Observing the in vivo behavior of nanoparticles (NPs) remains a challenge, with limited NPs macroscopically “visible” in biological tissues. Fluorescence microscopy allows observation at the cellular level, and broader visibility within living tissues necessitates innovative approaches. Near-infrared (NIR) light, specifically wavelengths beyond 1000 nm, has emerged as a powerful tool because optical loss is minimal, which facilitates deep-tissue observation. NIR fluorescent NPs open avenues beyond behavioral insights, allowing the exploration of deep-tissue structures, conditions, and phenomena. Well-designed NIR fluorescent NPs can also be used to image temperature changes in deep biological tissues. Moreover, tomographic imaging is achievable through the back-projection of fluorescence images acquired from multiple angles. However, because NIR light does not have a linear propagation similar to that of X-rays, the effects of light refraction on NIR fluorescence tomography have been examined quantitatively, and the refractive index gap on object surfaces should be <2%. The organic molecular dye IR-1061 exhibited intriguing behavior after encapsulation in polymeric micellar NPs, particularly under heat treatment. Aggregation or dimer formation impedes the full exhibition of NIR fluorescence when an affinity mismatch occurs with surrounding molecules, such as the hydrophobic chains of polymers, allowing the assessment of the in vivo stability of polymeric micellar NPs for drug delivery. The findings and techniques reviewed here shed light on the challenges associated with NIR fluorescence tomography, while providing valuable insights for overcoming these challenges. These advancements promise a better quality of life through the tailored design of NPs for biomedical applications and the visualization of environmental NP behavior.

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