High resolution, multiplexed experiments are a staple in cellular imaging. Analogous experiments in animals are challenging, however, due to significant scattering and autofluorescence in tissue at visible (VIS, 350–700 nm) and near-infrared (NIR, 700–1000 nm) wavelengths. Here, we enable real-time, non-invasive multicolor imaging experiments in animals through the design of optical contrast agents for the shortwave infrared (SWIR, 1000–2000 nm) region and complementary advances in imaging technologies. We developed tunable, SWIR-emissive flavylium polymethine dyes and established structure-photophysical property relationships for this class of bright SWIR contrast agents. In parallel, we designed an imaging system with variable NIR/SWIR excitation and single-channel detection, facilitating video-rate multicolor SWIR imaging for optically guided surgery and imaging of awake and moving mice with multiplexed detection. Optimized dyes matched to 980 nm and 1064 nm lasers, combined with the clinically approved indocyanine green, enabled real-time, three-color imaging with high temporal and spatial resolutions.

Tissue is translucent to shortwave infrared (SWIR) light, rendering optical imaging superior in this region. However, the widespread use of optical SWIR imaging has been limited, in part, by the lack of bright, biocompatible contrast agents that absorb and emit light above 1000 nm. J-Aggregation offers a means to transform stable, near-infrared (NIR) fluorophores into red-shifted SWIR contrast agents. Here we demonstrate that J-aggregates of NIR fluorophore IR-140 can be prepared inside hollow mesoporous silica nanoparticles (HMSNs) to result in nanomaterials that absorb and emit SWIR light. The J-aggregates inside PEGylated HMSNs are stable for multiple weeks in buffer and enable high resolution imaging in vivo with 980 nm excitation. Optical imaging with shortwave infrared (SWIR, 1000-2000 nm) light has emerged as a powerful method of fluorescence imaging in animals due to the superior resolution and contrast one can achieve with low energy light (Figure 1A). 1 A primary challenge with SWIR imaging is the development of bright, biocompatible, SWIR contrast agents. 2 Originally, the advantageous qualities of imaging in the SWIR region were showcased with carbon nanotubes, 3 quantum dots, 4 and rare earth nanomaterials. 5 In efforts to set the stage for clinical translation, the past three years have seen a focus on the synthesis of nontoxic, SWIR-emissive organic fluorophores. 6 This work has significantly expanded the suite of fluorophores that emit above 1000 nm; however, challenges remain in the stability, delivery, *

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Tissue is translucent to shortwave infrared (SWIR) light, rendering optical imaging superior in this region. However, the widespread use of optical SWIR imaging has been limited, in part, by the lack of bright, biocompatible contrast agents that absorb and emit light above 1000 nm. J-aggregation offers a means to transform stable, near-infrared (NIR) fluorophores into red-shifted SWIR contrast agents. Here we demonstrate that hollow mesoporous silica nanoparticles (HMSNs) can template the J-aggregation of NIR fluorophore IR-140 to result in nanomaterials that absorb and emit SWIR light. The J-aggregates inside PEGylated HMSNs are stable for multiple weeks in buffer and enable high resolution imaging <i>in vivo</i>with 980 nm excitation.

Tissue is translucent to shortwave infrared (SWIR) light, rendering optical imaging superior in this region. However, the widespread use of optical SWIR imaging has been limited, in part, by the lack of bright, biocompatible contrast agents that absorb and emit light above 1000 nm. J-aggregation offers a means to transform stable, near-infrared (NIR) fluorophores into red-shifted SWIR contrast agents. Here we demonstrate that hollow mesoporous silica nanoparticles (HMSNs) can template the J-aggregation of NIR fluorophore IR-140 to result in nanomaterials that absorb and emit SWIR light. The J-aggregates inside PEGylated HMSNs are stable for multiple weeks in buffer and enable high resolution imaging <i>in vivo</i>with 980 nm excitation.

The recent developments of fluorophores having photoemission beyond 1000nm provide the opportunity to develop novel fluorescent microscopes sensitive to those wavelengths. The imaging at wavelengths beyond the visible spectrum allows to achieve imaging depths of hundreds of microns in intact tissue, making this attractive for volumetric imaging applications. Here, a novel shortwave-infrared line-scan confocal microscope is presented that is capable of deep imaging of biological specimens, as demonstrated by visualization of labelled glomeruli in a fixed uncleared kidney at depths of 300 um to 400 um. This development improves the acquisition speed of deep tissue fluorescence imaging.