Cell Imaging Using Two-Photon Excited CdS Fluorescent Quantum Dots Working within the Biological Window

In recent years, two-photon excited semiconductor quantum dots (QDs) have been the subject of intense investigation due to their long excitation wavelength which helps to achieve deeper penetration and higher image resolution in optical bioimaging. In this paper, water-soluble CdS QDs were synthesized using a hydrothermal method and applied to human liver hepatocellular carcinoma (HepG2) cells. The first-principles calculation suggested that the S-rich defected structure contributes to a narrower band gap compared to the pristine structure. The resulting fluorescence wavelength was significantly red shifted, which was attributed to the deep defect states emission. The large Stokes shifts (> 200 nm) of the QDs can eliminate the possible cross-talk between the excitation light and the emission light. Two-photon induced red fluorescence emission can avoid overlapping with the autofluorescence emission of biological samples. The uptake and cell viability measurements of the HepG2 cells showed a good biocompatibility and a low toxicity of CdS QDs. Two-photon excited scanning microscopy images revealed that the HepG2 cells incubated with CdS QDs emitted bright red upconversion fluorescence and the fluorescence brightness was 38.2 times of that of the control group. These results support CdS QDs as a good candidate for application in cellular imaging.


The preparation process of CdS QDs
100 mL of 0.02 mol/L CdCl2•2.5H2O solution was loaded into a 250 mL the three-necked flask. 0.35 mL of Thioglycolic acid (TGA) was added to three-necked flask during magnetic stirring. The pH value of the mixed solution was adjusted to be 10.5 by the dropwise addition of 1 mol/L NaOH solution. Then 0.2459 g of NaS•9H2O was dissolved into 5 mL of deionized water and added to the above mixture. Finally, the solution mixture was heated to 100 ° C in an oil bath and refluxed for 5 hours before being cooled to room temperature. A transparent pale yellow CdS QDs solution was obtained.

The measurement of the photoluminescence quantum yields and the two-photon absorption cross-section of the CdS QDs solution
We calculated the photoluminescence quantum yields (PL QYs) and the two-photon absorption cross-section (σ2PA) of the CdS QDs solution by the following equations (1) [1] and (2) [2], respectively. Rhodamine 6G (R6G) dissolved in methanol (QY=95%) was used as a reference [3]. In order to avoid reabsorption of the samples, the absorbance of the CdS QDs solution and the R6G solution were less than 0.1 at the same excitation wavelength [4].
In formula (1), F is the measured photoluminescence intensity, A is the absorbance at the excitation wavelength, and n is the refractive index of the solvent. In formula (2), C is the concentration of the sample, F is the two-photon luminescence intensity, and the η is the QY.

Experimental setup for the Two-photon luminescence
The experimental setup used for the two-photon excitation of CdS QDs is schematically shown in Figure S1.

The upconversion luminescence lifetimes of CdS QDs
We measured the upconversion luminescence lifetimes of CdS QDs using a fluorescence lifetime spectrometer that is based on the technique of time correlated single photon counting (TCSPC). The result of measurement is shown in Figure S2. Figure S2. Two-photon excited luminescence decay curves (dots) of the CdS QDs solution and the best fitting curves (red line).

Cell Viability and Cellular Uptake of CdS QDs
The techniques of cell culture were similar to those described in reference [6]. Cell viability was measured using the MTT assay. The measurement was based on the method previously reported in reference [6]. The absorbance of the 96-well plate at 490 nm was measured using an iMark Microplate (BioRad). The cell viability was calculated by the following formula (3): Where OD refers to the absorbance of each well. The subscripts "treated" and "control" refer to the HepG2 cells incubated with and without CdS QDs, respectively. The subscript "blank" refers to the cell medium in the absence of cells and CdS QDs. The morphology images of HepG2 cells incubated with CdS QDs at different concentrations are shown in Figure S3. The uptake of CdS QDs of individual HepG2 cells was measured by inductively coupled plasma-mass spectrometry (ICP-MS) (ICAP-qc, Thermo Fisher, Germany) [7]. We incubated HepG2 cells with the CdS QDs in a humidified incubator (37 ° C, 5% CO2) for 24 hours. Then, the cells were washed three times with Phosphate Buffered Solution (PBS) and centrifuged to form cell pellets. Finally, the cell pellets were fixed by glutaraldehyde (2.5%), embedded in resin, cut to ultra-thin slices, and stained by osmic acids. The samples were supported by Ni mesh coated with an ultra-thin carbon film. We obtained the distribution images and EDS data of CdS QDs in the cells using TEM (JEOL-1400HR, JEOL, Japan) equipped with energydispersive X-ray spectrometry (EDS) under an acceleration voltage of 200 kV. Figure S4 is the TEM image of a HepG2 cell incubated with CdS QDs for 24 hours.