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Lp920

Manufactured by Edinburgh Instruments
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The LP920 is a transient absorption spectrometer that measures the absorption and emission of excited molecules or materials over time. It is capable of measuring transient absorption and luminescence signals with a time resolution down to the nanosecond scale.

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Quanta ray, by Spectra-Physics (2 mentions) Iccd camera, by Oxford Instruments (1 mentions) Originpro 9, by OriginLab (1 mentions) Originpro 2019, by OriginLab (1 mentions) Uv 2450, by Shimadzu (1 mentions)

10 protocols using lp920

1

Nanosecond Transient Absorption Spectroscopy

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Nanosecond transient absorption difference spectra and kinetics were measured using an Edinburgh Instruments LP-920 laser flash photolysis system equipped with a pulsed 450 W Xe arc lamp and an iStar iCCD Camera (Andor) serving as the detector at the exit port of a spectrograph. The laser excitation pump source was a Vibrant LD 355 II ND: YAG/OPO system from OPOTEK. The differential transient absorption spectra were recorded in deaerated THF following pulsed nanosecond laser excitation (λex = 514 nm, 2 mJ per pulse, 5–7 ns fwhm). The optical density in each sample was maintained between 0.3–0.5 OD at the excitation wavelength. Single wavelength kinetic traces were measured by using a monochromator and PMT detector (R-928 Hamamatsu), with data acquisition controlled by the Edinburgh Instruments LP 900 software. The single wavelength absorbance transients of the Zr(iv) photosensitizer were single exponential in all instances. The recorded single wavelength kinetic decays of each triplet acceptor/annihilator following triplet sensitization were adequately fit to a model equation following parallel first-and second-order kinetics22,59 in Igor Pro 7.08 software. Please see the model equations (eqn (S1)–(S3)†) for more details.
Yang M., Sheykhi S., Zhang Y., Milsmann C, & Castellano F.N. (2021). Low power threshold photochemical upconversion using a zirconium(iv) LMCT photosensitizer. Chemical Science, 12(26), 9069-9077.
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2

Photophysical Characterization of Iridium(III) Complexes

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Spectrophotometric grade solvents were purchased from Alfa Aesar. The UV–vis absorption spectra of complexes Ir1 – Ir5 were collected on a Varian Cary 50 spectrophotometer, and steady-state emission measurements were carried out using a HORIBA FluoroMax 4 fluorometer/phosphorometer. The emission quantum yields of complexes Ir1 – Ir5 in argon-sparged CH3CN solution were determined by the relative actinometry method55 using [Ru(bpy)3]Cl2 (Φem = 0.097, λex = 436 nm)56 as the standard. The nanosecond transient absorption (TA) spectra and decays, triplet excited-state quantum yields, and triplet lifetimes were collected on argon-sparged (40 min) acetonitrile solutions on an Edinburgh LP920 laser flash photolysis spectrometer using the third harmonic output (355 nm) of a Nd:YAG laser (Quantel Brilliant, pulsewidth ~4.1 ns, repetition rate was set to 1 Hz). Molar extinction coefficients (εT1-Tn) for triplet excited states were determined by the singlet depletion method57 at the TA band maxima, and quantum yields for triplet excited state formation were measured according to the relative actinometry method58 using the εT1-Tn values, with SiNc in benzene as the reference (ε590 = 70,000 M−1cm−1, ΦT = 0.20).59
Liu B., Monro S., Lystrom L., Cameron C.G., Colon K., Yin H., Kilina S., McFarland S.A, & Sun W. (2018). Photophysical and Photobiological Properties of Dinuclear Iridium(III) Bis-tridentate Complexes. Inorganic chemistry, 57(16), 9859-9872.
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3

Kinetic Characterization of NOS-CaM Interaction

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The kinetics experiments were performed on an Edinburgh LP920 laser flash photolysis spectrometer, in combination with a Q-switched Continuum Surelite I-10 Nd:YAG laser and a Continuum Surelite OPO [25 (link)]. [CaM] is 2.3-fold of [nNOS] in all the IET experiments because two CaM molecules binds to one dimeric NOS molecule. Human Hsp90α protein was also added when necessary. The laser flash photolysis experiments were conducted at least twice on separate dates, and the averages of the observed IET rates are listed in tables.
Zheng H., Li J, & Feng C. (2020). Enhancement of the FMN–heme interdomain electron transfer in neuronal nitric oxide synthase by heat shock protein 90. FEBS letters, 594(17), 2904-2913.
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4

