Quantitative analysis of transferrin uptake into living cells at single-molecule level

• Published in: Talanta (Oxford) 2025-01, Vol.282, p.127031, Article 127031
• Main Authors: Pilz, Marta, Kalwarczyk, Tomasz, Burdzy, Krzysztof, Hołyst, Robert
• Format: Article
• Language: English
• Subjects: Endocytosis; Fluorescence correlation spectroscopy; HeLa Cells; Humans; Quantification of endocytosis; Quantitative imaging; Single Molecule Imaging - methods; Single-cell analysis; Transferrin - metabolism
• ISSN: 0039-9140; 1873-3573; 1873-3573
• DOI: 10.1016/j.talanta.2024.127031

Endocytosis, apart from providing a mechanism for cells to take up nutrients, is the principal route of entry for many nanomedicines. The evaluation of therapeutic delivery efficiency without providing quantitative parameters, like the number of molecules entering the cell and the time of their entry, is very limited. Despite advances in single-molecule methods for in-cell quantitative measurements, they have not become widely used in the study of endocytosis. Their application, however, is challenging owing to the appropriate experimental design and applicable analysis methods. Here, by using an integrated approach based on the multidimensional time-correlated single-photon counting (TCSPC) technique, we demonstrate the quantitative method to measure the time- and concentration-dependent uptake of TRITC-transferrin into living cells with single-molecule sensitivity. The methodology opens possibilities for quantitatively assessing the delivery efficiency of fluorescent molecules into living cells. [Display omitted] •Quantitative analysis of fluorescence images with single-molecule sensitivity.•An efficient and easy tool for studying the endocytosis of any fluorescent molecules.•An analytical formula to describe endocytosis timescales.