Fl920

Absorption measurements were performed with a Lambda 750 UV/VIS spectrometer (Perkin Elmer, Waltham, USA). To determine the absorption coefficients, absorption spectra in Ca²⁺-free and Ca²⁺-saturated buffer solutions were recorded. The dye concentration varied from 1.7 µM to 12.5 µM for ACR and from 0.9 µM to 1.8 µM for ACG. Fluorescence quantum yields of the Ca²⁺-saturated dye forms were determined absolutely with the C 9929 integration sphere system (Hamamatsu, Hamamatsu City, Japan). Since the fluorescence quantum yields of the Ca²⁺-free dye forms were below the detection limit of this system (Φ_F <0.01), fluorescence quantum yields of these dye forms were determined relative to the respective Ca²⁺-saturated form as a fluorescent reference [25] , [26] (link). Steady-state fluorescence spectra were recorded with FluoroMax 4 (Horiba, Kyoto, Japan). For time-resolved fluorescence measurements in the BSA-buffer, ACR was excited by a supercontinuum source (SC-400-PP, Fianium, Southhampton, UK) operating at λ_ex = 550 nm with a repetition rate of 20 MHz and a pulse width of ∼30 ps. The laser beam was fiber-guided towards the fluorescence lifetime spectrometer FL920 (Edinburgh Instruments, Edinburgh, UK), where the emitted fluorescence was detected by a multichannel plate (ELDY EM1-123/300, EuroPhoton, Berlin, Germany) in the time-correlated single photon counting (TCSPC) mode.

The fluorescence lifetimes of the mAb charge variants at a concentration of 0.8 mg ml⁻¹ were measured on a time-correlated single-photon counting (TCSPC) setup (FL920, Edinburgh Instruments, UK). The bevacizumab charge variants were excited with a wavelength of 280 nm using a light emitting diode, 200 ns FWHM with PPD and laser diode, and the respective decay profiles mAb charge variant were monitored at 337 nm at the magic angle (54.7 °C). A solution of colloidal silica (Ludox) was inserted into the buffer to acquire the instrument response function (IRF). The IRF was used to deconvolute the fluorescence time-resolved decay of the isolated charge variants. The decay times (Ʈ) were obtained by using Marquardt least-squares minimization and fitting a four-parameter fit to the decay profile of the charge variants. The decay function was assumed to be the sum of two exponentials given by the equation: $$F\left(t\right)=\sum _i{\propto }_{i}exp(t/{\tau }_i)$$ where F(t) is the experimental intensity decay, ${\propto }_{\mathit{i}}$ is the pre-exponential factor, and ${\mathit{\tau }}_{\mathit{i}}$ is fluorescence lifetime of the ith discrete component, both recovered upon fitting.
The fractional intensity (f_i) of each lifetime and the mean lifetimes < ${\mathit{\tau }}_{\mathit{i}}$ > are given by Eq. (3) and (4), as follows: $$f_i=\frac{{\propto }_i{\tau }_i}{{\sum }_i{\propto }_i{\tau }_i}$$ $$〈{\tau }_i〉=\sum _i{f}_i{\tau }_i$$

The PSi/C-dot hybrid's fluorescence emission spectra were recorded at different excitation wavelengths using a spectrofluorimeter (FL920, Edinburgh Instruments, UK). The emission spectra were measured at different excitation wavelengths ranging from 300 to 600 nm.

Powder X-ray diffraction measurements were conducted on a Philips diffractometer (X’pert pro MRD) using Cu Kα radiation. The SEM tests were performed by using a Zeiss Gemini SEM 300 field-emission scanning electron microscope. The PL spectra were measured using an Edinburgh FL920 with a Xenon lamp, with the excitation wavelength at 460 nm. TRPL at 810 nm was monitored under a 478 nm light pulse as excitation from the HORIBA Scientific Delta Pro fluorimeter. For the μτ, I–V, I–t measurements, the Keithley 6571B Source Meter was used to apply the bias voltage and record the response current. A 365 nm LED light (model M365L, Zolix, Beijing) was used as the excitation source for μτ measurement. The power of 365 nm light was 420 mW. A rotational viscometer (Model NDJ−5s, purchased from LICHEN, shanghai.) was used to test the viscosity of suspensions at 30 rpm.

TRFS was done using FL920 Edinburgh spectrofluorimeter which was equipped with a xenon arc lamp, a polarizing device and a 375-nm laser diode (NanoLED 375 L, Horiba Ltd.) operated with a pulse frequency of 1 MHz. During the experiment photobleaching was avoided by using extremely low power (6–7 μW).
Decays in nanosecond timescales of DPH were measured in time correlated single photon counting (TCSPC) setup (FL920, Edinburgh Instruments, UK). Samples were excited at 375 nm using picosecond diode laser (pulse width ~100 ps). Fluorescence were dispersed in a monochromator and then collected by a MCP-PMT detector. The time-resolution of TCSPC setup of ~100 ps was determined by measuring the Instrument Response Function (IRF) using LUDOX solution. DPH labeled cells were placed in a quartz cuvette. Fluorescence was emitted at 426 nm and the time 100 ns was split into 4096 channels. Decay was measured at magical angle for 5000 peak counts. The G factor was determined by measuring I_HH and I_HV between the ranges 400–600 nm at five repeats. The anisotropy decay was determined by convolving the I_VH and I_VV.