Ellen May Sletten

J-aggregates of cyanine dyes are optimal organic materials for polariton formation, enabling access to concentrated solution-processable thin films, extreme transition dipole strength due to superradiance, and ultranarrow line widths. In the polaritonic literature, J-aggregates are often viewed as large molecules with narrow line widths. However, we emphasize that this is not the case, as intermolecular excitonic coupling produces a unique band structure that reshapes cavity coupling to the J-aggregate exciton. Here, we elucidate the effects of intermolecular dipolar coupling on polariton formation by comparing cavity coupling of a monomeric and J-aggregated cyanine dye at similar concentrations. The cavities display similar apparent Rabi splitting of 168 ± 4 meV (monomer) and 203 ± 1 meV (J-aggregate), yet possess distinctly different spectra, rendering the monomer intermediately coupled and the J-aggregate strongly coupled. Our results suggest that excitonic coupling facilitates polariton formation (even in highly disordered organic thin films), reinforcing J-aggregates as favorable molecular substrates for strong coupling.

Low-cost, safe materials that absorb and emit light at near-infrared (NIR) and shortwave infrared (SWIR) wavelengths are of interest for optical communications, sensors, and diagnostics. One promising avenue to access the NIR/SWIR regions is to self-assemble chromophores into J-aggregates. However, the molecular design of two-dimensional (2D) or tubular J-aggregates with NIR and SWIR emissions is challenging, as small monomeric changes can drastically affect the resulting aggregates' morphologies and photophysical properties. Here, by synthesizing four new heptamethine benzothiazole dyes with increasing steric bulk on the polymethine chain, we rationally design SWIR-emissive 2D J-aggregates. Adding steric bulk drastically changes the self-assembled mesophases and photophysical properties, leading to an unprecedented 3-4 distinct aggregates for each dye. We isolate a total of thirteen new NIR/SWIR aggregates, out of which nine exhibit room-temperature emission. Excitingly, one of the aggregates also shows a tubular morphology─the first SWIR tubular aggregate characterized. A series of interconversion studies, structural characterizations, and modeling provide insights into the molecular packing arrangements of each mesophase. This thorough understanding of the J-aggregate mesophases allowed us to perform four-color imaging using a single chromophore scaffold by selective excitation of the monomeric fluorophore and distinct aggregate structures. Overall, this work demonstrates a rational design strategy for accessing NIR/SWIR emission via supramolecular aggregation and highlights opportunities in energy transport and multiplexed imaging derived from this fundamental understanding of long-wavelength J-aggregates.

The shortwave infrared (SWIR, 1000-2000 nm) region offers significant advantages for in vivo fluorescence imaging, including reduced scattering, minimal autofluorescence, and deep-tissue penetration. However, the development of small-molecule fluorophores with high brightness and maximum emission above 1100 nm remains a key challenge. Here, we report a new class of silicon-containing flavylium (SiliFlav) polymethine dyes that address this limitation. By incorporating a silicon heteroatom into the established Flav scaffold, we achieve unprecedented bathochromic shifts in emission up to 200 nm, while maintaining respectable fluorescence quantum yields (up to 0.30%). A modular synthetic route enables diverse functionalization at the 1-, 2-, and 7-positions of the heterocycle, allowing for systematic tuning of photophysical properties and improved dye performance. The lead fluorophore, SiliFlav5, formulated into canola oil nanoemulsions, enables bright SWIR emission above 1300 nm. In vivo imaging in mice demonstrates high-resolution visualization of the vasculature and spleen, confirming the utility of SiliFlav polymethine dyes as a new platform for deep-tissue SWIR imaging.

The shortwave infrared (SWIR, 1000–2000 nm) region offers significant advantages for in vivo fluorescence imaging, including reduced scattering, minimal autofluorescence, and deep tissue penetration. However, the development of small molecule fluorophores with high brightness and maximum emission above 1100 nm remains a key challenge. Here, we report a new class of silicon-containing flavylium (SiliFlav) polymethine dyes that address this limitation. By incorporating a silicon heteroatom into the established Flav scaffold, we achieve unprecedented bathochromic shifts in emission up to 200 nm, while maintaining respectable fluorescence quantum yields (up to 0.30%). A modular synthetic route enables diverse functionalization at the 1-, 2-, and 7-positions of the heterocycle, allowing for systematic tuning of photophysical properties and improved dye performance. The lead fluorophore, SiliFlav5, formulated into canola oil nanoemulsions, enables bright SWIR emission above 1300 nm. In vivo imaging in mice demonstrates high-resolution visualization of the vasculature and spleen, confirming the utility of SiliFlav polymethine dyes as a new platform for deep-tissue SWIR imaging.

