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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 ASF/SF2-GFP: (A) A high magnification of nuclear speckles is shown. Same cell area was scanned with 10 min intervals, and the FLIM images were generated. (B) Charts demonstrate changes in average fluorescence lifetime (black) and corresponding protein concentrations (blue) in the nuclear speckles shown at (A).
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Figure 4: Monitoring of the fluorescence lifetime of ASF/SF2-GFP: (A) A high magnification of nuclear speckles is shown. Same cell area was scanned with 10 min intervals, and the FLIM images were generated. (B) Charts demonstrate changes in average fluorescence lifetime (black) and corresponding protein concentrations (blue) in the nuclear speckles shown at (A).

Mentions: A different type of molecular dynamics was found in the nuclear speckles. In these nuclear domains, the molecular concentration appeared more homogeneous than in the nucleolus. At the same time, the average fluorescence lifetime periodically fluctuated. These changes were synchronous for different nuclear speckles located in the same cell nucleus (Fig. 4). Overall, our time-lapse experiments revealed that the fluorescence lifetime within the same nuclear domain is not static, but is dynamically changed over the time, which indicates that nuclear compartments undergo through constant changes in concentration of macromolecules.


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 ASF/SF2-GFP: (A) A high magnification of nuclear speckles is shown. Same cell area was scanned with 10 min intervals, and the FLIM images were generated. (B) Charts demonstrate changes in average fluorescence lifetime (black) and corresponding protein concentrations (blue) in the nuclear speckles shown at (A).
© Copyright Policy
Related In: Results  -  Collection

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

Figure 4: Monitoring of the fluorescence lifetime of ASF/SF2-GFP: (A) A high magnification of nuclear speckles is shown. Same cell area was scanned with 10 min intervals, and the FLIM images were generated. (B) Charts demonstrate changes in average fluorescence lifetime (black) and corresponding protein concentrations (blue) in the nuclear speckles shown at (A).
Mentions: A different type of molecular dynamics was found in the nuclear speckles. In these nuclear domains, the molecular concentration appeared more homogeneous than in the nucleolus. At the same time, the average fluorescence lifetime periodically fluctuated. These changes were synchronous for different nuclear speckles located in the same cell nucleus (Fig. 4). Overall, our time-lapse experiments revealed that the fluorescence lifetime within the same nuclear domain is not static, but is dynamically changed over the time, which indicates that nuclear compartments undergo through constant changes in concentration of macromolecules.

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