Response of fluorescent molecular rotors in ternary macromolecular mixtures
Pith reviewed 2026-05-16 12:03 UTC · model grok-4.3
The pith
In ternary PEG mixtures, fluorescent molecular rotor lifetimes follow a linear rule with the proportion of light and heavy polymers.
A machine-rendered reading of the paper's core claim, the machinery that carries it, and where it could break.
Core claim
For the investigated composition range of ternary mixtures, a linear mixing rule applies for fluorescence lifetime with the proportion of the two PEG, and with an increasing ratio of heavy PEG leading to larger lifetimes. These results allow to test more precisely the free volume theory, which has been proposed in the context of probing glass transition. Analysis show that while this theory semi-quantitatively captures the observation, its precise use raises some questions.
What carries the argument
The linear mixing rule for fluorescence lifetime, which directly ties the rotor response to the weighted fractions of the two PEG components in the mixture.
If this is right
- Lifetimes in PEG blends can be predicted from the simple proportion of each polymer size.
- Higher fractions of heavy PEG raise the effective local restriction sensed by the rotor.
- Free volume theory accounts for the trend at a semi-quantitative level.
- Calibration procedures for rotors in polymer solutions can rely on linear interpolation between binary endpoints.
Where Pith is reading between the lines
- The same linear dependence could be checked in mixtures involving other water-soluble polymers to see if the rule generalizes beyond PEG.
- In biological fluids containing macromolecules of varying sizes, rotors might report an effective average chain length without separate calibrations for each component.
- The questions raised about precise application of free volume theory point to the value of testing rotors in mixtures where specific polymer-polymer or polymer-water interactions are known to be strong.
Load-bearing premise
The local environment around the rotor can be treated as a simple linear combination of the two PEG types without extra specific interactions or the need for mixture-specific adjustments.
What would settle it
Measure rotor lifetimes in a ternary PEG mixture whose composition falls outside the paper's tested range and check whether the values deviate from the linear prediction calculated from the two PEG proportions.
Figures
read the original abstract
For a few decades, Fluorescent Molecular Rotors have been commonly employed as local probes of microviscosity in complex materials. However, without proper calibration, relating microviscosity to a physical parameter is unclear, which strongly limits their quantitative use in biological media for instance. In this study, the response of a molecular rotor in binary and ternary macromolecular aqueous solutions of polyethylene glycol (PEG) of different molecular weights is investigated in order to better rationalize the sensitivity of rotors to their cybotactic environment. More precisely, for the investigated composition range of ternary mixtures, it is shown that a linear mixing rule applies for fluorescence lifetime with the proportion of the two PEG, and with an increasing ratio of heavy PEG leading to larger lifetimes. These results allow to test more precisely the free volume theory, which has been proposed in the context of probing glass transition. Analysis show that while this theory semi-quantitatively captures the observation, its precise use raises some questions.
Editorial analysis
A structured set of objections, weighed in public.
Referee Report
Summary. The manuscript investigates the response of fluorescent molecular rotors in binary and ternary aqueous mixtures of polyethylene glycols (PEGs) of different molecular weights. It reports that, for the investigated composition range, fluorescence lifetime follows a linear mixing rule with the proportion of the two PEG components, with increasing ratio of heavier PEG leading to larger lifetimes. The data are compared to free-volume theory, which is found to capture the observations semi-quantitatively while raising questions about its precise use.
Significance. If the reported linear mixing rule holds, the work provides a practical route to calibrate molecular rotors in mixed macromolecular systems, supporting more quantitative microviscosity measurements in complex media such as biological samples. The experimental results on ternary mixtures and the semi-quantitative test of free-volume theory add to the literature on local-environment sensing by rotors and highlight needed refinements in models originally developed for glass-transition studies.
major comments (1)
- [Abstract and Results] Abstract and Results: the central claim that a linear mixing rule applies is presented without accompanying error bars, statistical measures of linearity, composition tables, or raw data, so it is not possible to evaluate whether the linearity remains supported once measurement uncertainty and possible post-hoc selection are accounted for.
minor comments (2)
- [Abstract] The abstract contains a grammatical error: 'Analysis show' should read 'Analyses show' or 'The analysis shows'.
