The Pursuit of Shortwave Infrared-Emitting Nanoparticles with Bright Fluorescence through Molecular Design and Excited-State Engineering of Molecular Aggregates

Shortwave infrared (SWIR) fluorescence detection gradually becomes a pivotal real-time imaging modality, allowing one to elucidate biological complexity in deep tissues with subcellular resolution. The key challenge for the further growth of this imaging modality is the design of new brighter biocompatible fluorescent probes. This review summarizes the recent progress in the development of organic-based nanomaterials with an emphasis on new strategies that extend the fluorescence wavelength from the near-infrared to the SWIR spectral range and amplify the fluorescence brightness. We first introduce the most representative molecular design strategies to obtain near-infrared-SWIR wavelength fluorescence emission from small organic molecules. We then discuss how the formation of nanoparticles based on small organic molecules contributes to the improvement of fluorescence brightness and the shift of fluorescence to SWIR, with a special emphasis on the excited-state engineering of molecular probes in an aggregate state and spatial packing of the molecules in nanoparticles. We build our discussion based on a historical perspective on the photophysics of molecular aggregates. We extend this discussion to nanoparticles made of conjugated polymers and discuss how fluorescence characteristics could be improved by molecular design and chain conformation of the polymer molecules in nanoparticles. We conclude the article with future directions necessary to expand this imaging modality to wider bioimaging applications including single-particle deep tissue imaging. Issues related to the characterization of SWIR fluorophores, including fluorescence quantum yield unification, are also mentioned.


Definition of J-aggregates
Despite years of study and a number of scientific reports devoted to mechanisms of self-assembly and structure-related photophysics of J-aggregates, many recent papers that claimed the formation of J-aggregates in fluorescent nanoparticles used observed red shift of the fluorescence upon aggregation as an evidence of J-aggregates formation. According to the theory, the formation of J-aggregates is characterized by increased radiative rate (superradiant emission) that can results in enhanced quantum yield and reduced fluorescence lifetime and an appearance of a new red shifted narrow 'J-band'. The red shift of fluorescence emission is not the evidence for J-aggregates formation, which could be attributed to an increase in charge transfer (CT) character due to a geometry change, reduction in the band gap between the ground and excited states due to the enhanced van der Waals interactions, hydrogen bonding, and so on. In fact, in some cases, the red shift of the fluorescence maximum can be observed in aggregates with the H-type arrangement 1 . Moreover, the twisted charge-transfer π-conjugated systems can show blue-shifted emission upon J-aggregation 2 . Therefore, assigning red shifted fluorescence spectra as J-aggregates without analyzing absorption spectral narrowing and change in the radiative rate constant is misleading.

Definition of aggregation-induced emission
Most of new SWIR dye designs are based on the insertion of propeller shaped free-rotating units (e.g. TPA, TPE) into the molecule framework. This is inspired by the fascinating feature of aggregation-induced emission (AIE)-dyes where free-rotating units cause fluorescence quenching of monomeric dyes in an organic solvent through the opening of alternative/dominant nonradiative channels that depopulate excited state. By hindering the motions of free-rotating units in the aggregate state (RIR mechanisms), the appearance or enhancement of fluorescence emission is observed (AIE, AIEE effects).
We found that some recent articles reported new SWIR-emitting AIEgens based on the introduction of rotating units into fluorescent molecules, where such a new fluorophore possesses decent fluorescence in an organic solvent that is quenched upon aggregation in a water environment (e.g. Figure 12c). The observed quenched fluorescence in aggregated forms were called AIE because of the 1) introduction of well-known AIEgen generating groups and 2) observation of partial recovery of fluorescence at different organic-to-solvent ratios could be attributed to the suppression of TICT. Since the term AIE has a connotation of fluorescence enhancement, describing the partial recovery of the fluorescence intensity in the aggregate states as AIE is a misuse for the term even the partial recovery of the fluorescence intensity could be interpreted by the frozen motion of the TPA/TPE units and the inhibition of π-π stacking due to these bulky groups. The partial recovery of the fluorescence upon aggregation formation should be described as anti-quenching effect.

Reevaluation of fluorescence quantum yield standards in SWIR spectral region
Here, we point out an urgent need for a critical reevaluation of Φfl values of NIR/SWIR-emissive nanomaterials. The most common method to determine Φfl is to compare their fluorescence intensity with a standard material that has a known Φfl. The first choice of fluoresce standard in the SWIR spectral range is IR-26 in dichloroethane (DCE). IR-26 is a relatively stable dye with absorption and fluorescence in a desirable spectral range, yet it has a narrow absorption spectrum with a very low Φfl, which led to a poorly characterized Φfl. The most commonly referred values (Φfl = 0.5 %) somehow appeared during the development of new NIR/SWIR emitters 3,4 . These articles refer to the Φfl value of IR-26 determined by both lifetime measurements and absolute measurement, citing original work from early 1980s that reported the Φfl value of three IR dyes (no. 5, no. 15, no. 9860). In addition, these articles pointed that Φfl of IR-26 is similar to that of IR dye no. 5. However, the Φfl value of IR dye no. 5 reported in the original work is Φfl = 0.05% in DCE) 5 . In another source 6 , Φfl of IR-26 (Φfl = τF/τrad) was calculated based on previously reported fluorescence lifetime (τF) and radiative lifetime (τrad) derived from the S0-S1 absorption cross-section integral and the relative fluorescence quantum distribution.
In 2010, Φfl of IR-26 was determined using integrating sphere that provided most reliable value Φfl = 0.048% ± 0.002 for IR-26 in DCE directly 7 , suggesting that reported Φfl values of many SWIR materials were overestimated by a factor of ca. ̴ 10 if the old value (Φfl = 0.5%) has been used. Lately, a large number of researchers started to use IR1061 in DCE as an alternate standard. The authors referred to Φfl (Φfl = 1.7%) determined by a direct comparison of the emission intensity of the IR1061 (SWIR) dye in dichloromethane relative to the fluorescence intensity of the rhodamine B (visible) dye in ethanol 8 . Recently, Φfl of IR-1061 in DCE was redetermined with an integrating sphere instrument, giving the value Φfl = 0.32%, which renders all Φfl measured relative to this dye overestimated by a factor of ca. ̴ 5.3 times 9 .
Fluorescence quantum yields of IR-26 and IR-1061 determined using integrated sphere