Krypton

For simultaneous mutagenesis at several positions of the mTagBFP gene the overlap-extension approach has been applied [19] (link). After mutagenesis a mixture of the mutants was electroporated into LMG194 host cells (Invitrogen)
Protein expression in the library was induced overnight at 37°C with 0.002% arabinose. Next morning, expressing bacteria were washed with PBS and then diluted with PBS for FACS sorting to optical density of 0.02 at 600 nm. MoFlo cell sorter (Dako) equipped with standard argon, krypton and argon-krypton mixed-gas lasers was used. To select the most photostable clones the library was illuminated before FACS using LED array at 405 nm for 15 min. About 10 sizes of the library were sorted on the FACS with 407 nm of krypton excitation line and 450/65 nm emission filter. The collected bright blue bacterial cells were rescued in rich SOC medium at 37°C for one hour, and then plated on Petri dishes with 0.02% arabinose. The next day, the dishes were analyzed with Leica MZ16F fluorescence stereomicroscope using a custom blue filter set (390/40 nm exciter, 460/40 nm emitter) from Chroma. To select the most photostable colonies, dishes were illuminated with 405 nm LEDs (80 mW/cm²) for 15 min, and images were acquired before and after illumination. For further analysis, 20 to 50 brightest and photostable clones were selected and analyzed using Olympus IX81 inverted microscope equipped with a 200 W metal-halide lamp (Prior), 100×1.4 NA oil objective lens (UPlanSApo, Olympus), and 390/40 nm excitation and 460/40 nm emission filters. At this stage photobleaching of the blue bacterial clones was checked. The best clones were applied for sequencing.

For the solution data, sampling and isotope analysis was done in a prior study and is detailed in Britton et al.^{23 (link)}. To summarise, the second and third molars (M₂ and M₃) were extracted from the mandibles, brush-cleaned with water and left to dry overnight. The whole teeth were mechanically abraded to remove surficial enamel. Sampling was done on the buccal face of the anterior loph as it presented a thicker enamel, with the face removed and cleaned from adhering dentine using a tungsten carbide burr. Enamel faces were then marked for horizontal sectioning at ∼ 1.5 mm intervals, ultrasonicated in deionize water (DI H₂O, 18.3 MΩ) and dried, before being cut into strips using diamond-coated superfine circular drill bits. Samples were then individually ultrasonicated in DI H₂O, dried and split longitudinally with ∼ 5 mg of enamel being reserved for ⁸⁷Sr/⁸⁶Sr solution analysis and the remainder being retained for carbon and oxygen isotope analysis. In that study, sections were numerically assigned from the ERJ to the OS (M₂-1, M₂-2, M₂-3,…).
Strontium was isolated from enamel in clean laboratory facilities at the Department of Human Evolution, Max Planck Institute for Evolutionary Anthropology (MPI-EVA), Leipzig, Germany using a modified version of the method from Deniel and Pin^{28 (link)} described in detail in Copeland et al.^{34 (link)}. The following description of the analytical procedure is reproduced from Britton et al.^{23 (link)}, and is also presented there in full. The ∼ 5 mg samples were dissolved in 1 ml 14.3 M high purity HNO₃ then evaporated to dryness. The obtained residue was then re-dissolved in 1 ml 3 M HNO₃ before loading into pre-conditioned columns containing Sr Resin (Eichrom Technologies, Lisle, IL, USA), being passed through three times. Strontium was then eluted using ultrapure deionized water (18.2 MΩ), dried and re-dissolved in 3% HNO₃ and analysis of ⁸⁷Sr/⁸⁶Sr ratios was undertaken using a Thermo Fisher Neptune™ (MC-ICP-MS). All the acids solutions used in the procedure were purified through a PicoTrace double-distilled sub-boiling distillation system. The subsequent ⁸⁷Sr/⁸⁶Sr measurements on standards and samples were corrected for interferences from krypton (Kr) and rubidium (Rb) and normalized for instrumental mass bias to ⁸⁸Sr/⁸⁶Sr = 8.375209 (exponential law). Analysis of the international strontium isotope standard NIST SRM987 (National Institute of Standards and Technology, Gaithersburg, USA) during each analytical session was used for external normalisation of data (long-term ⁸⁷Sr/⁸⁶Sr value = 0.710273 ± 0.000033 (2σ) (n = 97)). All ⁸⁷Sr/⁸⁶Sr values reported here were adjusted so SRM987 = 0.710240⁷², involving a data correction factor of − 0.00002. Strontium concentrations of the enamel samples were determined using the method described in^{34 (link)}, which is accurate to within ± 31 ppm.