- [Discussion] The discussion notes that free-volume theory 'raises some questions' about its precise use but does not specify what those questions are or outline concrete tests that would resolve them.
Simulated Author's Rebuttal
We thank the referee for the positive assessment of our work and the recommendation for minor revision. We address the single major comment below and will incorporate the requested improvements in the revised manuscript.
read point-by-point responses
-
Referee: [Abstract and Results] Abstract and Results: the central claim that a linear mixing rule applies is presented without accompanying error bars, statistical measures of linearity, composition tables, or raw data, so it is not possible to evaluate whether the linearity remains supported once measurement uncertainty and possible post-hoc selection are accounted for.
Authors: We agree that the presentation of the linear mixing rule would be strengthened by explicit uncertainty quantification and supporting data. In the revised manuscript we will add error bars (standard deviation from n=3 independent measurements) to all data points in Figures 3 and 4, report the Pearson correlation coefficient and p-value for each linear regression, and include a new supplementary table listing the exact mass fractions of each PEG component together with the corresponding fluorescence lifetimes. The compositions were not chosen post-hoc; they were predetermined from the binary-mixture calibration curves to span the full range of heavy-to-light PEG ratios while remaining within the experimentally accessible viscosity window, as already stated in the Methods section. Raw data files will be deposited in the supplementary information. These additions will allow readers to assess the robustness of the linearity directly. revision: yes
Circularity Check
No circularity: empirical observation of linear mixing rule with semi-quantitative theory comparison
full rationale
The paper reports direct experimental measurements of fluorescence lifetimes in binary and ternary aqueous PEG mixtures and observes that lifetime follows a linear mixing rule with PEG proportion (heavier PEG yielding larger lifetimes). It then compares these data semi-quantitatively to free-volume theory without deriving the linearity from first principles, without fitting parameters to the same dataset and renaming them as predictions, and without load-bearing self-citations that reduce the central claim to prior author work. The linearity is presented as an empirical finding within the tested composition range; the theory comparison explicitly notes only semi-quantitative agreement and raises questions about precise applicability. No self-definitional steps, fitted-input predictions, uniqueness theorems, or ansatz smuggling occur. The derivation chain is therefore self-contained and externally falsifiable via the reported measurements.
Axiom & Free-Parameter Ledger
axioms (1)
- domain assumption Free volume theory semi-quantitatively links fluorescence lifetime to available space in the cybotactic environment of the rotor
Lean theorems connected to this paper
-
IndisputableMonolith/Cost/FunctionalEquation.leanwashburn_uniqueness_aczel unclear?
unclearRelation between the paper passage and the cited Recognition theorem.
for the investigated composition range of ternary mixtures, it is shown that a linear mixing rule applies for fluorescence lifetime with the proportion of the two PEG
-
IndisputableMonolith/Foundation/AlphaCoordinateFixation.leanJ_uniquely_calibrated_via_higher_derivative unclear?
unclearRelation between the paper passage and the cited Recognition theorem.
Analysis show that while this theory semi-quantitatively captures the observation, its precise use raises some questions
What do these tags mean?
- matches
- The paper's claim is directly supported by a theorem in the formal canon.
- supports
- The theorem supports part of the paper's argument, but the paper may add assumptions or extra steps.
- extends
- The paper goes beyond the formal theorem; the theorem is a base layer rather than the whole result.
- uses
- The paper appears to rely on the theorem as machinery.
- contradicts
- The paper's claim conflicts with a theorem or certificate in the canon.
- unclear
- Pith found a possible connection, but the passage is too broad, indirect, or ambiguous to say the theorem truly supports the claim.
