Then, the U87 and EAHY cells were incubated with nanorods for 6h and 24h and further co-stained with LysoTracker green and a membrane probe MemBright-640. First, we noticed that nanorods attached to glass surface were labelled with MemBright-640 (Figure ), indicating that this amphiphilic probe directly interacts with nanorods, probably because of its hydrophobic interface. As MemBright-640 cannot really cross plasma membrane, it could be used to distinguish the nanorods on the cells surface (positive staining with MemBright-640) from those inside the cells (not stained with MemBright-640). We found that at 6h incubation a significant amount of the nanorods were still co-localized with MemBright-640, indicating that a significant fraction of nanorods were bound to the cells surface (Figure ). However, after 24h, significant fraction of nanorods were observed without co-staining with MemBright-640 (Figure ), which implies that they entered the cells after this sufficiently long incubation time. Surprisingly, emission of nanorods was not co-localized at all with that of LysoTracker green (Figure ). To understand better this counter-intuitive result, we incubated all 4 cells lines capable to internalize nanorods for 24h, and then, after their fixation and permeabilization, immunostaining of lysosomes was done by LAMP-3, while actin filaments were co-stained with phalloidine-AF488. Remarkably, we could observe colocalization of nanorods with LAMP-3 for all four cell lines (Figure ). Although we cannot exclude a slight cross-talk between the two channels, the merged images clearly show that nanorods outside the cells appear in red, while those inside the cells are systematically yellow due to LAMP-3 staining (Figure ). These results suggest that nanorods are trapped inside lysosomes. Then, the failure of LysoTracker green to stain lysosomes entrapping nanorods could probably originate from the incapacity of these hypertrophic compartments to generate sufficiently low pH, required for successful staining by LysoTracker green. As we observed a stronger accumulation of nanorods inside the cells compared to NPs, we wanted to make an absolute quantification of the internalized nanomaterial inside the cells. For this purpose, U87 cells were incubated for 24h with nanorods or NPs at the same mass concentration and then detached and fixed in suspension. One should note that the detachment of the cells by trypsinization also led to detachment of the non-internalized nanorods form the cell surface (data not shown). A set of meshes of 100 µm and 1 µm were used to separate cells from non-internalized nanorods or NPs. The cell concentration was determined by cell counting under microscope, where we could also clearly observe much stronger signal from cells incubated with nanorods compared to NPs (Figures ), in line with the initial microscopy observations (Figure ). Then, the amount of fluorescent nanomaterials inside the cells was determined by absorbance measurement in organic solvent (1,4-dioxane) that solubilized the fluorescent dye. The measured absorbance value of 1 ml solution was 0.144 for nanorods per million cells counted, whereas for NPs this value was ~100-fold lower, namely 0.0014 (Figure ). These values correspond to 1.2 fmol and 11 amol of dyes per cell for nanorods and NPs, respectively. Considering the molar mass of the fluorophore and the dye loading used is a 5 wt%, it corresponds to 1.65 pg of dye and 33 pg of nanofibers per cell. Considering that the mass of a mammalian cell is around 1-2 ng, the internalized nanorods presented 1.5-3 % of the mass of the cells. Thus, nanorods display impressively high capacity to internalize the cells, which is 100-fold higher than that of NPs. Next, we made a quantitative analysis of the capacity of the five cells lines to internalize nanorods using flow cytometry in comparison to NPs. Four studied cell lines (U87, EAHY, LNZ308 and HEK293T) showed strong signal from the cells, where the signal from KB cells was relatively low (Figure ), in line with our observations by fluorescence microscopy (see above). NPs incubated at the same conditions displayed nearly two order of magnitude lower signal (Figure ), in line with our absolute quantitative analysis for U87 cells using absorption spectroscopy. Moreover, for all cells lines except KB, the signal from cells was higher compared to that of individual nanorods, suggested that multiple rods were present per cell in each line. As the signal from KB cells was between the control and individual nanofibers, we can conclude that nanorods were unable to enter KB cells, and the detected signal could be assigned to a fraction of small fragments of nanorods, which