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. 2026 Aug 28;6(5):863–868. doi: 10.1021/acsnanoscienceau.6c00066

Addition–Fragmentation–Transfer Monitored by Fluorescence Lifetime Imaging Microscopy

M K Jutze †, Ellie Haljun †, Mao Komura †, Nanthicha Thajai ‡, Reika Katsumata †,*
PMCID: PMC13693185  PMID: 42921425

Abstract

Radically activated dynamic covalent bonds can enable reprocessable, thus more sustainable, polymeric materials, but conventional characterization approaches limit understanding of atomic-scale rearrangements. Specifically, the addition–fragmentation–transfer (AFT) pathway has been characterized by bulk-scale changes in mechanical properties; attempts to probe the exchange on a atomic scale are limited. Traditional spectroscopic methods cannot detect AFT because the overall bonding environment is unchanged before and after exchange with a very short-lived intermediate. To this end, this study uses fluorescence lifetime imaging microscopy (FLIM) to probe local AFT bond exchange via nondestructive characterization. Herein, allyl sulfide AFT in acrylate polymer thin films is monitored by FLIM over time, leveraging the sensitivity of fluorescence lifetime to nearby bond rearrangement through a series of control systems. Notably, FLIM reveals a continuing AFT cascade beyond the typical time scale when mechanical property change saturates. We anticipate applying these findings to other hard-to-access phenomena, as well as a mechanistic understanding of the environmental dependence of fluorescence lifetime.

Keywords: dynamic covalent bond, AFT, polymer, fluorescence lifetime, FLIM, allyl sulfide

graphic file with name ng6c00066_0006.webp

graphic file with name ng6c00066_0005.webp

Addition–fragmentation–transfer (AFT) bond exchange is expected to be one of the most promising candidates for reconfigurable materials with minimal creep susceptibility, as the radical required for AFT can be introduced via various approaches that avoid conflict with the expected use environment. Many dynamic bond chemistries, such as disulfide exchange, transesterification, , Diels–Alder reactions, , reversible imine formation, , and thiol–thioester exchange, have been developed. Among them, the AFT of allyl sulfides (Figure a) occurs independently of environmental temperature or light; rearrangement is triggered by the formation of a free radical. , Allyl sulfide-based AFT chemistry has already been incorporated into various functional materials. For example, the pioneering work of Scott et al. demonstrated virtually complete stress and strain recovery and controlled actuation in cross-linked samples containing ultraviolet (UV)-active photoinitiator, owing to the associative nature of the cross-linked network. , Beyond tensile stress, this chemistry can also relax film stress and shrinkage stress originating from curing. , Allyl sulfide AFT chemistry is also demonstrated to be compatible with both additive manufacturing of reconfigurable materials , and hydrogels for cell culture with spatially controlled modulus. , Recently, Millan et al. developed allyl sulfide-based vitrimers without the incorporation of initiators via thermally induced disulfide cleavage, drastically improving the robustness. To establish design rules of these widespread applications, efficient and accurate monitoring of AFT is strongly desired.

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a) Free radical (R•)-initiated allyl sulfide AFT mechanism. b) Depiction of allyl sulfide AFT-based bond exchange along a polymer backbone near a sensing fluorophore, compared to a system without allyl sulfides.

AFT, like most other dynamic bond exchange pathways, has been traditionally characterized macroscopically, usually by monitoring the viscoelastic or stress/strain response ,, in a material as dynamic bonds are activated. However, bulk-scale measurements often convolute contributions from bond rearrangement, chain or segmental motion, and interchain friction. ,, Additionally, AFT occurs on an extremely short time scale, even relative to observed changes in mechanical properties that appear after tens of seconds. Chemically identical “before” and “after” states render typical ensemble spectroscopic methods, such as dielectric, infrared (IR), or nuclear magnetic resonance (NMR), unable to capture the short-lived intermediate. Simulated dynamic bond exchange has been studied in temperature-sensitive vitrimer systems using techniques such as molecular dynamics-Monte Carlo or finite element analysis; however, there has been no simulated investigation of allyl sulfide AFT as far as we know. Electron paramagnetic resonance and time-resolved spectroscopy enabled by pulse radiolysis , have been shown to access short-lived intermediate states. However, the advanced instrumentation needed for these techniques can be prohibitive.

