Neuroinflammation is a devastating predisposing factor for Alzheimer’s disease (AD). A number of clinical findings have reported peripheral disorders among AD patients. Amyloid beta (Aβ) is a toxic physiological aggregate that serves as a triggering factor for hepatic and cardiac disorders related to neurotoxicity. As a drawback of Aβ excessive accumulation in the brain, part of Aβ is believed to readily cross the blood–brain barrier (BBB) into the peripheral circulation resulting in serious inflammatory and toxic cascades acting as a direct bridge to cardiac and hepatic pathophysiology. The main aim is to find out whether neuroinflammation-related AD may result in cardiac and liver dysfunctions. Potential therapeutic interventions are also suggested to alleviate AD’s cardiac and hepatic defects. Male rats were divided into: control group I, lipopolysaccharide (LPS)-neuroinflammatory-induced group II, LPS-neuroinflammatory-induced group treated with sodium hydrogen sulphide donor (NaHS) (group III), and LPS-neuroinflammatory-induced group treated with mesenchymal stem cells (MSCs) (group IV). Behavior and histopathological studies were conducted in addition to the estimation of different biological biomarkers. It was revealed that the increased toxic Aβ level in blood resulted in cardiac and hepatic malfunctions as a drawback of exaggerated inflammatory cascades. The administration of NaHS and MSCs proved their efficiency in combating neuroinflammatory drawbacks by hindering cardiac and hepatic dysfunctions. The consistent direct association of decreased heart and liver functions with increased Aβ levels highlights the direct involvement of AD in other organ complications. Thereby, these findings will open new avenues for combating neuroinflammatory-related AD and long-term asymptomatic toxicity. Alzheimer’s disease (AD) is one of the most common types of aging-related neurological disorders that manifests in the form of personal behavior changes, irritability, memory loss, and cognitive dysfunctions  is a severe disease category that centers around impairments arising within the heart and blood vessels reducing heart functional capacity  is a type of novel gaseous endogenous signaling molecule mainly synthesized from L-cysteine by the action of both cystathionine γ-lyase (CSE) and cystathionine β-synthase (CBS) mammalian enzymes tissue types  may act as a potent antioxidant, anti-inflammatory, and anti-apoptotic mediator , are adult stem cells type capable of continuous self-renewal and tissue differentiation. They are mainly found in the bone marrow (BM) and can also be identified in several tissues including muscles, adipose tissue, peripheral blood, and the umbilical cord  donor known as sodium hydrogen sulphide (NaHS) were used to find out their hindering effect on cardiac injury and liver dysfunction related to AD.In this study, we aim to highlight the effect of neuroinflammatory AD type progression on cardiac and liver functions. Thereby, to achieve the goal of illustrating the detailed mechanism of neuroinflammatory-related AD pathogenesis and mechanism of impairments, Lipopolysaccharide (LPS) was directly used to induce AD neuroinflammatory type in male albino rats. Following the induction of AD and hallmarks depositions in the brain, cardiac and hepatic biological parameters were assessed in correlation with Aβ level in the blood. Additionally, a suggested therapeutic intervention using mesenchymal stem cells (MSCs) and exogenous hydrogen sulfide (H2S were both directly purchased from Sigma Aldrich (Merck KGaA).Lipopolysaccharide (LPS) extracted from Escherichia Coli 0111:B4 and sodium hydrogen sulfide (NaHS) as an exogenous donor type of H2 incubator at 37 °C. Obtained large colonies cells were repeatedly washed with PBS supplemented with trypsin (0.25%) for 4–5 min at 37 followed by direct centrifugation at 3000 rpm for 15 min . Bone marrow MSCs were directly placed in a sterilized medium containing the Dulbecco's modified Eagle's medium (DMEM) and 10% fetal bovine serum (FBS). Isolated cells were resuspended in a new culture medium containing 2% penicillin–streptomycin following isolation by density gradient. For 14 days, isolated cells were incubated in a 5% humidified COn = 6), LPS-neuroinflammatory-induced rats : where rats were intraperitoneally (IP) injected with a single dose of LPS (4 mg/kg) dissolved in o.5 ml PBS : where LPS-induced neuroinflammatory rats received NaHS as an exogenous hydrogen sulphide donor (IP) as one type of therapeutic intervention for hepatic and cardiac disorders related to AD at a single daily dose of 5 mg/kg reconstituted with deionized water for 20 days with slight dose modification , where LPS-induced neuroinflammatory rats received a single intravenous dose (I.V) of isolated MSCs 1 μL (500 × 103/μL) as a second therapeutic intervention type for hepatic-cardiac disorders related to AD  approval number (NODCAR/II/57/2022) following the 3Rs main principles . Twenty-four male Albino rats weighing from 150 to 180 gm (5–6 weeks old) were directly purchased from the animal facility house of . Rats were housed and kept maintained at a controlled temperature with free access to food and water (22 °C/12-h light/dark cycle). Whereas, the chosen dose for LPS induction assigned for each group of rats was highly considered for affecting the brain only and inducing Aβ hallmarks deposition to avoid confounding effects and to prove out the aims of the current study. The key findings of one of the published research papers proved out that circulating LPS is very rapidly cleared from the systemic circulation, with an approximate half-life of around 2–4 min, and a 5 min clearance rate (80%) following