Phasor Analysis of Fluorescence Lifetime Enables Quantitative Multiplexed Molecular Imaging of Three Probes

Abstract

The excited-state lifetime is an intrinsic property of fluorescent molecules that can be leveraged for multiplexed imaging. An advantage of fluorescence lifetime-based multiplexing is that signals from multiple probes can be gathered simultaneously, whereas traditional spectral fluorescence imaging typically requires multiple images at different excitation and emission wavelengths. Additionally, lifetime and spectra could both be utilized to expand the multiplexing capacity of fluorescence. However, resolving exogenous molecular probes based exclusively on the fluorescence lifetime has been limited by technical challenges in analyzing lifetime data. The phasor approach to lifetime analysis offers a simple, graphical solution that has increasingly been used to assess endogenous cellular autofluorescence to quantify metabolic factors. In this study, we employed the phasor analysis of FLIM to quantitatively resolve three exogenous, antibody-targeted fluorescent probes with similar spectral properties based on lifetime information alone. First, we demonstrated that three biomarkers that were spatially restricted to the cell membrane, cytosol, or nucleus could be accurately distinguished using FLIM and phasor analysis. Next, we successfully resolved and quantified three probes that were all targeted to cell surface biomarkers. Finally, we demonstrated that lifetime-based quantitation accuracy can be improved through intensity matching of various probe-biomarker combinations, which will expand the utility of this technique. Importantly, we reconstructed images for each individual probe, as well as an overlay of all three probes, from a single FLIM image. Our results demonstrate that FLIM and phasor analysis can be leveraged as a powerful tool for simultaneous detection of multiple biomarkers with high sensitivity and accuracy.

Figure 1.

Properties of the lifetime probe panel. (A) Emission spectrum and (B) phasor diagram locations for the Qdot 585, Bodipy TMR, and AF555 fluorescence lifetime probes. Lifetime measurements were obtained by the FLIM of probes in PBS buffer after conjugation to an antibody.

Figure 2.

Detection of spatially separated biomarkers on MCF7 cells. (A–C) Cells were labeled with (A) αEpCAM-Qdot 585, (B) αCK18-Bodipy TMR, (C) αKi67-AF555, or (D) the mixture of all three, and the results are shown for the (i) phasor diagram and (ii) total intensity image. Colored circles in the combined phasor in part (D(i)) correspond to the pure probe species from parts (A–C(i)). (E) Results from part (D) were painted with a color gradient to convey relative probe contributions. (F) Probe contributions were fully resolved and pure species images were generated for (i) αEpCAM-Qdot 585, (ii) αCK18-Bodipy TMR, and (iii) αKi67-AF555. (F(iv)) Pseudocolored overlay showing EpCAM in red, CK18 in green, and Ki67 in blue. Scale bars represent 20 μm.

Figure 3.

Detection of spatially colocalized biomarkers. Breast cancer cell lines (A) SK-BR-3, (B) MCF7, (C) MDA-MB-231, and (D) MCF10A were labeled with a mixture of αEpCAM-Qdot 585, αHER2-Bodipy TMR, and αTfR-AF555. Results are shown for a representative (i) phasor plot; (ii) total intensity image; resolved individual intensity images for (iii) EpCAM, (iv) HER2, and (v) TfR; and (vi) pseudocolored overlay image (red EpCAM, green HER2, blue TfR). Additionally, multiple images were analyzed to determine (vii) the mean fractional contribution (FC) of each probe. (E) Mean intensity for each biomarker probe was calculated by multiplying probe contribution by total mean intensity. (F) Results in part (E) were normalized by the mean intensity of single-stain images for the same biomarker–probe combination and cell line, shown in Figure S4 in the Supporting Information. Error bars represent the standard error from at least three independent experiments. Stars indicate p < 0.05 and double stars indicate p < 0.01 relative to the single-stain mean intensity for the same biomarker probe and cell line (see Figure S4 in the Supporting Information). Scale bars represent 20 μm.

Figure 4.

Intensity matching for SK-BR-3 cells. Signal intensities for αHER2-Bodipy TMR and αTfR-AF555 probes were lowered by the addition of the unlabeled antibody at labeled/unlabeled ratios of (A) 30:70 and (B) 10:90. αEpCAM-Qdot 585 was used without dilution. The results are shown for representative (i) phasor plot; (ii) total intensity image; resolved individual intensity images for (iii) EpCAM, (iv) HER2, and (v) TfR; and (vi) pseudocolored overlay image (red EpCAM, green HER2, blue TfR), as well as (vii) mean fractional contribution (FC) of each probe from multiple images. (C) Mean intensity for each probe, adjusted for dilution using calibrations given in Figure S5 in the Supporting Information. Nondiluted single stain (from Figure S4 in the Supporting Information) and resolved triple stain results (from Figure 3) are shown for comparison. (D) Adjusted mean intensity normalized by the nondiluted single stain. Error bars represent the standard error from at least three independent experiments. Stars indicate p < 0.05 and double stars indicate p < 0.01 relative to the mean intensity single stain for the same biomarker probe. Scale bars represent 20 μm.

Figure 5.

Intensity matching for other cell lines. (A) MCF7, (B) MDA-MB-231, and (C) MCF10A cell lines were labeled with αEpCAM-Qdot 585, αHER2-Bodipy TMR, and αTfR-AF555 (30:70 ratio TfR-labeled/unlabeled). The results are shown for representative (i) phasor plot; (ii) total intensity image; resolved individual intensity images for (iii) EpCAM, (iv) HER2, and (v) TfR; and (vi) pseudocolored overlay image (red EpCAM, green HER2, blue TfR), as well as (vii) mean fractional contribution (FC) of each probe from multiple images. (D) Mean intensity, adjusted for dilution using calibrations given in Figure S6 in the Supporting Information. (E) Adjusted mean intensity normalized by nondiluted single-stain images for the same biomarker–probe combination and cell line, shown in Figure S4 in the Supporting Information. Error bars represent the standard error from at least three independent experiments. Stars indicate p < 0.05 and double stars indicate p < 0.01 relative to the nondiluted single-stain mean intensity for the same biomarker–probe and cell line (see Figure S4 in theSupporting Information). Scale bars represent 20 μm.