In this work, we developed a methodology of preparation of ultrabright fluorescent polymeric nanorods by ultrasonication of electrospun polymeric nanofibers loaded with dye/bulky counterion ion pairs. Their studies in cultures of five cell lines revealed several outstanding features of these high aspect ratio nanomaterials, such as stronger capacity to internalize inside the cells compared to NPs and some cell selectivity. Further studies confirmed that the nanofibers internalize by endocytosis and remained trapped in hypertrophic lysosomes. More importantly, quantitative internalization analysis by two independent methods revealed these polymeric nanorods exhibited nearly 100-fold stronger accumulation inside the cells compared to nanoparticles made of similar polymer. In U87 cells they were able to accumulate up to 3% mass of the cells, which is an outstanding amount of polymeric material inside the cells. Despite this exceptional accumulation nanorods showed no signs of cytotoxicity, verified by three independent methods. Finally, taking advantage of this strong accumulation, we applied polymeric nanofibers loaded with near-infrared dyes for single-cell detection under chicken skin at distance of ~2 mm. Synthesis of fluorescent nanorods is based on dye-loaded polymeric nanofibers prepared by electrospinning method as we reported previously. The nanofiber mats, loaded with 5 and 23 wt% (30 wt% ratio vs polymer mass) of R18/F5-TPB, were suspended in milliQ water and then sonicated with a probe sonicator (Figure ). We observed that the initially heterogeneous suspension became homogeneous due to breaking of the long nanofibers into shorter nano-objects. Transmission electron microscopy (TEM) showed presence of short fibers with the thickness around ~0.4 µm and average length of 2-3 µm (Figure , Table ). The former is close to the diameter of nanofibers in the fiber mats, reported by us before. Thus, the sonication leads to breaking of the long fibers to shorter ones (nanorods). Even though there is some heterogeneity in the lengths of the fibers, we can notice that the fibers length for vast majority of nanorods was  1 µm. The latter implies that sonication can break nanofibers to rods but the process stops when the nanorod length reaches critical small size, below which the energy of sonication is not sufficient to break them. Nevertheless, there was no clear correlation between diameter and the length of individual nanorods, which was probably because the variation in the diameter was relatively small. Then, we studied optical properties by fluorescence spectroscopy. For both medium and high dye loading, namely 5 and 23 wt%, the emission spectra were composed of a single emission band, similar to that for R18/F5-TPB dye loaded at 5 wt% into PMMA-MA NPs (Figures and). The excitation spectra for 5 wt% loaded nanorods were nearly the same as for corresponding PMMA-MA NPs (Figures and). High absolute quantum yields were observed both types of nanorods: 40 and 25%, respectively. In contrast, R18 with small inorganic perchlorate counterion showed poor emission and low fluorescence quantum yield (3%) already at 5 wt% dye loading, suggesting strong ACQ already at medium loading. These data are in line with our previous reports on nanofiber mats and polymeric NPs, where bulky counterion was found essential to prevent self-quenching of cationic rhodamine dye R18 by playing a role of spacer preventing formation of dye H-aggregates. Then, the nanorods were deposited on glass surface simply by sedimentation and studied by wide-field fluorescence microscopy. Nanorods were observed as fluorescence nanostructures with high aspect ratio (Figure ). Their diameter could not be analyzed by this method because according to TEM it was close to the resolution of the optical microscopy (Table ). However, the fact that the brightness of nanofibers was rather homogenous, we can conclude that the diameter of the obtained fibers should be also homogenous, in line with TEM results. Moreover, we observed quite good homogeneity of the length of the nanofibers for both dye loadings (Figures and and Table ), with average length close to that obtained by TEM. The better homogeneity of length compared to TEM is probably related to the fact that sample preparation for fluorescence microscopy is much simpler, avoiding sample drying that lead to aggregation and inhomogeneous deposition on the surface. Therefore, we expect that the obtained distribution of lengths by fluorescence method reflects better the real state of the samples. We analyzed brightness of these nanorods in comparison with 60-nm PMMA-NPs. At the same experimental conditions, the signal detected from the center of nanorods was ~200-fold higher compared to NPs (Figure ). This difference is clearly related to the much larger diameter of the nanorods, so that much larger number of dyes contribute to the signal for those larger nanostructures. Then, we studied the interaction of nanorods with cells in comparison to fluorescent polymeric NPs made of similar polymer and loaded with the same dye R18/F5-TPB and loading degree (5 wt%). In case of NPs incubated with KB and U87 cells, we observed a perinuclear accumulation of the signal in the red channel (Figure ), which according to our previous studies with this type of NPs corresponds to lysosomes. In sharp contrast, nanorods showed practically no internalization inside KB cells (Figure ). A large fraction of nanorods could be found on the cell surface, as evidenced by colocalization with plasma membrane marker WGA-AF488. In case of U87 cells, the behavior was completely different as the cells presented strong intracellular fluorescence, where nanorods could be clearly identified by their morphology (Figure ). However, there were practically no nanorods detectable on the cell surface. Due to sharp discrepancy in the behavior of nanorods in these two cells lines, we studied three additional cell lines: LNZ-308, EA.hy926 (EAHY) and HEK293T. In all three cell lines a strong signal of nanorods inside the cells was systematically observed (Figure ), which indicates that nanorods tends to accumulate well inside the cells, while the behavior in KB cells is an exception. Thus, we can make two conclusions from this experiment. First, like NPs, nanorods internalized inside the cells. However, the inability of nanorods to enter KB cells remains a remarkable observation, which differ from NPs, indicating that nanorods could have a unique potential to distinguish some cell lines. The second conclusion is that the signal inside U87 was much higher compared to NPs, which suggests that for the same mass concentration outside the cells, the mass of nanorods (and the amount of encapsulated dyes) internalized inside the cells was much higher compared to that of NPs. Therefore, we explored further these two remarkable features of nanorods. To understand better the internalization of nanorods inside the cells, we studied the effect of low temperature (+4 °C), which is expected to block endocytosis. Moreover, we compared standard growth medium with physiological buffer HBSS. After 6h incubation in the standard growth medium, we could clearly observe internalization of nanorods inside U87 cells, whereas at +4 C no sign of internalization was observed (Figure ). This result suggests that nanorods enter the cells by active process (endocytosis) rather than by direct crossing of the plasma membrane. Remarkably, in HBSS medium, nanorods failed to enter cells: the majority of them remained on the glass surface, while some cells showed exclusively surface-bound nanorods (Figure ). We can speculate that growth medium stimulates internalization probably due to adsorption of serum proteins on the surface of PMMA nanorods and the presence of growth factors. Previous report showed that unmodified nanorods may internalize differently because of protein corona. Remarkably, when the nanorods were pretreated with Pluronic F127, the internalization inside U87 cells in complete growth medium was also inhibited (Figure ). This implied that Pluronic F127 converts hydrophobic PMMA surface into hydrophilic "stealth" surface that prevents interaction with serum proteins with nanorods, thus inhibiting their internalization.