direct infusion . A metal platform was submerged one inch under the opaque water and placed in the middle of the required target quadrant. The training was performed on 5 consecutive days where each trial was meant to not exceed 60 s. A final trial was conducted where the time taken by each rat to reach the submerged platform was directly calculated and recorded . Meanwhile, Serum Aβ level was detected among all groups. Since the main aim of the conducted study is to highlight the effect of neuroinflammation-related AD progression on the functionality of other organs including the heart and liver, it was vitally important to detect Aβ, and tau protein in LPS-induced neuroinflammatory groups in correlation with other cardiac and hepatic biological parameters. One the other hand, the main goal of the suggested therapeutic intervention represented in the treatment groups was to hinder the neuroinflammatory AD damaging effects on the heart and liver in correlation with Aβ serum level and the degree of hallmarks depositions.To detect the effect of neuroinflammatory AD progression on the heart and liver, several parameters were essentially required to be detected using the colorimetric method including: ammonia (Mybiosourse), lactate dehydrogenase (LDH), Creatine kinase-MB (CK-MB), total protein (TP), albumin, alkaline phosphatase (ALP), Aspartate aminotransferase (AST), Alanine transaminase (ALT) cholesterol and triglycerides (Biomed diagnostics). Meanwhile, troponin I, tumor necrosis factor-alpha (TNF-α), caspase-3, and interleukin 6 (IL-6) were detected using enzyme-linked immunosorbent assays (ELISA) kits purchased from (Mybiosourse) according to manufacturer instructions.Brain, heart, and liver were isolated from all groups and were directly fixed in 10% formalin followed by being embedded in paraffin blocks. 3–4 micron thickness were obtained and processed for Hematoxylin and Eosin (H&E) staining to detect histopathological alterations  as shown in Fig. Subsequently, the current study aims to highlight the drawbacks of neuroinflammatory AD progression in the heart and the liver. AD inflammatory type was initially induced in rats by a single interpretational injection of LPS. MWM was assessed to indicate the complete induction of neuroinflammatory AD type in order to study the deteriorative effects of neuroinflammation on the heart and liver. Our results indicated that the LPS-induced neuroinflammatory rats significantly consumed the longest time in MWM when compared with the control group (I)  as shown in Fig. Aβ and tau protein hallmarks deposition has been assigned in rat’s brain tissue to assure the complete induction of AD inflammatory type. Significant increase in Aβ and tau brain tissues level was observed in neuroinflammatory AD rats when compared with the control group (I) . A relevant decrease in Aβ serum level was observed in LPS-induced neuroinflammatory-treated MSCs group (IV) when compared with both control and AD group II . Meanwhile, the most significant effect on Aβ serum level was observed following the daily single administration of NaHS as hydrogen sulphide exogenous donor when compared with both control group (I) and LPS-induced neuroinflammatory group (II) respectively  as shown in Fig. A significant increase in Aβ serum level was observed following the induction of neuroinflammatory AD type in group (II) when compared with the control group (I) . The administration of MSCs to LPS-induced neuroinflammatory rats group (IV) resulted in a marked decrease in both cholesterol and triglycerides serum levels when compared with both control group (I) and LPS-induced neuroinflammatory group (II) . A significant and remarkable attenuating effect on cholesterol and triglycerides levels was noticed following the administration of NaHS to the LPS-induced neuroinflammatory rats group (III) when compared with both control group (I) and LPS-induced neuroinflammatory group (II)  as shown in Fig. Our results revealed that following Aβ serum level elevation in LPS-induced neuroinflammatory group (II), a significant increase in both cholesterol and triglycerides serum levels was observed as shown in Fig. P < 0.05, n = 6) as shown in Fig. P < 0.05, n = 6) as shown in Fig. The significant increase in Aβ serum level was displayed and found to be associated with a well-marked increase in TNF-α, IL-6, and caspase-3 release levels following the induction of neuroinflammatory AD type in group (II) when compared with the control group (I)  as shown in Fig. P < 0.05, n = 6) as shown in Fig. Upon neuroinflammatory AD-related pathology which resulted in increased Aβ serum level, elevated lipid profile, triggered inflammatory factors release, and disturbance in cardiac functionality have been observed in LPS-induced neuroinflammatory rats group (II) when compared with the control group (I). Thereby, a relevant observed increase in heart rate (HR), LDH, AST, CK-MB, and troponin I levels has been detected in the LPS-induced neuroinflammatory group (II) when compared with the control group (I) P < 0.0, n = 6 a2S donor effectively resulted in very relevant improvement in liver estimated parameters when compared with control group (I)  as shown in Fig. P < 0.05, n = 6) as shown in Fig. Significant hepatic dysfunctions have been observed in LPS-induced neuroinflammatory rats expressed in the form of increased ammonia, AST, ALT, and alkaline phosphates serum levels with a marked decrease in albumin, and TP serum levels when compared with the control group (I). Meanwhile, the administration of a single dose of NaHS as an exogenous HA correlation study was assessed to highlight the extent of the degree of significance, variability, and direct relationship