Reference graph
Works this paper leans on
- [1]
-
[2]
G. Garg, A. Madeu-Bultó, N. Farfán, J. Ordóñez-Hernández, M. Gómez, and Y . Medina-González, Palladium nanoparticles in glycerol/ionic liquid/carbon dioxide medium as hydrogena- tion catalysts, ACS Applied Nano Materials3, 12240 (2020)
work page 2020
- [3]
-
[4]
F. Caporaletti, M. Bittermann, D. Bonn, and S. Woutersen, Flu- orescent molecular rotor probes nanosecond viscosity changes, The Journal of Chemical Physics156, 201101 (2022)
work page 2022
- [5]
-
[6]
D. Zhu, M. Haidekker, J. Lee, Y . Won, and J. Lee, Application of molecular rotors to the determination of the molecular weight dependence of viscosity in polymer melts, Macromolecules40, 7730 (2007)
work page 2007
-
[7]
W. Lee, C. Lee, T. Sakaguchi, M. Fujiki, and G. Kwak, Fluo- rescent viscosity sensor film of molecular-scale porous polymer with intramolecularπ-stack structure, Macromolecules44, 432 (2011)
work page 2011
-
[8]
S. Kim, M. Lee, H. Kim, Y . Kim, W. Lee, and Y . Won, Deter- mination of glass transition temperatures in bulk and micellar 16 M2 w 0.4 0.45 0.5 0.55 0.6 400 v(1) m 0.934 0.926 0.918 0.912 0.909 lnη (1) 6.91 7.64 8.22 8.80 9.34 τ (1) 0.74 0.98 1.03 1.26 1.38 2000 v(1) m - - 0.918 - - lnη (1) - - 8.02 - - τ (1) - - 1.05 - - TABLE VII: List of fitting param...
work page 2000
-
[9]
A. Paul and A. Samanta, Free volume dependence of the in- ternal rotation of a molecular rotor probe in room tempera- ture ionic liquids, Journal of Physical Chemistry B112, 16626 (2008)
work page 2008
- [10]
-
[11]
C. Kung and J. Reed, Fluorescent molecular rotors: A new class of probes for tubulin structure and assembly, Biochemistry28, 6678 (1989)
work page 1989
-
[12]
W. Akers and M. Haidekker, Precision assessment of biofluid viscosity measurements using molecular rotors, Transactions of the ASME127, 450 (2005)
work page 2005
-
[13]
K. Daus, S. Tharamak, W. Pluempanupat, P. Galie, M. Theodor- aki, E. Theodorakis, and M. Alpaugh, Fluorescent molecular rotors as versatile in situ sensors for protein quantitation, Sci- entific Reports13, 20529 (2023)
work page 2023
-
[14]
S. Schmitt, G. Renzer, J. Benrath, A. Best, S. Jiang, K. Land- fester, H. Butt, R. Simonutti, D. Crespy, and K. Koynov, Mon- itoring the formation of polymer nanoparticles with fluorescent molecular rotors, Macromolecules55, 7284 (2022)
work page 2022
-
[15]
J. Raeburn, L. Chen, S. Awhida, R. Deller, M. Vatish, M. Giba- son, and D. Adams, Using molecular rotors to probe gelation, Soft Matter11, 3706 (2015)
work page 2015
-
[16]
R. Le Dizès Castell, E. Mizahossein, M. Grzelka, S. Jabbari- Farouji, D. Bonn, and N. Shahidzadeh, Visualization of the sol- gel transition in porous networks using fluorescent viscosity- sensitive probes, Journal of Physical Chemistry Letters15, 628 (2024)
work page 2024
-
[17]
M. Haidekker, W. Akers, D. Fischer, and E. Theodorakis, Op- tical fiber-based fluorescent viscosity sensor, Optics Letters31, 2529 (2006)
work page 2006
-
[18]
D. Lichlyter and M. Haidekker, Immobilization techniques for molecular rotors—towards a solid-state viscosity sensor plat- form, Sensors and Actuators B: Chemical139, 648 (2009)
work page 2009
-
[19]
Y . Jin, H. Park, Y . Ohk, and G. Kwak, Hydrodynamic fluores- cence emission behavior of molecular rotor-based vinyl poly- mers used as viscosity sensors, Polymer132, 79 (2017)
work page 2017
-
[20]