Laser Flash Photolysis of C12-PN-AzPyC2H5+ Compound

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The relaxation rate of the N-ethyl cis-azopyridinium moiety in C12-PN-AzPyC2H5+ (12K) was measured at rt with a
laser flash photolysis system
(LP 920, Edinburgh Instruments). The laser wavelength was 355 nm.
The pulse time was 10 ns with an energy of 10 mJ/pulse. The detection
wavelength was 365 nm. The data were fit with the exponential function
defined in eq 7 where ΔOD0 is the initial
optical density, τ is the relaxation time, and ΔOD is
the time-dependent optical density.
Ren H., Qiu X.P., Shi Y., Yang P, & Winnik F.M. (2019). pH-Dependent Morphology and Photoresponse of Azopyridine-Terminated Poly(N-isopropylacrylamide) Nanoparticles in Water. Macromolecules, 52(8), 2939-2948.
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5

Laser Flash Photolysis of CO-Ferrous Hemoglobin

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Laser flash photolysis (LFP) experiments were performed at 20°C using a laser photolysis system (Edinburgh Instruments LP920, UK) equipped with a frequency-doubled, Q-switched Nd:YAG laser (Quanta-Ray, Spectra Physics). The carbon monoxide (CO)–ferrous Hb complexes were prepared in sealed 4 × 10 mm quartz cuvettes with 1 ml of 100 mM potassium phosphate buffer (pH 7.0) containing 1 mM EDTA. In the case of LjGlb1-1 this buffer was supplemented with 200 mM NaCl to improve protein stability. The buffer was equilibrated with mixtures of CO and N2 in different ratios to obtain CO concentrations of 50–800 μM by using a gas mixer (High-Tech System; Bronkhorst, The Netherlands). Saturated sodium dithionite solution (10 μl) was added and the protein was injected to a final concentration of ∼4 μM. Formation of the CO–ferrous Hb complex was verified by UV/visible absorption spectroscopy. Recombination of the photo-dissociated CO-ligand was monitored at 417 nm.
Calvo-Begueria L., Cuypers B., Van Doorslaer S., Abbruzzetti S., Bruno S., Berghmans H., Dewilde S., Ramos J., Viappiani C, & Becana M. (2017). Characterization of the Heme Pocket Structure and Ligand Binding Kinetics of Non-symbiotic Hemoglobins from the Model Legume Lotus japonicus. Frontiers in Plant Science, 8, 407.
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6

Laser Photolysis of MaPgb*(II)-CO Mutants

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For laser photolysis, samples of MaPgb*(II)-CO mutants were prepared in a sealed 2 mm by 10 mm quartz cuvette. The ferric MaPgb* (MaPgb*(III)) mutant stock solutions were diluted with 100 mM phosphate buffer at pH 7.0 to a final concentration of 50 μM, equilibrated in either 1.0 atm or 0.1 atm CO and anaerobically reduced with sodium dithionite (2.0 mM). The oxidation state, the molar fraction of the protein-bound CO binding, and the heme concentration were determined spectrophotometrically.
Nanosecond flash-photolysis experiments and time-resolved difference absorbance spectra were measured as described [10 (link),12 (link)] or with a laser photolysis system (Edinburgh Instruments LP920, Livingston, UK) using a frequency-doubled Q-switched Nd:YAG laser (Spectra Physics Quanta-ray, Newport, CA) at 532 nm.
Tilleman L., Abbruzzetti S., Ciaccio C., De Sanctis G., Nardini M., Pesce A., Desmet F., Moens L., Van Doorslaer S., Bruno S., Bolognesi M., Ascenzi P., Coletta M., Viappiani C, & Dewilde S. (2015). Structural Bases for the Regulation of CO Binding in the Archaeal Protoglobin from Methanosarcina acetivorans. PLoS ONE, 10(6), e0125959.
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7

Laser-Based Kinetic Measurements of FMN-Heme IET

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The FMN – heme interdomain electron transfer kinetic measurements were conducted on an Edinburgh LP920 laser flash photolysis spectrometer, in combination with a Q-switched Continuum Surelite I-10 Nd:YAG laser and a Continuum Surelite OPO. The CO photolysis experiments were performed as described [20 (link)]. Briefly, sample contained ~ 20 μM dRF and 5 mM fresh semicarbazide in a pH 7.6 buffer (40 mM bis-Tris propane, 400 mM NaCl, 2 mM l-Arg, 20 μM H4B, 1 mM Ca2+ and 10 % glycerol). Sample without protein in a 1 cm cuvette sealed with a rubber septum was deaerated by bubbling with mixed CO/Ar (v/v 1:3) gas for 1 h. The gas was blown over the sample surface to remove traces of O2 added upon subsequent introduction of nNOS protein aliquots to the sample. The pre-degassed sample was illuminated for a certain period of time to obtain a partially reduced form of [Fe(II)–CO][FMNH•]. The sample was then flashed with 446 nm laser excitation to trigger the FMN – heme IET, which can be followed by the loss of absorbance of Fe2+ at 460 nm [21 (link)]. The experiments were conducted at least twice. The transient absorbance changes were averaged and analyzed using OriginPro 9.0 (OriginLab, Northampton, MA, USA).
Zheng H., He J., Li J., Yang J., Kirk M.L., Roman L.J, & Feng C. (2018). Generation and Characterization of Functional Phosphoserine Incorporated Neuronal Nitric Oxide Synthase Holoenzyme. Journal of biological inorganic chemistry : JBIC : a publication of the Society of Biological Inorganic Chemistry, 24(1), 1-9.
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8

