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.

Biophotonics in China

“…Qu's group from Shenzhen University focuses on cutting‐edge biomedical optical imaging techniques including fluorescence lifetime imaging microscopy (FLIM), super‐resolution optical imaging, nonlinear optical imaging and their applications in monitoring cell processes, cell‐drug interactions and phototherapy. With the support from the National Basic Research Program and the National Natural Science Foundation of China, Qu's group showed that fluorescence lifetime can be used to quantitatively map the local biomolecular concentration that is related to the functional and physiological state of a cell . Furthermore, they developed a rapid fluorescence lifetime imaging system (AOD‐FLIM) that is based on a pair of acousto‐optic deflectors (AOD) for the random regions of interest (ROI) study in the sample .…”

Fluorescence lifetime imaging of fluorescent proteins as an effective quantitative tool for noninvasive study of intracellular processes

“…In these nuclear compartments the average protein concentration was at 284 mg/mL and 150 mg/mL, respectively. 44 …”

“…This feature of FPs can be used for sensing changes in their environment such as intracellular pH, 39-41 temperature 42 or protein concentration. 43,44 As value of these parameters in speci¯c cellular compartments plays an important role in regulation of many intracellular processes, the reliable, accurate and noninvasive methods for Source: Reprinted by permission from Macmillan Publisher Ltd: Nature Methods, 37 Copyright 2016. determination of intracellular parameters are highly desirable in molecular biology and biomedicine. In this regard, genetically encoded FPs-based sensors have a big advantage, as they can be targeted to almost any cellular compartment without loading procedure that is required for conventional dyes.…”

“…56 In another study, the dependence of°uorescence lifetime on local refractive index was utilized for the measurement and monitoring of biomolecular concentrations in speci¯c subcellular compartments. 43,44 First, the°uorescence lifetime of GFP and tdTomato was employed for sensing of concentration changes in the nucleoplasm of HeLa cells during the cell cycle progression. 43 It was found that°uorescence lifetime values of GFP and tdTomato are notably shortened during the transition from mitosis to interphase, evidently due to rapid increase in the refractive index during the cell division.…”

Applications of fluorescence lifetime imaging in clinical medicine

“…These features allow for°u orescence lifetime imaging microscopy (FLIM) to accurately quantitatively assess changes of many biophysical parameters of a target molecular microenvironment, such as oxygen pressure, polarity, pH, ion concentration and other biochemical parameters. 6,7 At present, the gold standard for the diagnosis of organ tissue tumors is pathological diagnosis and for cardiovascular disease, especially coronary artery disease, is angiography. However, many diagnoses of nonneoplastic diseases do not have the so-called gold standard, such as psoriasis, retinal artery occlusion, liver disease, nervous system, digestive system diseases, Alzheimer's disease, etc.…”