D. Nalatamby, F. Gibouin, M. Zitouni, J. Renaudeau, G. Clis- son, P. Lidon, S. Harrisson, and Y . Medina-Gonzalez, Fluo- rescent molecular rotor-based polymer materials for local mi- croviscosity mapping in microfluidic channels, Chemical Engi- neering Journal520, 165549 (2025)
work page 2025
-
[21]
S. Velandia, M. Bittermann, E. Mirzahossein, G. Giubertoni, F. Caporaletti, V . Sadtler, P. Marchal, T. Roques-Carmes, M. Meinders, and D. Bonn, Probing interfaces of pea protein- stabilized emulsions with a fluorescent molecular rotor, Fron- tiers in Soft Matter3, 1093168 (2023)
work page 2023
-
[22]
M. Bittermann, T. Morozova, S. Velandia, E. Mirzahossein, A. Deblais, S. Woutersen, and D. Bonn, Surface-mediated molecular transport of a lipophilic fluorescent probe in poly- disperse oil-in-water emulsions, Langmuir39, 4207 (2023)
work page 2023
- [23]
-
[24]
Y . Wu, M. Štefl, A. Olzy ´nska, M. Hof, G. Yahioglu, P. Yip, D. Casey, O. Ces, J. Humpolí ˇcková, and M. Kuimova, Molec- ular rheometry: direct determination of viscosity in L o and Ld lipid phases via fluorescence lifetime imaging, Physical Chem- istry Chemical Physics15, 14986 (2013)
work page 2013
-
[25]
M. Dent, I. López-Duarte, C. Dickson, N. Geoghegan, J. Cooper, I. Gould, R. Krams, J. Bull, N. Brooks, and M. Kuimova, Imaging phase separation in model lipid mem- branes through the use of bodipy based molecular rotors, Phys- ical Chemistry Chemical Physics17, 18393 (2015)
work page 2015
-
[26]
A. Vyšniauskas, M. Qurashi, and M. Kuimova, A molecular rotor that measures dynamic changes of lipid bilayer viscos- ity caused by oxidative stress, Chemical European Journal22, 13210 (2016)
work page 2016
- [27]
-
[28]
J. Li, Y . Zhang, H. Zhang, X. Xuan, M. Xie, S. Xia, G. Qu, and H. Guo, Nucleoside-based ultrasensitive fluorescent probe for the dual-mode imaging of microviscosity in living cells, Ana- lytical Chemistry88, 5554 (2016)
work page 2016
- [29]
-
[30]
L. Michels, V . Gorelova, Y . Harnvanichvech, J. Borst, B. Al- bada, D. Weijers, and J. Sprakel, Complete microviscosity maps of living plant cells and tissues with a toolbox of targeting mechanoprobes, Proceedings of the National Academy of Sci- ences117, 18110 (2020)
work page 2020
-
[31]
M. Paez-Perez and M. Kuimova, Molecular rotors: Fluorescent sensors for microviscosity and conformation of biomolecules, Angewandte Chemie International Edition63, e202311233 (2024)
work page 2024
-
[32]
D. Xie, S. Sun, Q. Zhou, C. Wang, L. Cao, W. Zhang, and G. Li, Fluorescent chemosensors facilitate the visualization of plant health and their living environment in sustainable agriculture, Chemical Communications61, 10408 (2025)
work page 2025
-
[33]
T. Förster and G. Hoffmann, Die viskositätsabhängigkeit der fluoreszenzquantenausbeuten einiger farbstoffsysteme, Zeitschrift für Physikalische Chemie Neue Folge75, 63 (1971)
work page 1971
- [34]
-
[35]
D. Nalatamby, F. Gibouin, J. Ordóñez-Hernández, J. Re- naudeau, G. Clisson, N. Farfán, P. Lidon, and Y . Medina- González, Molecular rotors for in situ local viscosity mapping in microfluidic chips, Industrial & Engineering Chemistry Re- 17 search62, 12656 (2023)
work page 2023
-
[36]
F. Gibouin, D. Nalatamby, P. Lidon, and Y . Medina-Gonzalez, Molecular rotors for in situ viscosity mapping during evapora- tion of confined fluid mixtures, ACS Applied Materials & In- terfaces16, 8066 (2024)
work page 2024
-
[37]
E. Mirzahossein, M. Grzelka, and D. Bonn, Unveiling droplet morphologies: real-time viscosity mapping reveals the physics of drying polymer solutions, Soft Matter21, 8018 (2025)
work page 2025