between different tested variables. It was observed a significant positive correlation between Aβ Vs Trop I serum level, Aβ Vs CK-MB, Aβ Vs LDH serum level, Aβ serum level Vs HR, Aβ Vs Cholesterol serum level, Aβ Vs TNF-α serum level, Aβ Vs ALT serum level, Aβ Vs AST serum level, Aβ Vs ALP serum level, and Aβ VS Ammonia serum level as shown in Fig. Our observed results revealed that the control group (I) showed normal branching and anastomosing longitudinal muscle fibers, clear acidophilic sarcoplasm, central oval vesicular nuclei of the cardiac myocytes, and normal cardiac interstitial cells with elongated nuclei as shown in Fig. Masson's trichrome heart staining was conducted among all the groups to assess the degree of collagen fiber distribution within cardiac areas. Control group (I) showed normal limited bluish color around the blood vessels and was also found to be faint between the heart muscle cells as shown in Fig. Our observed results revealed that the control group (I) showed no histological deformities, as well as a distinctive central vein and hepatocyte organization. Normal Hepatic strands that travel from the lobule's border to the central vein, besides sinusoids with the portal region were observed without any related damage as shown in Fig. Collagen assessment using Masson's trichrome special staining revealed normal collagen fibers surrounding blood vessels in the central region among control group (I) (Blue color was used to stain collagen (arrow)) as shown in Fig. Lately, the number of neuroinflammatory AD subjects has been noticed to be rapidly increasing over the past few years. As a consequence, cognitive impairment and neuroinflammation have become huge medical and social problems associated with several drawbacks including other organs disorders and altered normal vital functions. Unlike other diseases, the correlation between neuroinflammatory AD type and heart-liver abnormalities remains undefined and unclear. Although several studies have previously reported the associated risk of cardiac and liver dysfunctions to AD, up to now a detailed mechanism of these associated risks and drawbacks has been scarcely explained. A Turkish study previously reported that 29 AD patients suffered from diastolic and cardiac dysfunctions when compared with normal control patients  and hyperphosphorylated tau protein. Based on that, the AD neuroinflammatory model was used in the current study in order to study the drawbacks of AD on the heart and liver by using lipopolysaccharide (LPS) as an AD inducer in rats. LPS is a potent endotoxin component of Gram-negative bacteria. It stimulates the release of proinflammatory cytokines resulting in neuroinflammation and ending with AD. Following ten days of IP LPS injection, behavioral stress known as sickness behavior represented in the form of fever, decreased food and water consumption, limited interactions, and reduced exploration was observed among AD rats in MWM . Concerning liver necrosis biomarkers associated with AD progression, a decreased albumin level along with increased ammonia, ALP, AST, and ALT were observed as illustrated in Fig. According to our observed results, decreased albumin level in LPS-induced group was followed by increased ammonia, ALP, AST, and ALT level when compared with the control group (I). In accordance with our results, decreased serum albumin as a drawback of neuroinflammatory AD-type progression may results in server liver dysfunctions due to systemic malnutrition inflammatory syndrome (Kaji et al. Nowadays, two valid drug types including memantine and cholinesterase inhibitors as a treatment for AD, are clinically proven to improve memory decline and alertness without hindering AD progression and life expectancy (Weller and Budson In conclusion, LPS-induced neuroinflammatory rats exhibit impaired cardiac and hepatic functionality when compared with control subjects. Liver and cardiac impairments were demonstrated by histopathological and special staining findings in correlation with observed disturbance in cardiac and hepatic normal biomarkers. Overall, these results are mainly proposed to be related to increased Aβ serum level triggering serious of inflammatory cascades, immune responses, and oxidative stress, The mechanisms responsible for exhibiting Aβ-related cardiac and hepatic dysfunctions was clearly identified and proven along with providing a clue to combat neuroinflammation drawbacks on other organs in order to improve the quality of life of AD patients by suggesting a dual way of therapeutic interventions. Our suggested therapeutic approach included the administration of NaHS and MSCs which proved their efficiency in alleviating cardiac and hepatic dysfunctions by hindering the extent of increased Aβ serum level drawbacks and thereby restoring normal heart and liver functions. Based on our observations and other reasonable evidence supporting the association between AD and hepatic-cardiac dysfunctions, the current study advocates for a major shift toward a greater interest in providing various types of successful therapeutic interventions for combating AD drawbacks whether on the brain or on other organs in order to hinder the overall neuroinflammatory AD status progression.


