Carbon dots (C-Dots) and ZnS-doped carbon dots (CZnS-Dots) were investigated for in vivo optical imaging. These nanomaterials are highly fluorescent, non-toxic, biocompatible, and stable, making them promising alternatives to traditional quantum dots (QDs) for in vivo imaging. C-Dots, which are small carbon nanoparticles surface-passivated with organic or biomolecules, exhibit strong fluorescence and are non-blinking. CZnS-Dots, which have an inorganic salt (ZnS) doped into the core, show enhanced fluorescence brightness. Both types of dots were successfully used for in vitro imaging with one- and two-photon excitation. In vivo studies showed that C-Dots and CZnS-Dots were well-behaved as contrast agents in live mice. Subcutaneous injection of C-Dots or CZnS-Dots resulted in bright fluorescence, with CZnS-Dots showing stronger emissions. The dots diffused slowly, with fluorescence fading about 24 hours post-injection. Excitation at longer wavelengths enabled red fluorescence, and both C-Dots and CZnS-Dots exhibited significant fluorescence under these conditions. Interdermal injection of C-Dots allowed tracking of their migration through lymph vessels. Unlike semiconductor QDs, which migrate to axillary lymph nodes quickly, C-Dots migrated more slowly, possibly due to their small size and surface functionalization with PEG. Axillary lymph nodes harvested 24 hours post-injection showed detectable fluorescence from the dots. Intravenous injection of C-Dots led to urine excretion, consistent with the known excretion pathway for PEGylated nanoparticles. Organs harvested 4 hours post-injection showed fluorescence in the kidneys and liver, with the kidneys showing brighter fluorescence. The relatively weak fluorescence in the liver suggested low accumulation of the dots. All experiments were conducted following IACUC-approved protocols, with no signs of acute toxicity observed in the mice. The results demonstrate that C-Dots, when injected into mice, remain strongly fluorescent in vivo, with biocompatibility and non-toxicity, offering great potential for optical imaging and related biomedical applications.Carbon dots (C-Dots) and ZnS-doped carbon dots (CZnS-Dots) were investigated for in vivo optical imaging. These nanomaterials are highly fluorescent, non-toxic, biocompatible, and stable, making them promising alternatives to traditional quantum dots (QDs) for in vivo imaging. C-Dots, which are small carbon nanoparticles surface-passivated with organic or biomolecules, exhibit strong fluorescence and are non-blinking. CZnS-Dots, which have an inorganic salt (ZnS) doped into the core, show enhanced fluorescence brightness. Both types of dots were successfully used for in vitro imaging with one- and two-photon excitation. In vivo studies showed that C-Dots and CZnS-Dots were well-behaved as contrast agents in live mice. Subcutaneous injection of C-Dots or CZnS-Dots resulted in bright fluorescence, with CZnS-Dots showing stronger emissions. The dots diffused slowly, with fluorescence fading about 24 hours post-injection. Excitation at longer wavelengths enabled red fluorescence, and both C-Dots and CZnS-Dots exhibited significant fluorescence under these conditions. Interdermal injection of C-Dots allowed tracking of their migration through lymph vessels. Unlike semiconductor QDs, which migrate to axillary lymph nodes quickly, C-Dots migrated more slowly, possibly due to their small size and surface functionalization with PEG. Axillary lymph nodes harvested 24 hours post-injection showed detectable fluorescence from the dots. Intravenous injection of C-Dots led to urine excretion, consistent with the known excretion pathway for PEGylated nanoparticles. Organs harvested 4 hours post-injection showed fluorescence in the kidneys and liver, with the kidneys showing brighter fluorescence. The relatively weak fluorescence in the liver suggested low accumulation of the dots. All experiments were conducted following IACUC-approved protocols, with no signs of acute toxicity observed in the mice. The results demonstrate that C-Dots, when injected into mice, remain strongly fluorescent in vivo, with biocompatibility and non-toxicity, offering great potential for optical imaging and related biomedical applications.