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13 protocols using fls1000 spectrofluorometer

1

Fluorescence Spectroscopy of Molecular Rotors

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2-(4-Dimethylamino)styryl)-1-methylpyridinium
iodide (DASPMI) was purchased from Molecular Probes Inc., Life Technologies
and used without further purification. 4,4′-Difluoro-4bora-3a,4a-diaza-s-indacene meso-substituted with para-dodecylphenyl moiety
(BPC12) was synthesized in our group according to the literature methods.46 (link) Gibco Dulbecco’s phosphate-buffered saline
(DPBS) pH 7.25 was purchased from Thermo Fisher Scientific. Steady-state
and time-resolved fluorescence of the molecular rotors DASPMI and
BPC12 were obtained using a FLS-1000 spectrofluorometer (Edinburgh
Instruments) equipped with a thermocontrolled cuvette holder. Fluorescence
intensity decay curves were measured using time-correlated single
photon counting (TCSPC) system (PicoQuant, GmBH) described earlier.24 (link) Viscosities were calculated at 5, 12, 22, and
37 °C by eq S1.
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2

Acridine-Modified ODN Probe Characterization

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Acridine-modified ODN probes (L_1_2*, L_7_2* and S_1_2*) were tested on FLS1000 spectrofluorometer (Edinburgh Instruments, Edinburgh, UK). A 1400-μl fluorescence quartz cuvette was used for determination of melting temperature. PCR buffer with ODN labelled with BHQ-1 and the probe modified by acridine (1000 μl, 1 μM of both ODN strands) was transferred to the cuvette. The mixture was heated at 95°C for 3 min and then allowed to cool slowly to room temperature for 10 min. The cuvette was placed in a holder tempered to 20°C (for S probes) or 40°C (for L probes) and fluorescence at λem = 500 nm was measured after 90 seconds of stabilisation (λex = 400 nm). Then, the temperature was slowly raised by increments of 0.5°C until it reached 60°C (for S probe) or 80°C (for L probes). Fluorescence was measured 90 seconds after every 0.5°C increment of the temperature was reached. Final values of fluorescence were plotted against temperature to get melting curves. Prism 9 (GraphPad) software was used for melting peak analysis. All experiments were performed in triplicate.
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3

Temperature-dependent Emission Spectroscopy

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Temperature maps of emission spectra (420–700 nm) were measured on FLS1000 spectrofluorometer (Edinburgh Instruments). A 1400-μl fluorescence quartz cuvette was used for measurements of temperature maps. TL (1 μl of 1 mM stock solution) was added to the solution of L_7_2* (1 μM) in PCR buffer (1000 μl). The mixtures were heated at 95°C for 3 min and then allowed to cool slowly to room temperature for 10 min. The cuvette was placed in a holder cooled to 40°C, and emission spectra (420–700 nm) were measured after 90 s of stabilisation (λex = 400 nm). Then, the temperature was raised by increments of 1.0°C until it reached 80°C. Emission spectra were measured 90 s after every 1.0°C increment of the temperature was reached.
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4

Detailed Characterization of Doped Upconversion Nanoparticles

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Transmission electron microscopy (TEM) images were collected using a JEM-1400 Plus (JEOL, Japan) operating at 120 kV. Element mapping analyses were performed with a Talos F200X (FEI Company, USA) equipped with a Chemi STEM EDS detector operating at 200 kV. X-ray diffraction (XRD) pattern was measured on a Bruker D8 Advance diffractometer (Bruker, Germany) with Cu Kα radiation (λ = 1.5406 Å) from 10° to 70° at a step of 0.02°/s. The luminescence spectra were obtained by a Horiba Fluoromax Plus fluorescence spectrometer, coupled with an 808 nm continuous-wave laser (CNI MDL-III-808, China). The absorption spectra of Nd3+ doped DSNPs, Yb3+ doped DSNPs, and whole blood were measured by a Persee Tu-1810 spectrophotometer (Beijing Persee Co. Ltd., China). The visible luminescence photographs were collected using a Canon EOS 60D SLR camera. The NIR luminescence photographs were taken with a C-RED 2 InGaAs camera (First Light Imaging). The NIR luminescence lifetime were measured with an Edinburgh FLS-1000 spectrofluorometer equipped with a liquid-nitrogen-cooled near-infrared photomultiplier tube (NIR-PMT), and the 808 nm excitation laser (CNI MDL-III-808, China) was modulated by an electronic pulse modulator to generate excitation pulse.
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5

Comprehensive Characterization of Novel Materials

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Powder X-ray diffraction (PXRD) data were collected on an UltimaIV diffractometer from Japan Tokyo. The single-crystal X-ray diffraction (SC-XRD) data collection was carried out on a Bruker AXS D8-VENTURE diffractometer (Mo Kα radiation, λ = 0.71073 Å) (Karlsruhe, Germany). Morphologies were observed using a scanning electron microscope (SEM, JSM-7610F, Tokyo, Japan). Thermogravimetric analysis (TGA) was performed using STA449F5 (Netzsch, Selb Germany). An F-4700 fluorescence spectrophotometer (HITACHI, Tokyo, Japan) was used for the collection of luminescence and excitation spectra. Decay curves and PL quantum yields were collected on an FLS 1000 spectrofluorometer (Edinburgh Instruments, Edinburgh, UK). The ultraviolet-visible (UV–vis) absorption spectra were collected on a UV 2600 (Tianmei, Shanghai, China) spectrophotometer.
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6