Experimentaldeflection ratios are compared with values calculated from moleculardynamics simulations. NaNH3 and NaH2O are deflectedas a spin 1/2 system, consistent with spin transitions occurring ona time scale significantly longer than 100 μs. For all otherclusters, reduced deflection is observed. The observed magnetic deflectionbehavior is correlated to the number of thermally populated rotationalstates in the clusters. We discuss that spin–rotational couplingsallow for avoided crossings and a reduction in the effective magneticmoment of the cluster. This work attempts to understand the evolutionof magnetic properties in isolated weakly bound clusters and is relevantto diamagnetic and paramagnetic species expected to exist in solvatedelectron systems.Using a pulsed Stern–Gerlach deflection experiment,we presentthe results of a comparative study on the magnetic properties of neutralsodium-doped solvent clusters Na(Sol) The formation of solvated, spin-pairedelectrons as a function of alkali metal concentration in sodium-dopednanodroplets15 and liquid bulk solutions16 is of current interest. Probing spin-pairingeffects via photoelectron spectroscopy (PES) is limited in its applicationdue to the similar electron kinetic energies of the involved species.However, investigating differences in the magnetic properties of sodium-dopedclusters might be a favorable approach to probe spin-pairing effects.Distinguishing diamagnetic (singlet) and paramagnetic (triplet) statesof singly sodium-doped clusters using Stern–Gerlach (SG) deflectionis suggested here as a first experimental step toward future studiesof more complex systems.Alkali metal-doped solventclusters provide ideal experimentaland theoretical model systems to study properties of excess electronsas a function of system size18 In atoms,the total angular momentum J is composed of the orbitalangular momentum L and its intrinsic spin angularmomentum S, neglecting the nuclear spin. In an SGexperiment, each Zeeman level corresponds to an individual deflectingbeamlet.19 In polyatomic molecules, additionaldegrees of freedom may contribute to the total angular momentum. Themolecular Zeeman effect is analogous to that in atoms, with additionalcontributions of angular momenta .Theoretical investigations of the diatomic case were performed byHill20 and later Schadee.21 An overview of the theory in diatomic molecules is givenby Berdyugina and Solanki,22 where analyticalexpressions of the Zeeman levels are given. Gedanken et al.24 showed the importance of spin–rotation interaction in SGdeflection experiments of oxygen and nitrogen oxide radicals.The original SG experiment was designedto determine the quantizationof the electron spin in silver atoms.26 found that paramagnetic moleculesand stable radicals were deflected less than predicted. They attributed the discrepancybetween experiment and prediction to be due to fast intramolecularspin relaxation induced by spin–orbit coupling, causing a lossof orientation of the magnetic moment and reduced deflection. However,Gedanken et al.23 found that magnetic deflectionspectra of TEMPO combined with line-profile calculations did not supportthe interpretation of spin relaxation processes occurring while traversingthe deflector. For large paramagnetic molecular and cluster systems,the density of Zeeman-like levels becomes so high that quantum chemicalcalculations are prohibitively expensive. Various groups29 developed more simplified theoretical models to explain SG deflectionexperiments within terms of intramolecular spin relaxation effectscaused by several spin transitions while conserving the total angularmomentum. De Heer and co-workers33 observed one-sided deflectionfor Fem, Com and Nim clusters (m = 10–1000), which was interpreted as a rapid intraclusterspin relaxation (ISR) process.27 Recentstudies show similar deflection behavior for metal–organicsandwich clusters37 and paramagnetic superatoms.41Amirav and Navonn with 1 ≤ n ≤4 . Interpretation of the SG deflection of molecularclusters is still a challenge. Singly sodium-doped solvent clustersare promising model systems as several characteristic properties canbe exploited in order to understand their magnetic properties as afunction of system size and type of solvent molecules. In the caseof singly sodium-doped clusters with sm = ±1/2, zero field splitting terms are vanishing, and high-symmetryhighest occupied molecular orbitals (HOMOs) suggest that contributionsof spin–orbit coupling may be minor in some cases.11 In the presented work, hyperfine effects from couplings to nuclearspins are neglected, although Fuchs et al.42 demonstrated that nuclear spin can diminish electron spin coherencein metal clusters. The magnetic deflection behavior of various sodium-dopedclusters is discussed in terms of thermally accessible rovibrationalstates. Spin–rotational couplings can produce avoided crossingsbetween Zeeman-like levels of the same J. We observethat the SG deflection behavior of the studied clusters appears tobe linked to thermally accessible rovibrational states and that themeasured magnetic deflection can be inversely related to the densityof the clusters’ rotational state manifolds.Here, we employ an SG deflection experimentto study the magneticproperties of neutral sodium-doped solvent cluster Na(Sol)22.143 Nevertheless, a short description of the experimentaland theoretical methods is given here. A sketch of the experimentalsetup is shown in The experimentalsetup to study size-dependent magnetic properties of neutral sodium-dopedsolvent clusters has been previously described in detail.2 into a vacuum (A). Thecluster formation conditions of the individual substances are summarizedin Table S1 of the Supporting Information (SI). The expansion is skimmed at S1, and the resulting clusterbeam was doped with sodium as it traverses the temperature-controlledoven in the chamber (B). See Table S1 forthe Na-oven temperatures used in the presented work (SI).Molecular clusters are generated via supersonicexpansion of eitherneat gas or mixtures with He or NB/dz in the deflector flight channel acts on the paramagneticclusters, causing the cluster beam to diverge. We have