Fluorescence lifetime imaging microscopy (FLIM) is a powerful tool for in-situ monitoring of small-scale fluctuations with imaging capabilities. This technique leverages the sensitivity of fluorescence energy exchange to a fluorophore’s local environment, manifested as changes in the time of the excitation and emission processes, or lifetime. Fluorescence occurs within nanoseconds (ns), providing access to short-lived and small-scale phenomena. FLIM is a ubiquitous characterization tool, with capability for monitoring cellular conditions, , aggregation phenomena, and nondestructive sensing. , In polymeric systems, FLIM has notably been utilized to characterize the glass transition temperature, − self-assembly, fracture mechanics, and molecular weight during polymerization. A fluorophore’s fluorescence lifetime is also sensitive to local heterogeneity, whereas bulk methods such as rheology or differential scanning calorimetry (DSC) are not. Additionally, as FLIM is an imaging technique, there are powerful implications for spatial mapping of samples during characterization within a single instrument. ,, FLIM has also proven useful for the study of chemical reactions and their conditions, such as emulsion and catalytic reactions, but has not yet been applied to monitor AFT.

Small-scale sensitivity in a noninvasive, well-controlled experimental setup makes FLIM an ideal method to characterize dynamic bond exchange. This work applies FLIM to monitor AFT in allyl sulfide-containing polymers (Figure b). Poly­(methyl acrylate) (PMA) was chosen as a model system because of its optical transparency and low glass transition temperature (T g); the melt state enhances chain and radical diffusion. Allyl sulfide moieties are introduced into the PMA backbone via ring-opening copolymerization of methyl acrylate (MA) with the recently developed comonomer 3,7-bis­(methylene)-1,5-dithiacyclooctane (BMDTO). The photoinitiator phenylbis­(2,4,6-trimethylbenzoyl)­phosphine oxide (BAPO) was incorporated as a well-controlled radical source for the AFT. The fluorescence lifetime of nitrobenzoxadiazole (NBD), which is covalently attached to the polymers to prevent undesired aggregation reported in untethered systems, was monitored in solid-state films with positive and negative controls of allyl sulfide and photoinitiator incorporation.

Four sample systems were designed to test for and rule out potential confounding factors, isolating the effects of AFT bond exchange on NBD lifetime. All polymers were synthesized via atom-transfer radical polymerization (ATRP), with either NBD-bromoisobutyrate (NBD-BiB) or ethyl-α-bromoisobutyrate (EBiB) as initiator (generating either labeled or unlabeled chains, respectively) (Figure a). ATRP initiated by NBD-BiB ensures labeling with one fluorophore per chain, located at the chain end. Extensive investigation by Kim, Paeng, and co-workers found no change in the temperature dependence of either free, end-, or midchain-tethered rotational probes. − Additionally, Ellison and Torkelson report end-tethered fluorophores accurately monitoring segmental dynamics. Polymer properties are listed in Table . All four polymers have T g well below the experimental temperature (22.5 ± 1.1 °C). Detailed synthesis and sample preparation procedures, along with characterization and instrument details, are provided in the Supporting Information.

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a) ATRP of PMA (R = CH3, [BMDTO] = 0), NBD-PMA (R = NBD label, [BMDTO] = 0), PMAB (R = CH3, [BMDTO] > 0), and/or NBD-PMAB (R = NBD label, [BMDTO] > 0). b) Light-initiated cleavage of BAPO to generate free radicals. c) Schematic of experimental system design and sample composition.

