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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

Mapping of the intracellular protein concentrations by using FLIM approach: (A) Fluorescence intensity images, fluorescence lifetime images, and corresponding lifetime histograms of fibrillarin-GFP and ASF/SF2-GFP (in picoseconds). (B) Estimated average protein concentration for the nucleolus, nuclear speckles and the nucleoplasm. Error bars show the standard deviations.
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Figure 2: Mapping of the intracellular protein concentrations by using FLIM approach: (A) Fluorescence intensity images, fluorescence lifetime images, and corresponding lifetime histograms of fibrillarin-GFP and ASF/SF2-GFP (in picoseconds). (B) Estimated average protein concentration for the nucleolus, nuclear speckles and the nucleoplasm. Error bars show the standard deviations.

Mentions: We found that the ASF/SF2-GFP molecules have approximately the same fluorescence lifetime, both in the nucleoplasm and in the nuclear speckles. The measurements in different cells were ranging from ~2180 to ~2250 picoseconds (Fig. 2A), which correspond to protein concentrations of ~230 mg/ml and 120 mg/ml in these regions of the cell nucleus, according to our calibration (Fig. 1C). The average protein concentration was ~168 mg/ml in the nucleoplasm and ~152 mg/ml inside the nuclear speckles; however this difference was not statistically significant. In contrast to ASF/SF2, which had relatively uniform fluorescence lifetime values across the cell nucleus, we found that the lifetime of fibrillarin-GFP strongly depends on its intranuclear location. In the nucleoplasm, the fluorescence lifetime for this protein was ranging from ~2180 ps to ~2270 ps (RI equivalent of 230 mg/ml and 90 mg/ml, respectively), which was close to data obtained with ASF/SF2-GFP. At the same time, fluorescence lifetime of fibrillarin-GFP was significantly shorter inside the nucleolus measuring 2110 ps to 2190 ps, which represents protein concentrations of 354 mg/ml and 217 mg/ml, respectively. The average fluorescence lifetime of fibrillarin-GFP in nucleolus was 2150 ps, which corresponds to ~284 mg/ml of protein concentration (Fig. 2B).


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)

Mapping of the intracellular protein concentrations by using FLIM approach: (A) Fluorescence intensity images, fluorescence lifetime images, and corresponding lifetime histograms of fibrillarin-GFP and ASF/SF2-GFP (in picoseconds). (B) Estimated average protein concentration for the nucleolus, nuclear speckles and the nucleoplasm. Error bars show the standard deviations.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 2: Mapping of the intracellular protein concentrations by using FLIM approach: (A) Fluorescence intensity images, fluorescence lifetime images, and corresponding lifetime histograms of fibrillarin-GFP and ASF/SF2-GFP (in picoseconds). (B) Estimated average protein concentration for the nucleolus, nuclear speckles and the nucleoplasm. Error bars show the standard deviations.
Mentions: We found that the ASF/SF2-GFP molecules have approximately the same fluorescence lifetime, both in the nucleoplasm and in the nuclear speckles. The measurements in different cells were ranging from ~2180 to ~2250 picoseconds (Fig. 2A), which correspond to protein concentrations of ~230 mg/ml and 120 mg/ml in these regions of the cell nucleus, according to our calibration (Fig. 1C). The average protein concentration was ~168 mg/ml in the nucleoplasm and ~152 mg/ml inside the nuclear speckles; however this difference was not statistically significant. In contrast to ASF/SF2, which had relatively uniform fluorescence lifetime values across the cell nucleus, we found that the lifetime of fibrillarin-GFP strongly depends on its intranuclear location. In the nucleoplasm, the fluorescence lifetime for this protein was ranging from ~2180 ps to ~2270 ps (RI equivalent of 230 mg/ml and 90 mg/ml, respectively), which was close to data obtained with ASF/SF2-GFP. At the same time, fluorescence lifetime of fibrillarin-GFP was significantly shorter inside the nucleolus measuring 2110 ps to 2190 ps, which represents protein concentrations of 354 mg/ml and 217 mg/ml, respectively. The average fluorescence lifetime of fibrillarin-GFP in nucleolus was 2150 ps, which corresponds to ~284 mg/ml of protein concentration (Fig. 2B).

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