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2015 ; 5
(9
): 919-30
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Single Cell Assay for Molecular Diagnostics and Medicine: Monitoring
Intracellular Concentrations of Macromolecules by Two-photon Fluorescence
Lifetime Imaging
#MMPMID26155309
Pliss A
; Peng X
; Liu L
; Kuzmin A
; Wang Y
; Qu J
; Li Y
; Prasad PN
Theranostics
2015[]; 5
(9
): 919-30
PMID26155309
show ga
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.