included adescription of the magnetic field pulses in the SI. The magnetic field gradient obtained from COMSOL simulationsat 1000 A deflector current is shown in Figure 1d in ref (SI. Selection of clusters after the SG deflectoris performed by a 2 mm diameter skimmer (S3) at the entrance of thedetection chamber (D). With the existing experimental setup, we areable to measure clusters that are transmitted only through S3.The sodium-doped solvent clusters pass a 1.5 mmdiameter skimmer(S2) to enter the deflection chamber (C) which houses the pulsed SGdeflector. The magnetic field gradient dd in ref  and in tvy. The displacement from the image center r is related to the neutral clusters’ velocity vy byVR is thevoltage on the repeller, m is the cluster mass, and C is a setup-dependent calibration constant. Velocity distributionsof the sodium-doped solvent clusters obtained in this study are listedin Upon reaching the ionization region inside the extraction optics,the neutral clusters are ionized with a pulsed (∼7 ns) 266nm (4.66 eV) Nd:YAG laser. The resulting photoions are extracted perpendicularto the molecular beam axis and detected by either time-of-flight (TOF)mass spectrometry or velocity map imaging (VMI). Ion-resolved VMIwas achieved by gating the microchannel plates of the imaging detectorto only be high when the ions of interest arrived at the detector.The perpendicular configuration of the molecular beam axis and TOFaxis allows us to determine molecular beam velocities t1, t2, and t3 to thelaser timing tL. These relative time delaysare optimized to the velocity distribution of each cluster beam studied in orde45 Furthermore, as an experimental verification that the magnetic deflectordid not alter the cluster signal, we collect mass spectra and VMI’swith the deflector “off” and with the deflector “on”but with the ionizing laser pulse not synchronized to interest theclusters that experienced the magnetic field. The signals showed nosignificant deviations.We do not expect fragmentation or monomer evaporationof theseweakly bound clusters to be significant upon photoionization. We areionizing the Na atom and not the monomer units of the cluster, andthe outgoing photoelectron is carrying away the excess energy of thephoton as the kinetic energy. Na-doping and UV ionization has beenestablished as an ultrasoft method for cluster size determination.2.243 Here,we summarize the main aspects of our approach and point out improvementsfrom earlier work. Cluster trajectories are simulated from the entryof the deflector to the ionization region. The initial coordinates and momentum of each particle are defined in the cluster beam, where themass is defined as a given cluster mass. The velocity in the molecularbeam direction  at ionizationand corresponding momentum  for particles of spin states−1/2, 0, and +1/2. Simulations of S = 0 produceresults equivalent to those of deflector “off”. Insteadof taking the topology of the rovibrational Zeeman diagram into accountto simulate the magnetic deflection behavior of the clusters, we haveintroduced an optional additional scaling factor of the magnetic momentto account for possible ISR effects. We scale the magnetic momentμ0 of a single ms = ±1/2 particle with an exponential decay, defined bythe interaction time with the magnetic field tm and a characteristic relaxation time τ.tm = sdef/vy is defined by the deflector length sdef and molecular beam velocity vy. By sampling various values of τ and comparingsimulations to experimental deflection results, we estimate the characteristicspin relaxation times τ for the investigated clusters. Withmodeled effective magnetic moments μeff, we quantitativelycompare the deflection behavior of the clusters.A detaileddescription of the molecular dynamics (MD) approach to model the deflectionprocess has been given previously.33.13.1.12O velocity distributions (obtained from VMIs) for deflector “off”and “on” measurements at an operating current of 700A and optimized deflector coil timings43 for the sampled velocity distribution  = 150m/s) to achieve maximal possible deflection. Under these conditions,we measured a deflection ratio, γd, of 0.66(10) asshown in d values for all sampled Id. Deflection (γd > 0) is observed for Id ≥ 200 A and agrees with the MD simulationsfor each sampled deflector current. We note that the experimentalγd is slightly and systematically higher than thesimulations for a spin 1/2 particle and suspect collisions of thecluster beam with background gas molecules  while the deflector is switched “on”to be a possible explanation for the systematic deviations. The heatingof the deflector arises from thermal loads generated by the in-vacuumcoils that cannot be completely dissipated with our cooling system.A total loss of the signal occurs at a chamber pressure above 5 ×10–4 mbar, which we attribute to obstruction ofthe flight channel n clusters with n >1 exhibit a similar cluster size dependence in magnetic deflection.In a previous study, we observed deflection behaviorof an 3.1.2Id = 700 A) and a broad velocitydistribution of the cluster beam (∼800–1500 m/s) areshown in 2O)2, (b) Na(H2O)3, and (c) Na(H2O)4. The distinct peaks of thevelocity profiles are caused by mechanical recoils of the pulsed Even–Lavievalve46 used to generate the beam of waterclusters. The simulations predict full deflection (θrel = 0) for velocities <1100 m/s. This, however, is not observedin the experiment and means that these clusters do not interact withthe magnetic field as free spin 1/2 systems. The results for Na(H2O)2, Na(H2O)3, and Na(H2O)4 are consistent with spin transitions takingplace on significantly faster time scales than the experiment. Inthe simulated data for a