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

Pliss A, Peng X, Liu L, Kuzmin A, Wang Y, Qu J, Li Y, Prasad PN - Theranostics (2015)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

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.

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.

No MeSH data available.


Related in: MedlinePlus

Inhibition of RNA synthesis by actinomycin D induces changes in subnuclear protein density: (A) The fluorescence intensity image, the fluorescence lifetime image and the fluorescence lifetime histogram are shown for the cell expressing ASF/SF2-GFP and treated by actinomycin D for 3h. White arrows point to doughnut-shaped structures, which emerge in response to the drug treatment. (B) Averaged protein concentrations in the nuclear speckles before the drug treatment and in drug-treated cells (both in nuclear speckles and in the doughnut-shaped structures). Error bars show the standard deviations. (C) FLIM monitoring of the nuclear speckles in a course of actinomycin D treatment. (D) Dynamic changes of fluorescence lifetime values (black) and corresponding concentrations of proteins (blue) in the nuclear speckles during actinomycin D treatment. The plot is generated from data shown in (C).
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Figure 5: Inhibition of RNA synthesis by actinomycin D induces changes in subnuclear protein density: (A) The fluorescence intensity image, the fluorescence lifetime image and the fluorescence lifetime histogram are shown for the cell expressing ASF/SF2-GFP and treated by actinomycin D for 3h. White arrows point to doughnut-shaped structures, which emerge in response to the drug treatment. (B) Averaged protein concentrations in the nuclear speckles before the drug treatment and in drug-treated cells (both in nuclear speckles and in the doughnut-shaped structures). Error bars show the standard deviations. (C) FLIM monitoring of the nuclear speckles in a course of actinomycin D treatment. (D) Dynamic changes of fluorescence lifetime values (black) and corresponding concentrations of proteins (blue) in the nuclear speckles during actinomycin D treatment. The plot is generated from data shown in (C).

Mentions: The fluorescence lifetime values indicate that the drug treatment leads to a substantial increase in the protein molecular density in the nuclear speckles from ~150 mg/ml found in non-treated cells to an average ~525 mg/ml following the actinomycin D treatment (Fig. 5). Furthermore, the doughnut-shaped structures in the treated cells were found to accommodate very high concentrations of proteins at nearly ~600 mg/ml. This concentration range is not expected for soluble proteins, however, protein clustering is known during the drug-induced apoptosis development 39. Thus, emergence of highly concentrated agglomerates of proteins, likely represents a hallmark of apoptotic cells.


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

Pliss A, Peng X, Liu L, Kuzmin A, Wang Y, Qu J, Li Y, Prasad PN - Theranostics (2015)

Inhibition of RNA synthesis by actinomycin D induces changes in subnuclear protein density: (A) The fluorescence intensity image, the fluorescence lifetime image and the fluorescence lifetime histogram are shown for the cell expressing ASF/SF2-GFP and treated by actinomycin D for 3h. White arrows point to doughnut-shaped structures, which emerge in response to the drug treatment. (B) Averaged protein concentrations in the nuclear speckles before the drug treatment and in drug-treated cells (both in nuclear speckles and in the doughnut-shaped structures). Error bars show the standard deviations. (C) FLIM monitoring of the nuclear speckles in a course of actinomycin D treatment. (D) Dynamic changes of fluorescence lifetime values (black) and corresponding concentrations of proteins (blue) in the nuclear speckles during actinomycin D treatment. The plot is generated from data shown in (C).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4493531&req=5

Figure 5: Inhibition of RNA synthesis by actinomycin D induces changes in subnuclear protein density: (A) The fluorescence intensity image, the fluorescence lifetime image and the fluorescence lifetime histogram are shown for the cell expressing ASF/SF2-GFP and treated by actinomycin D for 3h. White arrows point to doughnut-shaped structures, which emerge in response to the drug treatment. (B) Averaged protein concentrations in the nuclear speckles before the drug treatment and in drug-treated cells (both in nuclear speckles and in the doughnut-shaped structures). Error bars show the standard deviations. (C) FLIM monitoring of the nuclear speckles in a course of actinomycin D treatment. (D) Dynamic changes of fluorescence lifetime values (black) and corresponding concentrations of proteins (blue) in the nuclear speckles during actinomycin D treatment. The plot is generated from data shown in (C).
Mentions: The fluorescence lifetime values indicate that the drug treatment leads to a substantial increase in the protein molecular density in the nuclear speckles from ~150 mg/ml found in non-treated cells to an average ~525 mg/ml following the actinomycin D treatment (Fig. 5). Furthermore, the doughnut-shaped structures in the treated cells were found to accommodate very high concentrations of proteins at nearly ~600 mg/ml. This concentration range is not expected for soluble proteins, however, protein clustering is known during the drug-induced apoptosis development 39. Thus, emergence of highly concentrated agglomerates of proteins, likely represents a hallmark of apoptotic cells.

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

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.

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.

No MeSH data available.


Related in: MedlinePlus