Shortwave Infrared Fluorofluorophores for Multicolor In Vivo Imaging**

Abstract

Developing chemical tools to detect and influence biological processes is a cornerstone of chemical biology. Here we combine two tools which rely on orthogonality-perfluorocarbons and multiplexed shortwave infrared (SWIR) fluorescence imaging-to visualize nanoemulsions in real time in living mice. Drawing inspiration from fluorous and SWIR fluorophore development, we prepared two SWIR-emissive, fluoroussoluble chromenylium polymethine dyes. These are the most red-shifted fluorous fluorophores-"fluorofluorophores"-to date. After characterizing the dyes, their utility was demonstrated by tracking perfluorocarbon nanoemulsion biodistribution in vivo. Using an excitation-multiplexed approach to image two variables simultaneously, we gained insight into the importance of size and surfactant identity on biodistribution.

Quantitative multiplexed SWIR imaging to study nanomedicine surface coatings in vivo

“…In recent studies, there have been some studies demonstrating multiplexing in SWIR, but very few have pushed the boundary beyond multiplexing of two distinct contrast agents. 27,28,30,32 Even among the successes, none has used fluorophores exclusively in SWIR and differentiate the signals based solely on spectral differences. 26,29 Therefore, there is the need to push the boundary and demonstrate the potential of multicolor multiplexing with SWIR imaging.…”

“…Past studies have achieved multiplexing with spectral separation of SWIR emitting dyes resulted from different excitation wavelength. [26][27][28] But with limitation from the photophysics of the organic dyes, the imaging window was constrained in the shorter wavelength range without reaching pass the 1400 nm water absorbance feature. Another study engineered lanthanide-doped nanoparticles to have different lifetime profiles for signal separation with the use of an additional time-gated imaging module.…”

Multiplexed Short-wave Infrared Imaging Highlights Anatomical Structures in Mice

“…42 This enables multiplexing with much higher signal-to-noise for straightforward fluorophore differentiation. Nonetheless, current examples of SWIR multiplexing remain limited to dual-color imaging, 28,29,31,33 and none uses multiple fluorophores with discrete SWIR emission peaks to differentiate signals based solely on emission wavelength. 27,30…”

“…Multiplexing with SWIR emitting dyes is achieved using discrete excitation wavelengths to distinguish the fluorescence from different emitters, which is all collected using the same long-pass emission filter. [27][28][29] The emission peaks of the fluorescent dyes and a newly developed fluorescent protein, however, are typically in the NIR-I; SWIR imaging of these emitters relies on collecting photons from the long wavelength tail of the emission peak, precluding spectral separation of the emission peaks and relying on the dimmest portion of the contrast agent emission. Another study engineered lanthanidedoped nanoparticles with distinct luminescence lifetimes for signal separation via time-gated imaging.…”

OME-Zarr: a cloud-optimized bioimaging file format with international community support

“…This format can be used to download entire datasets for local processing or for streaming to applications for interactive viewing. Data shown includes (A) idr0076 (Ali et al 2020), (B) idr0101 (Payne et al 2021), (C) idr0077 (Valuchova et al 2020), (D) S-BIAD548 (Lim et al 2023), (E) S-BIAD217 (P. de Boer et al 2020), and (F) S-BIAD501 (Igarashi et al 2015).…”