spin 1/2 particle (red traces) of The deflection results for the highest possible magnetic fieldgradient (3.23.2.1Id = 700 A. NaMeOH exhibitsdeflection with θrel = 0.5(1) over the full velocitydistribution, and minimal relative signal (θrel =0.2(1)) in the velocity range of 800–1100 m/s. The residualsignals to the slow and fast side of the velocity distribution area result of the applied magnetic field pulses, which are too shortin time to ensure significant magnetic field gradients across thesampled velocity distribution. For the data shown in ms =±1/2 system. d as a function of the deflectorcurrent, where partial deflection is observed for Id > 500 A. Again, the MD simulations are not in agreementwith the experimental observations.3.2.2Id = 700 A areshown in 2, (b) Na(MeOH)3, and(c) Na(MeOH)4. None of these clusters exhibits deflectionacross their sampled velocity distributions. The deflector “off”trace and deflector “on” traces are the same withinexperimental error. The obtained simulations, however, predict deflectionfor all three cluster sizes. These deviations of experiment and simulationagain show that the magnetic properties of the investigated clusterscannot be described by the magnetic moment of sm = ±1/2 particles.Thedeflection results for 3)n (see ref (2O)n n. The presented results can be explained by ISR processes takingplace on significantly faster time scales than the experiment. Inorder to discuss possible ISR processes of partially and nondeflectingclusters, we analyze thermally accessible rovibrational states asa function of cluster size, n see (For Na(NH(see ref ) andNa(2O)n see 3.1, we ne, n see 3.5.3.33.3.1Id = 700 A. We determinedθrel = 0.49(5) for the sampled velocity distribution.The corresponding simulated data exhibits full deflection (θrel = 0) for velocities between 600 and 900 m/s, but incompletedeflection (θrel ≈ 0.20) for slower ≤600m/s and faster ≥900 m/s particles. However, the simulationdid not match our experimental observation. d as a functionof deflector current, where deflection is observed for Id > 100 A. Quantitative agreement between experimentandsimulation is not found for any of the studied deflector currents,although deflection increases with increasing deflector current inboth.Velocity-calibrated photoionVMIs of NaDME are shown in For NaDME, the deviation between simulations andexperiment maybe due to ISR processes, which occur on similar time scales to theinteraction time with the magnetic field, causing a reduction of thecluster’s magnetic moment.3.3.22, analogous to that of NaDME, is presentedin Id = 700 A (rel = 0.82(7).According to the simulations, full deflection is expected in the velocityrange of 600–800 m/s and residual signals for slower ≤600m/s and faster ≥800 m/s particles. We classify Na(DME)2 as partially deflected at Id =700 A, whereas at lower deflector currents, no significant deflectionwas observed (see Magnetic deflectiondata of Na(DME) = 700 A 7a, the drved see 7b.2 may bedue spin transitions occurring on a time scale similar to the interactiontime with the magnetic field gradient. It is interesting to note thatdeflection of Na(DME)2 becomes observable at Id > 600 A. The higher deflector current would producea stronger magnetic field gradient and a larger Zeeman splitting,which could make more avoided crossings of Zeeman-like states. For Id > 600 A, it appears that the magnetic fieldgradient is large enough to deflect some Na(DME)2 clusters,even though we essentially determine μeff = 0 atlower currents.The reduced deflection observed for Na(DME)3.3.33 at Id = 700 A are shown in 3 as nondeflected, althoughslight deflection might be visible in the region of the center velocities(650–850 m/s). We needed increased sensitivity to make a definitivestatement. The simulated signal in contrast reduces to θrel = 0.25 and as in the case of previous clusters exhibitingreduced deflection. Again, the deflection process is not describedby the interaction of ms = ±1/2 particles with the inhomogeneous magnetic field. Measurementsat lower deflector currents confirm this deflection behavior sinceγd = 0 was measured ured see 8b.n (n = 1–3) are discussedin terms of possible ISR processes as a function of cluster size n in The differences between experiment and simulationfound for Na(DME)3.4eff of NaH2O  are equ0 during the transit time tm with a characteristic relaxation time τ. PerformingMD simulations with μeff for various characteristicrelaxation times τ allows us to match the simulations to our experimental observations.For each τ value, the squared deviations 2 are evaluated by theirχ2 value given byi is the experimental error of each sampled velocity vi over a region of interest chosen fromthe experimental velocity distribution. The evaluated χ2(τ) values are fitted quadratically as a function ofτ to obtain the characteristic relaxation times. The evaluationof τ is carried out for each deflector current individuallyin order to be sensitive to any possible magnetic field dependence.For nondeflected clusters, the fitting procedure is not applicable,and we therefore report an upper limit on τ for such cases.The results are summarized in As stated in ai is the unitless average attenuation factor of the exponentialterm for a single particle in tm while traversing the deflector, defined by the deflector length sdef and molecular beam velocity vi.With the characteristic relaxation times τ,we determineIn vi andthe deflector lengthdictates the observable μeff within the applied model.A comparison of different μeff values is thereforeonly meaningful when compared at the same velocity or interactiontime with a magnetic field gradient. For a constant velocity vi at any value -optimizedcluster