1. Properties of all Polymers Synthesized. M n and Đ Measured by GPC, T g by DSC, and F BMDTO by 1H NMR.

  PMA PMAB NBD-PMA NBD-PMAB
M n, g/mol 8100 1900 7500 1300
Đ 1.05 1.38 1.13 1.41
T g, °C –0.7 –1.9 –3.3 –13.2
F BMDTO, mol % 0 3.13 0 3.85
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Films were spin-coated onto cleaned and UV-ozone-treated glass slides from a 10 wt % polymer solution in toluene. To avoid fluorophore self-quenching, 10 wt % NBD-labeled polymer was blended with 90 wt % unlabeled (Figure S11). The homopolymer blend (not containing BMDTO) will be referred to herein as “PMA” samples, and the copolymer blend as “PMAB” samples. The photoinitiator BAPO was used to generate AFT-initiating free radicals via cleavage under irradiation with 405 nm wavelength light (Figure b), which is higher energy than the 488 nm laser used for NBD excitation and FLIM data collection; basic fluorescence imaging at 488 nm does not activate BAPO, as confirmed by UV–vis spectroscopy (Figure S10a). The use of a photoinitiator also eliminates temperature changes associated with thermally generated radicals. The choice to use BAPO, therefore, ensures precise temperature-independent control over when and where radicals are generated. The films containing BAPO were cast from 0.001 M BAPO in toluene and will be referred to as “PMA-BAPO” and “PMAB-BAPO” samples. Three films were created for each set of conditions to ensure good repeatability (Figure c), with all thicknesses between 140 and 160 nm as measured by ellipsometry (Table S4). Composition, molar NBD and BMDTO content, and T g for all four systems are listed in Table . All T g values are nearly identical and fall below experimental temperature (22.5 ± 1.1 °C), and BMDTO and NBD contents are comparable across systems. Systems with the radical scavenger 2,2,6,6-tetramethylpiperidinyloxy (TEMPO) were also tested, but the results were inconclusive because TEMPO is known to reduce fluorescence lifetime due to quenching effects and strong absorption at 488 nm, as described in the Supporting Information.

2. Composition and Properties of the Polymer Blend Films .

  Film sample names
  PMA PMAB PMA-BAPO PMAB-BAPO
PMA, wt % 90.12   90.19  
PMAB, wt %   89.58   89.55
NBD-PMA, wt % 9.88   9.81  
NBD-PMAB, wt %   10.42   10.45
BAPO, mol %     8.30 1.87
T g, °C –6.4 –10.5 –4.1 –7.5
F BMDTO, mol % 0 3.2 0 3.2
F NBD, mol % 10.6 14.5 10.5 14.6
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a

BAPO mol % is calculated relative to polymer in the initial toluene solutions. T g is measured by DSC, and F BMDTO and F NBD by 1H NMR.

FLIM measurements were performed using a Nikon Eclipse Ti A1 scanning confocal laser microscope, with films held at constant temperature (22.5 ± 1.1 °C) for the duration of all experiments on a Tokai Hit TP-CHSQ-C temperature-controlled stage. The instrument had an axial resolution of ∼1.4 μm, meaning the entire film thickness was in focus for imaging and lifetime measurement. Nikon’s “Perfect Focus System” ensured there was no drift during experiments. The active field of view was first irradiated with a 405 nm wavelength laser at a dose of 0.8 mJ/cm2 for ∼6 s to generate free radicals via homolytic radical cleavage of BAPO. Following irradiation, FLIM scans were taken with a 488 nm excitation laser (at a dose of ∼0.24 J/cm2 per scan) every 5 min for an hour. The lifetime value for each scan was calculated as the average lifetime for all pixels in the scan (512 × 256 pixels, or ∼5.1 × 10–10 cm2, equaling 110,592 lifetime measurements), and is plotted in Figure for all 12 samples over the full experiment run. Despite these data consisting of lifetime values averaged from several scans over multiple seconds, each individual value provides an instantaneous measure of local density. Therefore, even though each scan is obtained on a slower time scale than a single AFT event, the effect of AFT cascade throughout the polymer system can still be monitored, as our use of FLIM does not rely upon direct reporting of changes in chemical identity or bonding environments.

