(2020
). 32.
Longeville
, S.
, and L.-R.
Stingaciu
, “Hemoglobin diffusion and the dynamics of oxygen capture by red blood cells
,” Sci. Rep.
7
, 10448
(2017
). 33.
Kaza
, N.
, A.
Ojaghi
, and F. E.
Robles
, “Hemoglobin quantification in red blood cells via dry mass mapping based on UV absorption
,” J. Biomed. Opt.
26
, 086501
(2021
). 34.
Briole
, A.
, T.
Podgorski
, and B.
Abou
, “Molecular rotors as intracellular probes of red blood cell stiffness
,” Soft Matter
17
, 4525
–4537
(2021
). 35.
Briole
, A.
, and B.
Abou
, “Molecular rotors in haemoglobin and bovine serum albumin proteins
,” J. R. Soc. Interface
19
, 20220709
(2022
). 36.
Gurtovenko
, A. A.
, and J.
Anwar
, “Modulating the structure and properties of cell membranes: The molecular mechanism of action of dimethyl sulfoxide
,” J. Phys. Chem. B
111
, 10453
–10460
(2007
). 37.
Diez-Silva
, M.
, M.
Dao
, J.
Han
, C.
Lim
, and S.
Suresh
, “Shape and biomechanical characteristics of human red blood cells in health and disease
,” MRS Bull.
35
, 382
–388
(2010
). 38.
Balach
, M. M.
, C. H.
Casale
, and A. N.
Campetelli
, “Erythrocyte plasma membrane potential: Past and current methods for its measurement
,” Biophys. Rev.
11
, 995
–1005
(2019
). 39.
Barbosa
, N. S.
, E. R.
Lima
, M.
Bostrom
, and F. W.
Tavares
, “Membrane potential and ion partitioning in an erythrocyte using the Poisson–Boltzmann equation
,” J. Phys. Chem. B
119
, 6379
–6388
(2015
). 40.
Gedde
, M. M.
, and W. H.
Huestis
, “Membrane potential and human erythrocyte shape
,” Biophys. J.
72
, 1220
–1233
(1997
). 41.
Einstein
, A.
, “Über die von der molekularkinetischen Theorie der Wärme geforderte Bewegung von in ruhenden Flüssigkeiten suspendierten Teilchen
,” Ann. Phys.
322
, 549
–560
(1905
). 42.
Kapusta
, P.
, “Absolute diffusion coefficients: Compilation of reference data for FCS calibration,” Picoquant GmbH, 2010.43.
Heinrich
, V.
, K.
Ritchie
, N.
Mohandas
, and E.
Evans
, “Elastic thickness compressibilty of the red cell membrane
,” Biophys. J.
81
, 1452
–1463
(2001
). 44.
Bennett
, V.
, and D. N.
Lorenzo
, “An adaptable spectrin/ankyrin-based mechanism for long-range organization of plasma membranes in vertebrate tissues
,” Curr. Top. Membr.
77
, 143
–184
(2016
).45.
Lux
, S. E.
, IV, “Anatomy of the red cell membrane skeleton: Unanswered questions
,” Blood
127
, 187
–199
(2016
).46.
Waugh
, R.
, N.
Mohandas
, C.
Jackson
, T.
Mueller
, T.
Suzuki
, and G.
Dale
, “Rheologic properties of senescent erythrocytes: Loss of surface area and volume with red blood cell age
,” Am. Soc. Hem.
79
, 1351
–1358
(1996
).You do not currently have access to this content.
