High-fidelity carbon dots polarity probes: revealing the heterogeneity of lipids in oncology

Abstract

Polarity is an integral microenvironment parameter in biological systems closely associated with a multitude of cellular processes. Abnormal polarity variations accompany the initiation and development of pathophysiological processes. Thus, monitoring the abnormal polarity is of scientific and practical importance. Current state-of-the-art monitoring techniques are primarily based on fluorescence imaging which relies on a single emission intensity and may cause inaccurate detection due to heterogeneous accumulation of the probes. Herein, we report carbon dots (CDs) with ultra-sensitive responses to polarity. The CDs exhibit two linear relationships: one between fluorescence intensity and polarity and the other between polarity and the maximum emission wavelength. The emission spectrum is an intrinsic property of the probes, independent of the excitation intensity or probe concentration. These features enable two-color imaging/quantitation of polarity changes in lipid droplets (LDs) and in the cytoplasm via in situ emission spectroscopy. The probes reveal the polarity heterogeneity in LDs which can be applied to make a distinction between cancer and normal cells, and reveal the polarity homogeneity in cytoplasm.

Fig. 1. Schematic representation of experiments.

Schematic representation of the lipid droplets and cytoplasm imaging with different emission windows and high-fidelity detection of polarity by in situ emission spectrum

Fig. 2. Morphological, structural characterization, absorption, and fluorescence spectra of PS-CDs.

a TEM and HRTEM images of PS-CDs with size distribution. b FT-IR spectroscopy of PS-CDs and CDs. c XPS survey spectrum of PS-CDs. d B 1s XPS spectrum of PS-CDs. e Different wavelengths of excitation influence the fluorescent emission spectra of PS-CDs. f The emission and excitation spectra of PS-CDs. g Emission spectrum of PS-CDs in various 1,4-dioxane/H₂O ratios (0–10%). h Linearity of the maximum emission wavelength, maximum emission fluorescence intensity, and the solvent’s Δƒ

Fig. 3. Subcellular colocalization assays of PS-CDs with commercial organelle dyes.

a Schematic diagram of LDs and cytoplasm imaging by PS-CDs with two fluorescent colors. b Confocal microscope images of SMMC 7721 pretreated with PS-CDs (50 μg mL⁻¹), λ_ex = 476 nm. c Confocal microscope images of HepG 2 cells pretreated with PS-CDs (50 μg mL⁻¹) and HCS LipidTOXTM Deep Red (1:1000 dilution), λ_ex(PS-CDs) = 476 nm, λ_ex(HCS) = 637 nm. d Confocal microscope images of SMMC 7721 cells pretreated with PS-CDs (50 μg mL⁻¹) and Hoechst 33342 (1 μL), λ_ex(PS-CDs) = 476 nm, λ_ex(HCS) = 405 nm. Scale bar, 10 μm in a, b; Scale bar, 20 μm in c

Fig. 4. Effect of voltage, gain and concentration on fluorescence intensity and λ_em.

Pseudocolored images of SMMC 7721 cells treated with 50 μg mL^–1 PS-CDs at different voltage (a), gain (b), and concentration of PS-CDs (c), λ_ex = 476 nm, λ_em = 485–525 nm (channel 1). Scale bar: 10 μm. In situ emission spectrum of LDs at a different voltage (d), gain (e), and concentration of PS-CDs (f). Scale bar: 10 μm

Fig. 5. Confocal images and in situ emission spectra of different cells.

a Imaging of SMMC 7721 cells, Huh–7 cells, HepG 2 cells, HeLa cells, MCF–7 cells, 4T1 cells, HEK 293 cells, and HL 7702 cells, λ_ex = 476 nm, λ_em = 485–525 nm. b In situ emission spectrum of LDs in different cell lines with 50 μg mL^–1 PS-CDs. c Imaging of SMMC 7721 cells, Huh–7 cells, HepG 2 cells, HeLa cells, MCF–7 cells, 4T1 cells, HEK 293 cells, and HL 7702 cells, λ_ex = 476 nm, λ_em = 585–625 nm. d In situ emission spectra of cytoplasm in different cell lines with 50 μg mL^–1 PS-CDs. Scale bar in a, c: 10 μm