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Plotted lifetime over accumulated exposure to the FLIM imaging laser (488 nm) for all three PMA (black open circles), PMA-BAPO (purple open circles), PMAB (black closed circles), and PMAB-BAPO (purple closed circles) films. Error bars represent a 95% confidence interval for the average lifetime value of all pixels in that scan.

Both the PMA and PMA-BAPO systems showed no change in lifetime over time, while the lifetime of the PMAB and PMAB-BAPO systems decreased. The very small changes in the absolute values of measured lifetimes imposed a limitation on our use of the FLIM technique, as false-color lifetime maps of the data do not show a visible color change between scans (Figure S12). Across all samples, the PMA and PMA-BAPO systems had almost 1 ns longer lifetimes than the PMAB and PMAB-BAPO systems, likely due to the PMAB systems’ decrease in T g and therefore higher segmental mobility while held at the same temperature. This is consistent with former works reporting that allyl sulfide incorporation reduces T g. , Both PMAB systems showed a slight decrease in lifetime after irradiation, though the PMAB-BAPO samples’ decrease was larger (∼2× steeper slope than that of the PMAB samples).

Figure compares the lifetime vs exposure slope values from Figure for each system. For the control PMA system, no change was seen, confirming the lack of interaction between the irradiation and imaging lasers used and both the homopolymer and the NBD fluorophore. Further, any possible photobleaching caused by repeated imaging does not appear to affect the measured lifetime. Similarly, the PMA-BAPO system showed no significant change in lifetime after irradiation, providing strong evidence for the lack of interaction between the NBD fluorophore and the free radicals generated by BAPO cleavage; introduction of BAPO does not influence the fluorescence lifetime.

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Average slope of the relationship between lifetime and cumulative exposure to the 488 nm FLIM laser for all systems, with results from three individual sample films for each system overlaid as scatter points. Error bars represent a 95% confidence interval for the average slope of all samples in that system. Statistical Analysis of Covariance (ANCOVA) was performed using MATLAB as described in the Supporting Information.

The PMAB system showed a slight decrease in the lifetime over time following irradiation. The temperature was held constant, only slightly above room temperature, and samples were placed on the heated stage to acclimatize before the experiment was initiated; therefore, it is unlikely that any change in temperature could have caused the decrease over time. If the samples were slowly heated over the course of the experiment, a change would be seen in the PMA systems as well. Notably, the PMAB system had the lowest T g value of all four systems (Table ), indicating the highest degree of segmental mobility. However, as previously stated, the T g discrepancy between the PMAB systems and the PMA systems manifests as a difference in the measured lifetime range and not in the rate of lifetime decrease upon irradiation. Interestingly, UV–vis spectroscopy showed higher absorbance in PMAB and PMAB-BAPO films at 488 nm than PMA or PMA-BAPO films (Figure S10b) even after normalizing by molar NBD content. This result suggests a photochemical reaction involving small amounts of C–S bond cleavage and potential further reactions, a promising pathway for initiator-free AFT of allyl sulfides.

The highest rate of change in lifetime after free radical generation occurs in the PMAB-BAPO system. Lack of change in lifetime in the PMA and PMA-BAPO systems rules out potential artifacts arising from instrumentation or quenching by interaction with free radicals. The PMAB system accounted for a slight decrease in lifetime with repeated 488 nm irradiation. However, the PMAB-BAPO system’s slope is still statistically significantly different from the PMAB system, being twice as steep. This system, being the only one containing both dynamic allyl sulfides and a source of free radicals, was hypothesized to be the only one in which dynamic bond exchange could occur. The steeper slope, therefore, likely represents a combination of the 488 nm laser and BAPO; with two sources of AFT initiation, more bond exchange will occur, resulting in a further decrease in lifetime.

The question remains whether the decrease in lifetime is due to direct sensing of an AFT event or to higher mobility resulting from rearrangement following that event. BMDTO content in the PMAB systems is around 3.2 mol %, as calculated from 1H NMR integration and relative polymer wt % in the films (Table S1). From this, we hypothesize that the local environment of only around 6% of fluorophores would contain an allyl sulfide moiety (a single BMDTO ring-opens to form two backbone allyl sulfides); the majority of fluorophores would simply sense changes in chain mobility, and so those changes are the more likely source of the decrease in lifetime. While complementary T g measurement before and after FLIM could further support this observation, such measurements are extremely challenging because only a small area (∼550 μm × 225 μm) is exposed to 488 nm light during the FLIM measurement.