geometries were carried out with the Gaussian program package.ms = ±1/2system is split into two eigenstates according to their spin orientation.In the case of ms = ±1/2,zero field splitting of spin microstates can be neglected as onlyone unpaired electron is present. Available rotational states increasethe number of diabatic Zeeman-like levels to 2J +1 = (2S + 1)·(2R + 1), where S is the spin, R the rotational, and J is the total angular momentum . If S and R are uncoupled, then degeneracies of the Zeeman-like statesare allowed. However, if S and R are even weakly coupled, then crossings of the Zeeman-like statesare avoided for adiabatic states with equal J. Atan avoided crossing, ms changes sign in both of the associated Zeeman-like states, whilethe total angular momentum J = R + S is conserved. For ms = ±1/2 systems, this leads to Δms = 1, which only allows forspin transitions between adiabatic Zeeman-like states with ΔMR = 1. In terms of the avoided crossing model,29 clusters with a higher density of populatedrovibrational states are expected to undergo more spin transitionsand on average exhibit less deflection, when compared to clusterswith lower densities of thermally accessible adiabatic Zeeman-likelevels.The Zeemandiagram of an En,R. We choose to describe En,R with respectto the vibrational energies of a harmonic oscillator and the rotationalenergies of a rigid rotor.n is the vibrationalquantum number, υ0 is the vibrational eigenfrequency, R is the rotational quantum number, and Brot is the rotational constant. With estimated vibrational Tvib and rotational Trot temperatures, we express the average number of thermally accessiblerovibrational states in terms of the partition function. The densityof rovibrational states is expressed as the average number of populatedstates within the maximum achievable Zeeman energy splitting ΔEZeeman = 2μBBmax ≈ 2 cm–1, where μB is the Bohr magneton and Bmax ≈ 2T. The following discussions are basedon the assumption of thermally equilibrated clusters, where the populationof states is described by a Maxwell–Boltzmann distributionat estimated thermal energies kBT.In order to discuss our experimental findings and relativedeflectiontrends, we analyze rovibrational eigenstates S and R-coupledadiabatic Zeeman-like levels, states with ms = ±1/2 contributions are able to undergo avoidedcrossings if neighboring states with ΔMR = 1 have an energy difference smaller than the Zeeman energysplitting. We chose the maximal Zeeman energy splitting for a spin1/2 system (ΔEZeeman ≈ 2cm–1) as an upper limit for all investigated clusters.Harmonic frequency calculations of the investigated clusters and theirstructural isomers (see the SI) show thatdifferences in vibrational energies of thermally accessible modesare larger than the Zeeman energy splitting. We find that rotationalenergy level differences, for a rigid rotor approximated by Erot = ⟨B⟩rotJ(J + 1) with the averagerotational constant ⟨B⟩rot = (Arot + Brot + Crot)/3 , areless than ΔEZeeman for all studiedclusters except NaH2O and NaNH3, which are thetwo clusters deflected as spin 1/2 particles. Thus, multiple rotationallevels within ΔEZeeman are to beexpected in the cases in which we measure reduced deflection. We takethe number of avoided crossings a cluster undergoes to be governedby the density of rotational energy levels within ΔEZeeman and rather independent of the population of vibrationalmodes, as they are spaced by more than ΔEZeeman. We, therefore, discuss avoided crossings in terms ofrotational states, assuming no additional contributions from excitedvibrational states.In the case of 3.5.1EZeeman through the rotational partition function Qrot. We use Qrot to represent the average number of populated states. Then, Qrot divided by the average rotational energy kBTrot and multipliedby ΔEZeeman corresponds to the averagenumber of states within ΔEZeeman. Our analysis is based upon a DFT-optimized cluster with variousisomers (ωB97XD/6-31+G*). In the high-temperature approximation,the rotational partition function of a nonlinear rotor is expressedbysym is the symmetrynumber, Trot the rotational temperature,and Arot, Brot, and Crot are the rotational constantsof the rigid rotor. Since the rotational temperatures of free clustersare not well-defined, we assume a temperature range 10 K ≤ Trot ≤ 50 K. This range comes from thecomparison of the vibrational and rotational energy transfer crosssections σvib and σrot, which followσvib ≪ σrot.5252 In an adiabatic expansion, the differences in effectiveenergy transfer lead to Trot < Tvib. Fuchs et al.53 determined Trot < Tvib with 5 K ≤ Trot ≤ 20 K for small metal clusters generated in a supersonicexpansion at a nozzle temperature of 16 K. In our experiments, nozzletemperatures are significantly higher (10 °C ≤ Tnozzle ≤ 150 °C) and collisionswith sodium atoms in the oven are expected to affect the cluster temperatures.Although it is unclear how much this influences the cluster temperatures,as the cluster can evaporate monomer units to dissipate the acquiredenergy, we take a rotational temperature range of 10 K ≤ Trot ≤ 50 K for the analysis.We represent theaverage number of rotational eigenstates within ΔQrot via n ≤ 4 where the letters  refer to different isomers of a cluster with a given numberof monomer units (n) . This analysiswas performed to see if the average number of rotational states withinΔEZeeman (represented by Qrot) correlated with the trend in the magneticdeflection behavior we measured in this study. If so, we should findthat an increase in thermally accessible rotational states coincideswith a reduction in deflection.47Withestimated rotational temperatures and rotational constants, we determinethe