Enhanced Visualization of Erythrocytes Through Photoluminescence Using NaYbF4:Yb3+,Er3+ Nanoparticles

Abstract

Rare-earth nanoparticles (RE-NPs), particularly NaYF₄:Yb³⁺,Er³⁺, have emerged as a promising class of photoluminescent probes for bioimaging and sensing applications. These nanomaterials are characterized by their ability to absorb low-energy photons and emit higher-energy photons through an upconversion luminescence process. This process can be triggered by continuous-wave (CW) light excitation, providing a unique optical feature that is not exhibited by native biomolecules. However, the application of upconversion nanoparticles (UCNPs) in bioimaging requires systematic optimization to maximize the signal and ensure biological compatibility. In this work, we synthesized hexagonal-phase UCNPs (average diameter: 29 ± 3 nm) coated with polyacrylic acid (PAA) and established the optimal conditions for imaging human erythrocytes. The best results were obtained after a 4-h incubation in 100 mM HEPES buffer, using a nanoparticle concentration of 0.01 mg/mL and a laser current intensity of 250-300 mA. Under these conditions, the UCNPs exhibited minimal cytotoxicity and were found to predominantly localize at the erythrocyte membrane periphery, indicating surface adsorption rather than internalization. Additionally, a machine learning model (Random Forest) was implemented that classified the photoluminescent signal with 80% accuracy and 83% precision, with the signal intensity identified as the most relevant feature. This study establishes a quantitative and validated protocol that balances signal strength with cell integrity, enabling robust and automated image analysis.

Keywords: biocompatibility; photoluminescence; rare-earth nanoparticles; red blood cells.

Figure 1

(A) Transmission electron microscopy (TEM) image of the synthesized NaYF₄:Yb³⁺/Er³⁺ NPs. The inset shows the particle size distribution. (B) Photoluminescence (PL) spectrum of NaYF₄:Yb³⁺,Er³⁺ NPs under excitation at 980 nm, (C) X-ray diffraction (XRD) pattern of NaYF₄:Yb³⁺,Er³⁺ -NPs and reference (JCPDS card 28-1192) for the hexagonal β-phase of NaYF₄. (D) Representative scheme for the mechanism of upconversion photoluminescence by energy transfer.

Figure 2

(A) MTT assay results showing the viability of Vero cells exposed to different concentrations of NaYF₄:Yb³⁺,Er³⁺@PAA NPs. The negative control corresponds to untreated Vero cells, and the positive control is doxorubicin. (B) Hemolysis assay results for RBCs at 2% hematocrit exposed to different concentrations of NaYF₄:Yb³⁺,Er³⁺@PAA NPs. The negative control corresponds to untreated erythrocytes, while the positive control is 0.1 M Triton X-100.

Figure 3

Photoluminescence imaging of erythrocytes incubated with and without rare-earth nanoparticles (NaYF₄:Yb³⁺,Er³⁺@PAA-NPs). Bright-field image of erythrocytes (A) incubated with RE-NPs and (B) after excitation with a 980 nm laser. Bright-field image of (C) control erythrocytes without RE-NPs and (D) after 980 nm laser excitation. All fluorescence images were acquired with a 200 ms exposure time.

Figure 4

Micrographs of 2% erythrocytes after being incubated for 3 h with NaYF₄:Yb³⁺,Er³⁺@PAA NPs 0.01 mg/mL. Images (A,C,E,G) were obtained using a bright field microscope, while images (B,D,F,H) show the photoluminescence (green) of the nanoparticles after excitation with a 980 nm laser. All fluorescence images were acquired with a 200 ms exposure time.

Figure 5

Photoluminescence images (green) of 2% erythrocytes incubated with NaYF4:Yb³⁺,Er³⁺@PAA nanoparticles (0.01 mg/mL) in HEPES buffer (100 mM) under near-infrared excitation (980 nm) at different incubation times (1, 3, 4, 6, and 24 h). The images corresponding to each time point are presented in pairs: bright-field images (A,C,E,G,I) and the corresponding photoluminescence images (B,D,F,H,J). All fluorescence images were acquired with a 200 ms exposure time.

Figure 6

Images of erythrocytes at 2% hematocrit incubated for 4 h with NaYF₄:Yb³⁺,Er³⁺@PAA NPs (0.01 mg/mL) and 100 mM HEPES (pH 7.2). Image (A) was obtained using a bright-field microscope. Images (B–J) show the effect of increasing the laser current intensity on the photoluminescence signal (green) from the nanoparticles after excitation with a 980 nm laser. All fluorescence images were acquired with a 200 ms exposure time.

Figure 7

Representative images of erythrocytes after exposure to different concentrations of NaYF₄:Yb³⁺,Er³⁺@PAA nanoparticles, obtained by bright-field microscopy (A,C,E,G) and PL microscopy after excitation with a 980 nm laser (B,D,F,H). Images were acquired at a laser current intensity of 300 mA and incubation time of 4 h. All fluorescence images (green) were acquired with a 200 ms exposure time.

Figure 8

Photoluminescence images (green) of erythrocytes at 2% hematocrit after incubation for 4 h with NaYF₄:Yb³⁺,Er³⁺@PAA NPs (0.01 mg/mL). The images correspond to different exposure times to excitation with a 980 nm laser: (A) 0 min, (B) 5 min, (C) 10 min, and (D) 20 min. All fluorescence images were acquired with a 200 ms exposure time.

Figure 9

Boxplots showing the distribution of (A) mean intensity and (B) maximum intensity.

Figure 10

Confusion matrix illustrating the classification performance of the Random Forest model on the test set. The matrix shows the number of true positives (21), true negatives (5), false positives (6), and false negatives (4).

Figure 11

Histogram showing the distribution of predicted probability scores generated by the Random Forest model, separated by true class label. ROIs with a positive photoluminescent signal (red) are concentrated at high probability values (>0.85), while negative cases (blue) are distributed mostly below 0.15.