Interestingly, previous investigations of AFT in photoinitiator-containing systems found responses in bulk properties only while the system remained exposed to the light, i.e., only while radicals were being actively generated. ,, During periods without irradiation, mechanical properties such as residual network curing stress and strain recovery essentially plateaued. This would suggest quite the limitation on AFT’s ability to cascade to another allyl sulfide moiety following the initial rearrangement. We have established that a portion of the observed lifetime decrease is due to repeated allyl sulfide cleavage with each 488 nm FLIM scan. However, despite only being irradiated with 405 nm light for ∼6 s, the PMAB-BAPO samples exhibited a decrease in lifetime at twice the rate of the non-BAPO-containing PMAB samples over the span of an hour of FLIM imaging. It is quite likely that, on a scale inaccessible to bulk measurement, long-term AFT cascade and rearrangement occur, resulting in a steeper decrease in fluorophore lifetime, as observed.

In conclusion, we have shown, through a series of control systems, that fluorophore lifetime is sensitive to allyl sulfide AFT exchange. To our knowledge, this is the first application of FLIM to monitor dynamically exchanging covalent bonds. Further investigation is anticipated to both image the diffusion of the AFT cascade through a polymer environment and to confirm whether the decrease in lifetime observed herein is a result of increased mobility after nearby AFT events have occurred. We anticipate application of this method, and other advanced atomic-scale characterization, to dynamic bonding environments beyond model linear polymers, such as those in a cross-linked network.

Supplementary Material

ng6c00066_si_001.pdf (4.7MB, pdf)

Acknowledgments

R.K. thanks NSF CAREER Award DMR #2046606 and Japan Science and Technology Agency (JST) PRESTO Grant Number JPMJPR22N2. M.K.J. thanks the UMass College of Natural Sciences Equity Fellowship. N.T. thanks the National Research Council of Thailand (NRCT): (Contact No. N23G660017). M.K. thanks JSPS Overseas Research Fellowships. The authors respectfully acknowledge the Pocumtuc Nation & Norrwutuck community, on whose unceded homelands this work was performed. The authors deeply appreciate the University of Massachusetts Institute for Applied Life Sciences (IALS) Light Microscopy Facility (Nikon Center of Excellence) and the associated expertise and insight of Dr. James Chambers (IALS), Dimitri Nikolla (IALS), Dr. Jacob Ritz (IALS), Dr. Lylah Deady (Nikon), and Dr. Nicholas Deakin (Nikon). The authors additionally thank Grace Leone and Elizabeth Speck for assistance with the UV–vis spectroscopy measurements, and Dr. Ken Carter, Dr. Autumn Mineo, and Michelle Gross for insightful conversations and advice (UMass Amherst PSE).

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsnanoscienceau.6c00066.

  • Materials; detailed synthetic procedures; NMR, GPC, TGA, DSC, and UV–Vis characterization; sample film preparation and ellipsometry and self-quenching characterization; detailed FLIM procedure and calculations; and MATLAB scripts (PDF)

#.

Faculty of Agro-Industry, Chiang Mai University 50100, Thailand

CRediT: M. K. Jutze conceptualization, data curation, formal analysis, funding acquisition, investigation, methodology, resources, software, validation, visualization, writing - original draft, writing - review and editing; Ellie Haljun resources, writing - review and editing; Mao Komura funding acquisition, resources, writing - review and editing; Nanthicha Thajai formal analysis, funding acquisition, visualization, writing - review and editing; Reika Katsumata conceptualization, funding acquisition, investigation, methodology, project administration, supervision, writing - original draft, writing - review and editing.

The authors declare no competing financial interest.

Published as part of ACS Nanoscience Au special issue “2026 Rising Stars in Nanoscience”.

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