Optical Characterization of CABI Films

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UV–visible absorption spectra of
the CABI and CABI-xBr films were collected on a Shimadzu
UV-1800 absorption spectrometer (Shimadzu Corporation, Japan). An
FLS1000 spectrofluorometer (Edinburgh Instruments, UK) was employed
to measure steady-state PL spectra of the films. TCSPC measurements
to obtain the TRPL decays were performed using a Picoharp 300 controller
and a PDL 800-B driver for laser excitation. The excitation repetition
rate was 200 kHz and controlled with a Tektronix function generator.
A Hamamatsu R3809U-50 microchannel plate photomultiplier was used
for detection in a 90° configuration.
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7

Photoluminescence Characterization of GdVO4:Eu3+

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Emission spectra
and time-dependent PL intensity were measured with an Edinburgh FLS1000
spectrofluorometer under an excitation of λex = 276
nm. Time-dependent PL measurements were registered for the most intense
Eu3+ emission band at 620 nm. Absolute photoluminescence
quantum yield (PLQY) measurements were performed in an integrating
sphere using FLS1000. Our films were excited at 285 nm, and the emission
and scattering peaks measured in the integrating sphere in the spectral
range comprised between 270 and 850 nm. In addition, the scattering
and emission peaks of a scattering sample were also measured to serve
as a reference. Spatial-resolved microscopic PL measurements were
obtained using a confocal optical microscope (Leica Stellaris 8) using
an oil immersion objective and 465 nm laser light as excitation source.
Samples were illuminated through a glass coverslip employed as the
substrate. Spatial resolution is diffraction limited (ca. 250 nm ×
250 nm). The step size of the scanning is 70 nm. Emitted photons were
collected in the wavelength range comprised between 606 and 636 nm
that corresponds to the main emission band of GdVO4:Eu3+. Background measurements were taken in the wavelength range
comprised between 780 and 810 nm.
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8

Comprehensive Characterization of Novel Materials

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Powder X-ray diffraction (PXRD) data were collected on an UltimaIV diffractometer from Japan Tokyo. The single-crystal X-ray diffraction (SC-XRD) data collection was carried out on a Bruker AXS D8-VENTURE diffractometer (Mo Kα radiation, λ = 0.71073 Å) (Karlsruhe, Germany). Morphologies were observed using a scanning electron microscope (SEM, JSM-7610F, Tokyo, Japan). Thermogravimetric analysis (TGA) was performed using STA449F5 (Netzsch, Selb Germany). An F-4700 fluorescence spectrophotometer (HITACHI, Tokyo, Japan) was used for the collection of luminescence and excitation spectra. Decay curves and PL quantum yields were collected on an FLS 1000 spectrofluorometer (Edinburgh Instruments, Edinburgh, UK). The ultraviolet-visible (UV–vis) absorption spectra were collected on a UV 2600 (Tianmei, Shanghai, China) spectrophotometer.
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9

Characterization of Photophysical Properties

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UV–vis absorption spectra
were recorded on a Cary 50 Scan UV–vis spectrophotometer (Varian).
Fluorescence measurements were performed on an FLS1000 spectrofluorometer
(Edinburgh Instruments, U.K.). The femtosecond and nanosecond transient
absorption (fsTA and nsTA) setups were described elsewhere.33 (link) In short, we used 355 nm, a third harmonic of
Nd:YAG (adjusted to 1–4 mJ), and 500 nm (output from an optical
parametric amplifier, adjusted to 0.5 μJ) as an excitation pump
for nsTA and fsTA, respectively. The samples for nsTA measurements
were degassed by three to five freeze–pump–thaw cycles.
Quantum yields of triplets were measured using a relative actinometry
method compared with the formation of triplets of benzophenone in
acetonitrile (MeCN) (ΦT = 1.0).41 (link) Data sets were analyzed using OriginPro 2017 (OriginLab
Corporation).
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10

Temperature-Dependent Luminescence Spectroscopy

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Excitation spectra were collected by using an Edinburgh FLS1000 spectrofluorometer (Edinburgh Instruments Ltd) equipped with a continuous xenon lamp (450 W) as an excitation source; the optimal excitation wavelengths are 360 nm (Eu-NH2-BDC), 394 nm (Eu-OH-BDC), and 394 nm (Eu-NDC), respectively. The temperature-dependent solid state luminescence spectra were obtained on the same spectrophotometer at controlled variable temperature (80–400 K with an interval of 10 K) and were recorded with an Oxford Instruments liquid nitrogen cryostat accessory. The time decay curves were measured under the same conditions, with a temperature range of 100–300 K. The average lifetime was obtained from the double-exponential decays according to equation:where τ is the decay time of luminescence intensity and Ai is the amplitude of each component.
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