Du401a br dd 352

Micro-Raman spectra were obtained with an Alpha 500R confocal Raman microspectroscopy system (WITec GmbH, Germany) coupled with a helium-neon (He-Ne) continuous 633 nm laser beam (35 mW @ 633 nm, Research Electro-optics, Inc., USA). The excitation laser beam was collimated into a 20× objective lens (NA = 0.85, N-Achroplan, Zeiss, Germany) for Raman excitation. Raman photons were collected by the same objective lens and transmitted through a holographic edge filter to a multi-mode optical fiber (50 μm diameter) to the spectrometer (UHTS300, WITec GmbH, Germany), which was equipped with a resolution about 3 cm⁻¹ over a spectrum range of 0–2400 cm⁻¹. The spectra were recorded using a back-illuminated, deep depletion CCD camera containing 600 × 200 pixels (Du401A-BR-DD-352, Andor Technology, UK) working at −60 °C. The spectral data were acquired point-by-point over each kind of skin lesion with 3 s integration time. Before the experiment, a standard tungsten lamp (RS-3, EG&G Gamma Scientific, USA) was used for calibrating the spectral response of the system, and the Raman spectrum of silicon (520 cm⁻¹) was measured to calibrate the wavelength position.

UV-Vis spectra of the RF-CPDs and RF-CPD/PU composites were recorded on a Unispec2 LLG spectrophotometer. The measurement range was from 200 to 700 nm. The photoluminescence (PL) spectra of the RF-CPDs, RF-CPD/PU, and RB-CPDs lifetime of all samples were recorded on a Fluorolog spectrofluorometer (Horiba, Kyoto, Japan). UV-Vis and PL measurements were conducted in air at room temperature. The distribution of RF-CPDs in the PU was studied using a confocal Raman microscope (Alpha300 R+, WITec, Ulm, Germany) equipped with a WITec UHTS300 spectrometer (600 lines/mm grating) coupled to an EMCCD camera (DU401A-BR-DD-352, Andor, Abingdon, UK). The sample was excited at a laser wavelength and optical power of 785 nm and 100 µW using a 50× magnification objective (EC Epiplan-Neofluar Dic, NA = 0.8, Zeiss, Oberkochen, Germany) and a 50 mm diameter optical fiber.

The confocal Raman microscopy system used in this study has been described previously 25 . Briefly, a fiber coupled to a 532-nm semiconductor laser was collimated into A 63× water-immersion objective lens (NA=1.0, W Plan-APOCHROMAT, Zeiss, Germany) for Raman excitation and spectral measurements. Cells seeded CaF₂ slides were placed on a multi-axis piezo scanning stage (P-524K081, PI GmbH, Germany) for point-by-point spectral scanning. The spectrum from every scanning point was recorded by a spectrometer (UHTS300, WITec GmbH, Germany) incorporating a 600 mm^-1 grating blazed at 500 nm and a back-illuminated deep-depletion charge coupled device camera (CCD) (Du401A-BR-DD-352, Andor Technology, UK) working at -60 ˚C. All measurements were performed at room temperature after system wavelength and spectral intensity calibration.
During experiment, the Raman spectra from UT, 10T and 20T groups were measured randomly from 20 spots inside the cell structure. A total of 12 cells were measured in all groups (UT group, 4 cells; 10T group, 4 cells; 20T group, 4 cells), 4 biological replicants were performed (UT group, 1 replicant; 10T group, 1 replicant; 20T, 2 replicants), and totally 60 Raman spectra were considered (UT group, 20 Raman spectra; 10T group, 20 Raman spectra; 20T group, 20 Raman spectra).