Laser Flash Photolysis of Deazariboflavin

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Laser flash photolysis experiments were conducted at 21 °C on an Edinburgh LP920 laser flash photolysis spectrometer, as previously described.40 (link) Briefly, sample contained ~ 20 μM deazariboflavin (dRF) and 5 mM semicarbazide in a pH 7.6 buffer (40 mM bis-Tris propane, 400 mM NaCl, 1 mM Ca2+, 2 mM l-Arg, 20 μM H4Band 10% glycerol). The crowder (Ficoll 70 or Dextran 70) was added to the solution when necessary. The dRF solution without the protein in a cuvette was vigorously deaerated by a CO/Ar (v/v 1:3) mixed gas for ~ 90 minutes. The surface of introduced protein aliquot was then purged by the gas to remove traces of O2 before it was mixed into the dRF solution. The sample was illuminated for 3 – 4 minutes by white light to obtain the [Fe(II)−CO][FMNH•] form. The protein sample was subsequently flashed with 446 nm laser excitation to initiate the FMN ‒ heme IET, which can be monitored by the decrease of absorption of FMNH· and Fe(II) at 580 nm and 465 nm, respectively.41 (link) The CO photolysis experiments were conducted at least twice for one sample. The averaged kinetic traces were analyzed by OriginPro 2019 (OriginLab, MA).
Li J., Zheng H, & Feng C. (2019). The effect of macromolecular crowding on the FMN − heme intraprotein electron transfer in inducible NO synthase. Biochemistry, 58(28), 3087-3096.
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9

Measuring CO and O2 Binding Kinetics

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Laser flash photolysis experiments were performed to measure the CO and O 2 binding kinetics at 20 °C using a laser photolysis system (Edinburgh Instruments LP920, UK) equipped with a frequency-doubled, Q-switched Nd:YAG laser (Quanta-Ray, Spectra Physics). A laser flash of 532 nm was used.
The carbon monoxide (CO)-ferrous Hb complexes were prepared in sealed 4 × 10 mm quartz cuvettes with 1 ml of 100 mM potassium phosphate buffer (pH 7.0) containing 1 mM EDTA. The buffer was equilibrated with mixtures of CO and N 2 in different ratios to obtain CO concentrations of 50-800 µM by using a gas mixer (HighTech System; Downloaded by SYRACUSE UNIVERSITY from www.liebertpub.com at 10/21/19. For personal use only.
Germani F., Nardini M., De Schutter A., Cuypers B., Berghmans H., Van Hauwaert M.L., Bruno S., Mozzarelli A., Moens L., Van Doorslaer S., Bolognesi M., Pesce A, & Dewilde S. (2020). Structural and Functional Characterization of the Globin-Coupled Sensors of Azotobacter vinelandii and Bordetella pertussis. Antioxidants & redox signaling, 32(6).
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10

Spectroscopic Characterization of Materials

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Edinburgh Instruments model LP920 equipped with a pulsed Xe probe lamp was used in conjunction with a Nd:Yag laser pumped tunable optical parametric oscillator (Continuum model Surelite II and Surelite OPO Plus) as excitation source. The laser pulse width was 8 ns, and measurements were conducted either at 1 Hz or 10 Hz repetition rate. Samples were pressed into self-supporting wafers with 18 mg of material under 10 ton of force in a 13 mm die held under vacuum or Ar atmosphere in a quartz cuvette at 45 degree angle relative to perpendicular pump and probe beams.
Vibrational and optical spectroscopy: Samples for FT-IR and UV-vis transmission spectroscopy were placed in a home-built miniature stainless steel vacuum cell equipped with CaF 2 or KBr windows and evacuated for 2 hours. [1] Infrared spectra were recorded at 2 cm -1 resolution with Bruker spectrometers model Vector 33, IFS66V, or Vertex 80V equipped with liquid nitrogen cooled MCT detectors. FT-Raman spectra of powders were measured with the FRA-106 Raman accessory of the IFS66V spectrometer at 2 cm -1 resolution (1064 nm Nd:Yag emission, 120 mW). Optical absorption spectra of pressed wafers were recorded with a Shimadzu spectrometer model UV-2450.
Katsoukis G, & Frei H. (2018). Heterobinuclear Light Absorber Coupled to Molecular Wire for Charge Transport across Ultrathin Silica Membrane for Artificial Photosynthesis. ACS applied materials & interfaces, 10(37).
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