The shortwave infrared (SWIR or NIR-II) region of the electromagnetic spectrum is exceptional for performing fluorescence imaging through skin and tissue. These long, low-energy wavelengths of light provide higher contrast, sensitivity, and imaging depth compared to visible and near infrared light. Though the advantages of SWIR imaging are well established, imaging setups are often custom-built, and there are currently no FDA-approved SWIR fluorophores. It is, however, an exciting time for fluorescence imaging in the clinic. With several new FDA-approved fluorophores in recent years, there is growing interest in advancing the landscape of fluorescence imaging for both diagnostic and therapeutic pursuits. To translate SWIR imaging from fundamental science to clinical applications, progress in both imaging technology and contrast agent design are two crucial, intimately linked factors. This Account details our journey to design biocompatible SWIR-emissive chromenylium- and flavylium-based polymethine fluorophores. Classically, the low band gaps and extended structural conjugation required to achieve SWIR emission compromise the brightness, stability, and aqueous solubility of organic dyes. The driving hypothesis of these studies is that rigorous structural derivatization can illuminate key design principles to overcome these challenges and generate bright, water-soluble, and functional SWIR dyes. Our story begins with Flav7, the first SWIR fluorophore specifically designed for in vivo imaging. We then detail lessons in heterocycle and polymethine linker design principles. From this, 7-, 2-, and C4′-position modifications provided insights for modulating the peak absorption wavelength (λ_max,abs) and fluorescence quantum yield (Φ_F). Since chromenylium and flavylium polymethine dyes maintain high absorption coefficients (ε_max), their total brightness (ε_max × Φ_F) is excellent. Overall, the chromenylium dyes (e.g., Chrom7) proved to be a privileged scaffold for SWIR imaging. To maximize both fluorescence signal and multiplexing abilities, we focused on matching the λ_max,abs of fluorophores to commercial laser lines. This approach has enabled high resolution excitation-based multiplexed imaging with up to five fluorophores in mice in real time, at video frame rates. Building on these design principles, this Account then highlights our strategies to achieve water-soluble and functional SWIR-emissive dyes. We leverage late-stage click chemistry to install hydrophilic moieties via two distinct approaches: 1) small, charged groups or 2) short poly(2-methyl-2-oxazoline) polymer chains. The first strategy resulted in small-molecule dyes SulfoChrom7, AmmonChrom7, and PhosphoChrom7 with diverse functionalities, while the second gave a unique star polymer architecture named “chromenylium star” or “CStar” (CStar30). With optimized bright, functional, and water-soluble dyes in hand, we look toward clinical applications in vascular imaging with SulfoChrom7, lymphatic imaging with CStar30, bone imaging with PhosphoChrom7, and in vivo cell tracking with AmmonChrom7. Finally, we propose that excitation-multiplexed image-guided surgery in the SWIR region can advance existing clinical technologies. Broadly, we anticipate chromenylium and flavylium fluorophores to continue to light up the SWIR region, signaling a new era in optical imaging.

Fluorescence imaging across the near-infrared (NIR, 700–1000 nm) and shortwave infrared (SWIR, 1000–2000 nm) regions offers significant advantages for biomedical applications. Over the past decade, we have advanced a unique class of fluorophores–deemed fluorofluorophores–that are soluble in the fluorous phase. The unique properties of the fluorous phase, including low polarizability and high oxygen content, render this medium challenging for fluorophore brightness and photostability. However, the low dielectric constant of perfluorocarbon solvents causes the counterion to play a significant role in the resulting photophysical properties, offering a nontraditional avenue for fluorophore optimization. Here, we demonstrate that counterion exchange provides a straightforward strategy to enhance the brightness of two fluorous-soluble heptamethine cyanine dyes for NIR and SWIR imaging. Exchanging a chloride counterion with bulkier fluorinated aryl borate counterions boosts the brightness up to 10-fold and photostability up to 60-fold in perfluorooctyl bromide. We showcase the utility of these bright, NIR fluorofluorophores for imaging perfluorocarbon nanoemulsion uptake in macrophage cells, visualizing droplet actuation for mechanobiology studies in zebrafish, and mapping vasculature using high-resolution SWIR detection in mice. Overall, this simple modification provides a practical approach to optimize fluorofluorophores for in vivo imaging without the need to redesign the entire fluorophore scaffold.

The term polymethine dye (PMD) has been intimately connected to the dinuclear scaffold–two heterocycles linked together by a polymethine chain of varying length. Dinuclear PMDs have been a successful scaffold for noninvasive in vivo imaging in the biologically advantageous near infrared (NIR) and shortwave infrared (SWIR) regions of the electromagnetic spectrum. Trinuclear PMDs, resulting from the addition of a third heterocycle into the polymethine chain, possess the same photophysical properties that make dinuclear dyes excellent fluorescent probes, but have yet to be investigated for in vivo imaging. Herein, we expand upon the dinuclear and trinuclear heptamethine dye scaffolds by taking advantage of the increased reactivity of a cyclopentenyl linker and synthesizing flavylium‐ and chromenylium‐based SWIR‐emitting fluorophores. The trinuclear scaffold instills the fluorophores with increased steric bulk, leading to beneficial photophysical properties in micelles and outperforming their classic dinuclear counterparts. In this work, we apply trinuclear PMDs for in vivo SWIR imaging in mice and find them to be particularly efficient at lymph node labeling upon intravenous administration.

The term polymethine dye (PMD) has been intimately connected to the dinuclear scaffold–two heterocycles linked together by a polymethine chain of varying length. Dinuclear PMDs have been a successful scaffold for noninvasive in vivo imaging in the biologically advantageous near infrared (NIR) and shortwave infrared (SWIR) regions of the electromagnetic spectrum. Trinuclear PMDs, resulting from the addition of a third heterocycle into the polymethine chain, possess the same photophysical properties that make dinuclear dyes excellent fluorescent probes, but have yet to be investigated for in vivo imaging. Herein, we expand upon the dinuclear and trinuclear heptamethine dye scaffolds by taking advantage of the increased reactivity of a cyclopentenyl linker and synthesizing flavylium‐ and chromenylium‐based SWIR‐emitting fluorophores. The trinuclear scaffold instills the fluorophores with increased steric bulk, leading to beneficial photophysical properties in micelles and outperforming their classic dinuclear counterparts. In this work, we apply trinuclear PMDs for in vivo SWIR imaging in mice and find them to be particularly efficient at lymph node labeling upon intravenous administration.