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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

Monitoring of the fluorescence lifetime of Fibrillarin-GFP: (A) Intensity image of fibrillarin-GFP expressing cell. Nucleolus in white rectangle was acquired every 15 min. (B) Time sequenced FLIM images of nucleolus. A color-coded image demonstrates changing distribution of fluorescence lifetime values and corresponding concentrations of nucleolar proteins at each time point.
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Figure 3: Monitoring of the fluorescence lifetime of Fibrillarin-GFP: (A) Intensity image of fibrillarin-GFP expressing cell. Nucleolus in white rectangle was acquired every 15 min. (B) Time sequenced FLIM images of nucleolus. A color-coded image demonstrates changing distribution of fluorescence lifetime values and corresponding concentrations of nucleolar proteins at each time point.

Mentions: FLIM imaging has shown that the fluorescence lifetimes and the corresponding concentrations of proteins were relatively uniform in the nuclear speckles. At the same time, in the nucleolus, we could frequently distinguish sub-domains with different RI, evidently reflecting a higher-order heterogeneous molecular structure of this nuclear domain 38. To explore how the molecular structure of nucleolus may change in time, we performed a number of time-lapse studies wherein the FLIM data were sequentially acquired from the same cell with 10-20 min intervals. In cells expressing fibrillarin-GFP, our data has revealed that the intranucleolar distribution of proteins is highly dynamic. The fluorescence lifetime values indicated that the molecular density in various nucleolar domains changed considerably over time. An example of dynamic changes in distribution of proteins in the nucleolus is shown in Figure 3. We observed that concentration of proteins in different parts of the nucleolus changes by several tens of milligrams at subsequent FLIM image acquisitions.


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)

Monitoring of the fluorescence lifetime of Fibrillarin-GFP: (A) Intensity image of fibrillarin-GFP expressing cell. Nucleolus in white rectangle was acquired every 15 min. (B) Time sequenced FLIM images of nucleolus. A color-coded image demonstrates changing distribution of fluorescence lifetime values and corresponding concentrations of nucleolar proteins at each time point.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 3: Monitoring of the fluorescence lifetime of Fibrillarin-GFP: (A) Intensity image of fibrillarin-GFP expressing cell. Nucleolus in white rectangle was acquired every 15 min. (B) Time sequenced FLIM images of nucleolus. A color-coded image demonstrates changing distribution of fluorescence lifetime values and corresponding concentrations of nucleolar proteins at each time point.
Mentions: FLIM imaging has shown that the fluorescence lifetimes and the corresponding concentrations of proteins were relatively uniform in the nuclear speckles. At the same time, in the nucleolus, we could frequently distinguish sub-domains with different RI, evidently reflecting a higher-order heterogeneous molecular structure of this nuclear domain 38. To explore how the molecular structure of nucleolus may change in time, we performed a number of time-lapse studies wherein the FLIM data were sequentially acquired from the same cell with 10-20 min intervals. In cells expressing fibrillarin-GFP, our data has revealed that the intranucleolar distribution of proteins is highly dynamic. The fluorescence lifetime values indicated that the molecular density in various nucleolar domains changed considerably over time. An example of dynamic changes in distribution of proteins in the nucleolus is shown in Figure 3. We observed that concentration of proteins in different parts of the nucleolus changes by several tens of milligrams at subsequent FLIM image acquisitions.

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