was probably present in the sample. Thus, multiple independent measurements, namely qualitative fluorescence microscopy, quantitative optical spectroscopy and flow cytometry show dramatic difference in the internalization of nanorods compared to NPs for the same mass concentration of the nanomaterials during incubation. This highlights the unique property of nano-objects with high aspect ratio to accumulate inside the cells. We also cannot exclude that larger size/mass of nanorods compared to NPs also contributed to their higher internalization. This outstanding accumulation of nanorods required verification of their cytotoxicity. Therefore, MTT assay was performed, which estimates cytotoxicity through mitochondrial activity of cells. Remarkably, incubation of cells with nanorods for 24h resulted in no detectable cytotoxicity in comparison to control cells for all five cell lines tested (Figure ). Nanorods were not cytotoxic even at 500 µg/mL concentration, which corresponds to 50 times the concentration we used for microscopy experiments. In addition, we analyzed fluorescein diacetate labeling of U87 cells using flow cytometry, which is an alternative method to estimate cell viability, using intracellular esterase activity. Importantly, nanorods and NPs produced no change in the fluorescein signal compared to control cells (Figures 8B), indicating no sign of cytotoxicity of these two nanomaterials. Fluorescence microscopy imaging of multiple cells lines confirmed no effect of internalized nanorods on the intracellular signal of enzymatically cleaved fluorescein diacetate (Figure ). Moreover, the cells with internalized nanorods detached by standard trypsin treatment were able to adhere on a new surface and continue to grow and divide at same rate as the non-treated control cells (Figure ), so their key main biological functions were maintained. In addition, in these experiments, when cells were stained by trypan blue for automated counting, the rates of live cells were in the typical range of non-treated cells (eg. between 85 to 100 % for U87 cell line, Figure ), confirming the negligible toxicity and the maintenance of ATP related mechanism. Overall, nanorods despite their exceptional accumulation inside the cells are not cytotoxic, which could be explained by the fact that they are entrapped inside endosomes and thus do not interfere with biomolecular processes inside the cytosol. Importantly, after 3 and even 6 days of incubation, the strong signal from nanorods was still detected inside the cells (Figure ), suggesting that the nanorods remain inside the cells for prolonged time, allowing long-term imaging. This strong accumulation of nanorods could be used to label the cells with high quantity of a contrast agent in order to identify the cells in a complex media. Here, we explored a possibility to detect cells labelled with nanorods under model tissue -chicken skin. To ensure deeper tissue penetration, we prepared nanorods loaded with near-infrared dyes hydrophobic derivatives of Cy5.5 and Cy7.5 with bulky counterion F5-TPB. The obtained nanorods showed similar structural characteristics as their rhodamine analogues (Figure , Table ). Fluorescence spectra displayed emission bands typical for Cy5.5 and Cy7.5 dyes, with maxima centered at 720 nm and 835 nm, respectively (Figure ). Then, we incubated nanorods of two different colors independently in two populations of U87 cells for 24h. After trypsinization, these two cultures were mixed together and grown a glass coverslip for another 24h. Under fluorescent microscope, using two nearinfrared channels for Cy5.5 and Cy7.5 dyes, the obtained co-culture appeared as two cell populations with strong signal in the green (Cy5.5 channel) and red (Cy7.5) channels (Figure ). The individual green-and redcolored cells could be clearly identified in a merged image together with a bright field channel. Then, the coverslip was covered by a freshly cut piece of chicken thigh skin, with a thickness of ~2 mm according to literature, and imaged using the same fluorescence microscopy settings. Remarkably, the individual green and red signals could be identified directly under the chicken skin, suggesting that we could detect individual cells (Figures and). The yellow signal, which was generally of higher intensity, could be assigned to cell aggregates combining cells of two populations of different emission color. Thus, due to capacity of nanorods to internalize into cells at high quantities, individual cells could produce strong emission signal in the two near-infrared regions. This enabled us to detect single cells in two colors under skin through ~2 mm, using basic epi-fluorescence setup with 10x objective.