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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

Dependence of fluorescence lifetime on refractive index: (A) Fluorescence lifetime images of H2B-GFP in the cells, which were immersed into PBS or glycerol solutions of different concentration. From left to right: PBS, Glycerol 25%, Glycerol 50%, Glycerol 75% (% by weight). (B) Chart shows an inverse correlation between the square of the refractive index (n) and the fluorescence lifetime of GFP (τ) - (1/τ on the left and τ on the right Y axis). (C) Chart presents the dependence between the fluorescence lifetime of GFP and the estimated concentration of proteins. Error bars show the standard deviations.
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Figure 1: Dependence of fluorescence lifetime on refractive index: (A) Fluorescence lifetime images of H2B-GFP in the cells, which were immersed into PBS or glycerol solutions of different concentration. From left to right: PBS, Glycerol 25%, Glycerol 50%, Glycerol 75% (% by weight). (B) Chart shows an inverse correlation between the square of the refractive index (n) and the fluorescence lifetime of GFP (τ) - (1/τ on the left and τ on the right Y axis). (C) Chart presents the dependence between the fluorescence lifetime of GFP and the estimated concentration of proteins. Error bars show the standard deviations.

Mentions: In our initial experiments we performed a calibration of fluorescence lifetime of GFP in buffers with different RI. The calibration was performed using cells expressing the histone H2B-GFP protein; we selected this protein for it relatively uniform distribution pattern, which facilitates the analysis of fluorescence lifetime across the cell nucleus. The cells were fixed and placed into series of concentrations of glycerol to increase the RI in the immediate environment of H2B-GFP. Then, the lifetime data for GFP in solutions with different RI were acquired. As demonstrated by FLIM images at Figure 1A, an increase of RI of glycerol media leads to a reduction in the fluorescence lifetime of H2B-GFP, which is consistent with previous findings of others and our group 32, 34. To enable a simple matching of the fluorescence lifetime of GFP and RI of the media, we plotted the FLIM data onto a correlation chart (Fig. 1B). Using the approach of Handwerger et al., 9, we calculated the concentrations of proteins corresponding to each measured RI, as described in the Methods. Finally, the dependence between the fluorescence lifetime and sought concentration of proteins was plotted on the chart (Fig.1C).


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)

Dependence of fluorescence lifetime on refractive index: (A) Fluorescence lifetime images of H2B-GFP in the cells, which were immersed into PBS or glycerol solutions of different concentration. From left to right: PBS, Glycerol 25%, Glycerol 50%, Glycerol 75% (% by weight). (B) Chart shows an inverse correlation between the square of the refractive index (n) and the fluorescence lifetime of GFP (τ) - (1/τ on the left and τ on the right Y axis). (C) Chart presents the dependence between the fluorescence lifetime of GFP and the estimated concentration of proteins. Error bars show the standard deviations.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 1: Dependence of fluorescence lifetime on refractive index: (A) Fluorescence lifetime images of H2B-GFP in the cells, which were immersed into PBS or glycerol solutions of different concentration. From left to right: PBS, Glycerol 25%, Glycerol 50%, Glycerol 75% (% by weight). (B) Chart shows an inverse correlation between the square of the refractive index (n) and the fluorescence lifetime of GFP (τ) - (1/τ on the left and τ on the right Y axis). (C) Chart presents the dependence between the fluorescence lifetime of GFP and the estimated concentration of proteins. Error bars show the standard deviations.
Mentions: In our initial experiments we performed a calibration of fluorescence lifetime of GFP in buffers with different RI. The calibration was performed using cells expressing the histone H2B-GFP protein; we selected this protein for it relatively uniform distribution pattern, which facilitates the analysis of fluorescence lifetime across the cell nucleus. The cells were fixed and placed into series of concentrations of glycerol to increase the RI in the immediate environment of H2B-GFP. Then, the lifetime data for GFP in solutions with different RI were acquired. As demonstrated by FLIM images at Figure 1A, an increase of RI of glycerol media leads to a reduction in the fluorescence lifetime of H2B-GFP, which is consistent with previous findings of others and our group 32, 34. To enable a simple matching of the fluorescence lifetime of GFP and RI of the media, we plotted the FLIM data onto a correlation chart (Fig. 1B). Using the approach of Handwerger et al., 9, we calculated the concentrations of proteins corresponding to each measured RI, as described in the Methods. Finally, the dependence between the fluorescence lifetime and sought concentration of proteins was plotted on the chart (Fig.1C).

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