The main configurations of the VUV lamp-based photoionization mass spectrometer in Hefei and the microwave discharge flow tube reactor have been introduced in detail in our recent publications and only a brief description is presented here [27 (link),28 (link),31 (link)]. Briefly, the microwave discharge flow tube reactor is used to generate radicals and to study the self-reaction of C₂H₅O₂ under NOx-free conditions. The flow tube mainly consists of a main tube (450 mm length, 16 mm inner diameter) and a movable coaxial inner tube (600 mm length, 4 mm inner diameter). The inner surface of the main tube and the outer surface of the inner tube are coated with halocarbon wax to minimize the wall loss of the radicals. F₂ diluted to 5% in helium is discharged with a 2.45 GHz microwave discharge generator (GMS-200W, Sariem, France) to produce fluorine atoms and then to initiate the reactions. The pressure inside the flow tube is measured by a diaphragm vacuum gauge and precisely controlled by a closed-loop feedback throttle valve. In the present experiments, the pressure of the flow tube is fixed at 266 Pa. The total gas flux into the flow tube is 500 cm³ min⁻¹ and the reactants’ initial concentrations are 3.7 × 10¹³ molecules·cm⁻³ for F atoms, 3.1 × 10¹⁴ molecules·cm⁻³ for C₂H₆ and 2.6 × 10¹⁵ molecules·cm⁻³ for O₂, respectively.
A home-made VUV photoionization mass spectrometer is employed to online probe the intermediates and stabilized products inside the flow tube [28 (link)]. The photoionization mass spectrometer is composed of three vacuum chambers: a source chamber, an ionization chamber and a TOF chamber. The flow tube is connected with the source chamber directly, and a 1 mm diameter skimmer is adopted to sample the species inside the flow tube. Then, the species are photo-ionized with a krypton discharge lamp (PKS 106, Heraeus, Germany, hν = 10.0 and 10.6 eV) and the ions are analyzed with an orthogonal acceleration reflectron time-of-flight mass analyzer. The mass resolution of the VUV photoionization mass spectrometer is M/∆M = 2100 (FWHM, full width at half maximum). Very recently, the VUV photoionization mass spectrometer has been upgraded in some content to achieve a better detection limit, LOD < 0.001 µg/L [28 (link)]. In the kinetic experiments, the reaction time is varied by changing the distance between the inner tube and the skimmer.
The synchrotron photoionization experiments are performed at the VUV beamline at the Swiss Light Source. The detailed configurations of the synchrotron beamline and the i²PEPICO setup can be found in the literature [29 (link),30 (link)]. A 10 Hz pulsed Nd-YAG (213 nm, 16 mJ cm⁻²) laser is used to generate chlorine atoms through the photolysis of oxalylchloride (COCl)₂, which initiates the radical reactions in a side-sampled flow reactor. The reactor is a 57.4 cm long quartz tube with a 1.27 cm outer diameter, 1.05 cm inner diameter, and a 300 µm pinhole at the halfway-point along the tube. Ethane (0.03 sccm) and oxygen (75 sccm), as well as Ar carrier gas (15 sccm), are also added into the flow reactor (6 mbar) to produce ethyl peroxy radicals and the dimeric products C₂H₅OOC₂H₅. Photoelectrons and photoions are velocity map imaged onto two position-sensitive delay-line detectors, respectively, and detected in delayed coincidences [46 (link)]. In the present experiments, due to the weak signal, only the photoionization spectrum of C₂H₅OOC₂H₅ is acquired and presented here.
High-level theoretical calculations, consisting of the determinations of the structures of the neutral and ionic species of C₂H₅OOC₂H₅, the corresponding AIE, the Franck-Condon factors involved in the ionization and the reaction potential energy surfaces, have been performed. Briefly, the structures of the dimeric product C₂H₅OOC₂H₅ and its cation C₂H₅OOC₂H₅⁺ have been fully optimized at the explicitly correlated coupled cluster single-double and perturbative triple excitations approach, CCSD(T)-F12, in conjunction with the aug-cc-pVTZ basis set as implemented in the MOLPRO 2015 program [44 ]. Subsequently, the AIE of C₂H₅OOC₂H₅ has been theoretically calculated at the same level of theory. The Franck-Condon factors for the ionization transitions are calculated at the M062X/aug-cc-pVTZ level of theory using the time-independent adiabatic Hessian Franck-Condon model in the Gaussian 16 package. The reaction potential energy surfaces (PES) for the C₂H₅O₂ self-reaction, as well as the optimization of the structures of the reactants, intermediates, transition state and products are also calculated at the M06-2X/aug-cc-pVTZ level of theory with the Gaussian 16 package [45 ].

For the bacterial reduction experiments, one of the authors irradiated the inner surface of his hands five times with and without gloves. In each case, both hands or gloves (models “nitrile white” from VWR (Radnor, PA, USA)) were first rubbed over the face and upper body to ensure a high concentration of microorganisms even on the sterile gloves. Subsequently, both hands were rubbed together to achieve an even distribution of microorganisms on both hands and gloves.
A filtered 222 nm far-UVC krypton-chloride excimer lamp from Ushio (Tokyo, Japan) was used as the radiation source. First, the distance from the lamp to the plane, where the irradiance was 1 mW/cm², was determined with a photometer type X1-UV-3727 of Gigahertz-Optik (Tuerkenfeld, Germany). Then one contaminated hand was irradiated for 23 s to reach the dose of 23 mJ/cm² (without gloves) and 100 s to reach the dose of 100 mJ/cm² (with gloves). A maximum of one skin irradiation was performed per day to stay within the allowed limits in the EU. Three fingers of each of the irradiated and non-irradiated hands/gloves were pressed onto caso contact agar plates from VWR. The agar plates were then incubated for approximately 24 h at 37 °C. Afterwards, colonies were counted, and the ratio of colony numbers was determined.
For comparison, a similar disinfection study with contaminated gloves and fingers was performed with the commercial liquid alcohol-based hand disinfectant Sterilium of Bode Chemie (Hamburg, Germany). The application of the disinfectant was performed for 30 s according to WHO instructions [22 ], and with a following 2 min drying and waiting period before sampling.
In addition, the far-UVC transmission properties of some standard gloves were determined. First, pieces were first cut out of three different gloves. Among these glove models were the white nitrile gloves from VWR mentioned above, and also the “nitrile blue” models from Semperit (Vienna, Austria) and “latex white” from Braun (Schwalbach, Germany). Subsequently, the Gigahertz photometer was positioned in close proximity to the far-UVC lamp, and the irradiance on the detector was determined with and without a glove layer.