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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

FLIM imaging of fibrillarin-GFP in the DRB treated cell: (A) Fibrillarin-GFP: the fluorescence intensity image, the fluorescence lifetime image and the fluorescence lifetime histogram are shown. Fibrillarin GFP is accumulated in the necklace beads-like structures (indicated by white arrows) and in the nucleolar remnants (indicated by blue arrowhead). (B) Averaged fluorescence lifetimes in the nucleoli and in the bead-like structures before and after the DRB treatment, as indicated.
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Figure 6: FLIM imaging of fibrillarin-GFP in the DRB treated cell: (A) Fibrillarin-GFP: the fluorescence intensity image, the fluorescence lifetime image and the fluorescence lifetime histogram are shown. Fibrillarin GFP is accumulated in the necklace beads-like structures (indicated by white arrows) and in the nucleolar remnants (indicated by blue arrowhead). (B) Averaged fluorescence lifetimes in the nucleoli and in the bead-like structures before and after the DRB treatment, as indicated.

Mentions: In the second group of experiments the influence of 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB) was investigated. This drug inhibits pre-mRNA synthesis by RNA polymerase II, and induces reorganization of nucleolus, wherein DNA with ribosomal genes unwinds from nucleoli into necklace-beads like structure. Strikingly, in DRB treated cells, synthesis of ribosomal RNA continues in the “beads” of “necklace” 40. Our FLIM data shows roughly the same fluorescence lifetime values in remnants of the nucleoli of and in necklace-like structures of DRB- treated cells as in the nucleoli untreated cells (2150 and 2140 picoseconds respectively), roughly corresponding to 280-290 mg/ml of proteins concentration (Fig. 6). These data suggest that RI of nucleolus is dependent on synthesis of ribosomal RNA, while selective inhibition of mRNA synthesis by DRB has no significant effect on molecular density of nucleolus during first hours of drug treatment.


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)

FLIM imaging of fibrillarin-GFP in the DRB treated cell: (A) Fibrillarin-GFP: the fluorescence intensity image, the fluorescence lifetime image and the fluorescence lifetime histogram are shown. Fibrillarin GFP is accumulated in the necklace beads-like structures (indicated by white arrows) and in the nucleolar remnants (indicated by blue arrowhead). (B) Averaged fluorescence lifetimes in the nucleoli and in the bead-like structures before and after the DRB treatment, as indicated.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4493531&req=5

Figure 6: FLIM imaging of fibrillarin-GFP in the DRB treated cell: (A) Fibrillarin-GFP: the fluorescence intensity image, the fluorescence lifetime image and the fluorescence lifetime histogram are shown. Fibrillarin GFP is accumulated in the necklace beads-like structures (indicated by white arrows) and in the nucleolar remnants (indicated by blue arrowhead). (B) Averaged fluorescence lifetimes in the nucleoli and in the bead-like structures before and after the DRB treatment, as indicated.
Mentions: In the second group of experiments the influence of 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB) was investigated. This drug inhibits pre-mRNA synthesis by RNA polymerase II, and induces reorganization of nucleolus, wherein DNA with ribosomal genes unwinds from nucleoli into necklace-beads like structure. Strikingly, in DRB treated cells, synthesis of ribosomal RNA continues in the “beads” of “necklace” 40. Our FLIM data shows roughly the same fluorescence lifetime values in remnants of the nucleoli of and in necklace-like structures of DRB- treated cells as in the nucleoli untreated cells (2150 and 2140 picoseconds respectively), roughly corresponding to 280-290 mg/ml of proteins concentration (Fig. 6). These data suggest that RI of nucleolus is dependent on synthesis of ribosomal RNA, while selective inhibition of mRNA synthesis by DRB has no significant effect on molecular density of nucleolus during first hours of drug treatment.

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