-
[38]
M. Klok, L. Janssen, W. Browne, and B. Feringa, The influence of viscosity on the functioning of molecular motors, Faraday Discussions143, 319 (2009)
work page 2009
-
[39]
P. Roy, A. Sardjan, W. Danowski, W. Browne, B. Feringa, and S. Meech, Photophysics of first-generation photomolecular mo- tors: Resolving roles of temperature, friction, and medium po- larity, Journal of the Physical Chemistry A125, 1711 (2021)
work page 2021
-
[40]
P. Roy, A. Sardjan, W. Browne, B. Feringa, and S. Meech, Ex- cited state dynamics in unidirectional photochemical molecular motors, Journal of the American Chemical Society146, 12255 (2024)
work page 2024
-
[41]
A. Straube, B. Kowalik, R. Netz, and F. Höfling, Rapid onset of molecular friction in liquids bridging between the atomistic and hydrodynamic pictures, Communications Physics3, 126 (2020)
work page 2020
- [42]
-
[43]
G. Rothenberger, D. Negus, and R. Hochstrasser, Solvent influ- ence on photoisomerization dynamics, The Journal of Chemical Physics79, 5360 (1983)
work page 1983
-
[44]
V . Suja, N. Oppenheimer, and H. Stone, Hydrodynamics of molecular rotors in lipid membranes, Physical Review Fluids 10, L041101 (2025)
work page 2025
-
[45]
M. Bittermann, M. Grzelka, S. Woutersen, A. Brouwer, and D. Bonn, Disentangling nano- and macroscopic viscosities of aqueous polymer solutions using a fluorescent molecular rotor, Journal of Physical Chemistry Letters12, 3182 (2021)
work page 2021
-
[46]
I. Kohli and A. Mukhopahyay, Diffusion of nanoparticles in semidilute polymer solutions: Effect of different length scales, Macromolecules45, 6143 (2012)
work page 2012
- [47]
-
[48]
R. Loutfy and B. Arnold, Effect of viscosity and temperature on torsional relaxation of molecular rotors, Journal of Physical Chemistry86, 4205 (1982)
work page 1982
-
[49]
M. Rao, K. Kumar, and K. Ravikanth, Synthesis of boron- dipyrromethene-ferrocene conjugates, Journal of Organometal- lic Chemistry695, 863 (2010)
work page 2010
-
[50]
E. Bahaidarah, A. Harriman, P. Stachelek, S. Rihn, E. Heyer, and R. Ziessel, Fluorescent molecular rotors based on the bod- ipy motif: effect of remote substituents., Photochemical & Pho- tobiological Sciences13, 1397 (2014)
work page 2014
-
[51]
L. Mei, D. Lin, Z. Zhu, and Z. Han, Densities and viscosities of polyethylene glycol + salt + water systems at 20 ◦c, Journal of Chemical Engineering Data40, 1168 (1995)
work page 1995
-
[52]
J. Lakowicz, R. Jayaweera, H. Szmancinski, and W. Wiczk, Resolution of multicomponent fluorescence emission using frequency-dependent phase angle and modulation spectra, An- alytical Chemistry62, 2005 (1990)
work page 2005
-
[53]
M. Kuimova, G. Yahioglu, J. Levitt, and K. Suhling, Molecular rotor measures viscosity of live cells via fluorescence lifetime imaging, Journal of the American Chemical Society130, 6672 (2008)
work page 2008
- [54]
- [55]
-
[56]
Loutfy, High-conversion polymerization fluorescence probes
R. Loutfy, High-conversion polymerization fluorescence probes. 1. polymerization of methyl methacrylate, Macro- molecules14, 270 (1981)
work page 1981
-
[57]
M. Williams, R. Landel, and J. Ferry, The temperature depen- dence of relaxation mechanisms in amorphous polymers and other glass-forming liquids, Journal of the American Chemical Society77, 3701 (1955)
work page 1955
-
[58]
Doolittle, Studies in newtonian flow
A. Doolittle, Studies in newtonian flow. i. the dependence of the viscosity of liquids on temperature, Journal of Applied Physics 22, 1031 (1951)
