title: Single Cell Assay for Molecular Diagnostics and Medicine: Monitoring Intracellular Concentrations of Macromolecules by Two-photon Fluorescence Lifetime Imaging.
abstract: 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.
authors.affiliation: 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.
authors.forename: Artem
authors.initials: A
authors.lastname: Pliss
authors.affiliation: 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.
authors.forename: Xiao
authors.initials: X
authors.lastname: Peng
authors.affiliation: 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 ; 3. School of Physics and Optoelectronic Engineering, Xidian University, Xi'an, Shaanxi, China 710071.
authors.forename: Lixin
authors.initials: L
authors.lastname: Liu
authors.affiliation: 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.
authors.forename: Andrey
authors.initials: A
authors.lastname: Kuzmin
authors.affiliation: 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.
authors.forename: Yan
authors.initials: Y
authors.lastname: Wang
authors.affiliation: 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.
authors.forename: Junle
authors.initials: J
authors.lastname: Qu
authors.affiliation: 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 ; 4. School of information science and engineering, Lanzhou University, Lanzhou, Gansu, China, 730000.
authors.forename: Yuee
authors.initials: Y
authors.lastname: Li
authors.affiliation: 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 ; 5. Department of Chemistry, Korea University, Seoul 136-701, Korea.
authors.forename: Paras N
authors.initials: PN
authors.lastname: Prasad
mesh_terms: D006367:HeLa Cells; D006801:Humans; D042541:Intracellular Space / Q000737:chemistry*; D046911:Macromolecular Substances / Q000032:analysis*; D036641:Microscopy, Fluorescence, Multiphoton / Q000379:methods*; D061848:Optical Imaging / Q000379:methods*; D057089:Pathology, Molecular / Q000379:methods*; D059010:Single-Cell Analysis / Q000379:methods*; D000068936:Theranostic Nanomedicine / Q000379:methods
chemical_list: D046911:Macromolecular Substances
keywords: Fluorescence lifetime imaging (FLIM); macromolecular crowding; nuclear organization; nuclear speckles.; nucleolus; nucleoplasm; protein concentration; two-photon excited fluorescence