Theranostics 2015; 5(9):919-930. doi:10.7150/thno.11863

Research Paper

Single Cell Assay for Molecular Diagnostics and Medicine: Monitoring Intracellular Concentrations of Macromolecules by Two-photon Fluorescence Lifetime Imaging

Artem Pliss1, Xiao Peng2, Lixin Liu1,3, Andrey Kuzmin1, Yan Wang1, Junle Qu2✉, Yuee Li1,4, Paras N Prasad1,5✉

1. Institute for Lasers, Photonics and Biophotonics and the Department of Chemistry, University at Buffalo, the State University of New York, Buffalo, NY 14260, USA
2. College of Optoelectronic Engineering, Shenzhen University, Key Laboratory of Optoelectronic Devices and Systems of Ministry of Education and Guangdong Province, Shenzhen, Guangdong, China 518060
3. School of Physics and Optoelectronic Engineering, Xidian University, Xi'an, Shaanxi, China 710071
4. School of information science and engineering, Lanzhou University, Lanzhou, Gansu, China, 730000
5. Department of Chemistry, Korea University, Seoul 136-701, Korea

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Pliss A, Peng X, Liu L, Kuzmin A, Wang Y, Qu J, Li Y, Prasad PN. Single Cell Assay for Molecular Diagnostics and Medicine: Monitoring Intracellular Concentrations of Macromolecules by Two-photon Fluorescence Lifetime Imaging. Theranostics 2015; 5(9):919-930. doi:10.7150/thno.11863. Available from

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Molecular organization of a cell is dynamically transformed along the course of cellular physiological processes, pathologic developments or derived from interactions with drugs. The capability to measure and monitor concentrations of macromolecules in a single cell would greatly enhance studies of cellular processes in heterogeneous populations. In this communication, we introduce and experimentally validate a bio-analytical single-cell assay, wherein the overall concentration of macromolecules is estimated in specific subcellular domains, such as structure-function compartments of the cell nucleus as well as in nucleoplasm. We describe quantitative mapping of local biomolecular concentrations, either intrinsic relating to the functional and physiological state of a cell, or altered by a therapeutic drug action, using two-photon excited fluorescence lifetime imaging (FLIM). The proposed assay utilizes a correlation between the fluorescence lifetime of fluorophore and the refractive index of its microenvironment varying due to changes in the concentrations of macromolecules, mainly proteins. Two-photon excitation in Near-Infra Red biological transparency window reduced the photo-toxicity in live cells, as compared with a conventional single-photon approach. Using this new assay, we estimated average concentrations of proteins in the compartments of nuclear speckles and in the nucleoplasm at ~150 mg/ml, and in the nucleolus at ~284 mg/ml. Furthermore, we show a profound influence of pharmaceutical inhibitors of RNA synthesis on intracellular protein density. The approach proposed here will significantly advance theranostics, and studies of drug-cell interactions at the single-cell level, aiding development of personal molecular medicine.

Keywords: Fluorescence lifetime imaging (FLIM), two-photon excited fluorescence, macromolecular crowding, protein concentration, nuclear organization, nucleoplasm, nucleolus, nuclear speckles.