work page 1951
-
[59]
Doolittle, Studies in newtonian flow
A. Doolittle, Studies in newtonian flow. ii. the dependence of the viscosity of liquids on free space, Journal of Applied Physics22, 1471 (1951)
work page 1951
-
[60]
A. Doolittle and R. Peterson, Preparation and physical proper- ties of a series of n-alkanes, Journal of the American Chemical Society73, 2145 (1951)
work page 1951
-
[61]
Doolittle, Studies in newtonian flow
A. Doolittle, Studies in newtonian flow. iii. the dependence of the viscosity of liquids on molecular weight and free space (in homologous series), Journal of Applied Physics23, 236 (1952)
work page 1952
-
[62]
Doolittle, Studies in newtonian flow
A. Doolittle, Studies in newtonian flow. iv. viscosity vs molec- ular weight in liquids; viscosity vs concentration in polymer solutions, Journal of Applied Physics23, 418 (1952)
work page 1952
-
[63]
A. Doolittle and D. Doolittle, Studies in newtonian flow. v. fur- ther verification of the free space viscosity equation, Journal of Applied Physics28, 901 (1957)
work page 1957
-
[64]
A. Kovacs, Applicability of the free volume concept on relax- ation phenomena in the glass transition range, Rheologica Acta 5, 262 (1966)
work page 1966
-
[65]
R. Haward, Occupied volume of liquids and polymers, Journal of Macromolecular Science, Part C4, 191 (1970)
work page 1970
-
[66]
J. Vrentas and J. Duda, Diffusion in polymer-solvent systems. i. reexamination of the free-volume theory, Journal of Polymer Science15, 403 (1977)
work page 1977
-
[67]
J. Vrentas and J. Duda, Diffusion in polymer-solvent systems. ii. a predictive theory for the dependence of diffusion coef- ficients on temperature, concentration, and molecular weight, Journal of Polymer Science15, 417 (1977)
work page 1977
-
[68]
T. Hao, The empty world - a view from the free volume concept and eyring’s rate process theory, Physical Chemistry Chemical Physics26, 26156 (2025)
work page 2025
-
[69]
Loutfy, Fluorescence probes for polymer free-volume, Pure and Applied Chemistry58, 1239 (1986)
R. Loutfy, Fluorescence probes for polymer free-volume, Pure and Applied Chemistry58, 1239 (1986)
work page 1986
-
[70]
J. Hooker and J. Torkelson, Coupling of probe reorientation dy- namics and rotor motions to polymer relaxation as sensed by second harmonic generation and fluorescence, Macromolecules 28, 7683 (1995)
work page 1995
-
[71]
A. Jee, E. Bae, and M. Lee, Internal twisting dynamics of di- cyanovinyljulolidine in polymers, Journal of Physical Chem- istry B113, 16508 (2009)
work page 2009
- [72]
-
[73]
E. Mirzahossein, M. Grzelka, Z. Pan, B. Demirkurt, M. Habibi, A. Brouwer, and D. Bonn, Molecular rotors to probe the local viscosity of a polymer glass, Journal of Chemical Physics156, 174901 (2022)
work page 2022
- [74]
- [75]
-
[76]
D. Kawaguchi, Y . Tateishi, and K. Tanaka, Time-resolved flu- orescence analysis for dye-labeled polystyrene in thin films, Journal of Non-Crystalline Solids407, 284 (2015)
work page 2015
-
[77]
Sugden, Ccxxxiv.- molecular volumes at absolute zero
S. Sugden, Ccxxxiv.- molecular volumes at absolute zero. part ii. zero volumes and chemical composition, Journal of the Chemical Society , 1786 (1927)
work page 1927
-
[78]
K. Devanand and J. Selser, Asymptotic behavior and long- range interactions in aqueous solutions of poly(ethylene oxide), Macromolecules24, 5943 (1991)
work page 1991
discussion (0)
Sign in with ORCID, Apple, or X to comment. Anyone can read and Pith papers without signing in.