rotational partition functions Qrot inthe cluster systemsin the temperature range 10 K ≤ Trot ≤ 50 K shows the lowest values for NaH2O and NaNH3. Furthermore, the rotational state population is only weaklydependent on rotational temperature for NaH2O and NaNH3. This is consistent with our deflection measurements havinga similar behavior to that of a spin 1/2 particle or an effectivemagnetic moment similar to μ0. NaMeOH and NaDME exhibita similarly low Qrot at 10 K but a morepronounced increase of Qrot with temperatureand experimentally only reduced deflection compared to a simulatedspin 1/2 particle of the same mass.Comparison of n clusters are expectedto be colder than the otherclusters studied due to the relatively low nozzle temperature usedfor the DME expansion (see Table S1) andrelatively weak intermolecular forces binding DME molecules. With Trot(Na(NH3)n) > Trot(Na(DME)n), we estimate fewer populated rotational states for NaDMEthan forNa(NH3)2 and confirm the experimental observationof μeff(NaDME) > μeff(Na(NH3)2). Slight deflection of Na(DME)2 isconsistent with it having a lower rotational temperature, whereasNa(MeOH)2 shows an increase of populated rotational states,which could explain its lack of deflection. At the lowest temperaturesconsidered here, the change in the rotational state density betweenNaH2O and Na(H2O)2 is unlikely tobe the reason for the absence of deflection for Na(H2O)2. This leads us to the idea that the Na(H2O)n clusters may be hotter than the other sodium-dopedsolvent clusters studied.Estimates of thermal temperaturesshowed that Na(DME)3,4 and Na(DME)3 exhibit significant populationin many rotational states, whichsupports the observation of no significant deflection. The absenceof deflection for Na(H2O)3,4 is difficult toexplain by only comparing thermally accessible rotational states.Possible explanations include a higher rotational temperature or contributionsother than spin–rotational couplings. The structural isomersof Na(NH3)3,4 show substantially different populationsof rotational states. For Na(NH3)3, isomer 3bis 83 meV lower in energy than isomer 3a. The experimental observationof partial deflection for Na(NH3)3 may implya preferential production of isomer 3b and its lower rotational statedensity. Similar arguments hold for Na(NH3)4 which shows partial deflection, which may be explained by preferentialproduction of the tetrahedral isomer 4b with its significantly reducedpopulation of rotational states compared to isomer 4a. Previous workof our group10 found n = 4 to be a magic number for the photoelectron anisotropy in sodium-dopedammonia clusters. These complementary experiments support the ideathat Na(NH3)4 is produced and deflects a highlysymmetric tetrahedral structure with substantially lower rotationalstate density. For the Td structure σsym = 12, less symmetric isomers withlower σsym (IB in ref (54The larger clusters of Na(MeOH)Trot(Na(DME)n) < Trot(Na(NH3)n) < Trot(Na(MeOH)n) < Trot(Na(H2O)n). Althoughinfluences from contributions other than spin–rotational couplingmay also be important, further and more refined studies are needed.In general, we find a correlationbetween experimental deflectiontrends and rotational level spacings and populations. The most significantdeviations of the experiment and model are possibly due to differencesin cluster rotational temperatures, with a proposed order in our experimentsbeing 42O)n (n = 1–4), Na(NH3)n (n = 1–4),Na(MeOH)n (n = 1–4),and Na(DME)n (n = 1–3)clusters was characterized. We showed that the fully deflecting ms = ±1/2 character ofNaH2O and NaNH3 is consistent with these clustershaving large rotational level splitting compared with ΔEZeeman. In all other clusters studied, the observationof reduced deflection was found to be consistent with an increasedpopulation of closely spaced, excited rovibrational states. Magneticdeflection observed to be less than that of a free ms = ±1/2 system was modeled by theattenuation of the magnetic moment μ0. With thisapproach, we were able to retrieve characteristic spin relaxationtimes τ. The relative experimental deflection trends were discussedin terms of thermally accessible rovibrational states in an attemptto understand trends in their deflection behavior. Thermal energiesof the studied clusters were estimated using evaporative ensembletheory and quantum chemical calculations of vaporization enthalpies.The rovibrational states were analyzed in the harmonic oscillatorand rigid rotor approximations of geometry-optimized cluster geometries.The magnetic deflectionbehavior of Na; however, for larger systems, calculations will be challenging.Calculations of the Zeeman diagram for small sodium-doped clustersare of interest to evaluate contributions from other angular momentain magnetic deflection experiments.The magnetic deflection behavior observed in the weakly bound clustersystems studied here does not have an obvious explanation and willneed further investigations to give a clear understanding. For themoment, we point to spin–rotational couplings and avoided crossingsas a possible cause for a reduction in the effective magnetic momentand reduced deflection. More refined experimental and theoreticalwork is needed in order to understand other contributions to the attenuationof the magnetic moment while traversing a magnetic field gradient.In particular, well-defined and low temperatures for vibrational


Photo-elastic experiments are performed tracing the TE and TM shifts of the whispering gallery mode resonances upon application of an axial strain. The strain optical coefficient K’ for Silk I fibroin is found to be 0.059 ± 0.004, with the corresponding value for Silk II being 0.129 ± 0.004. Remarkably, the elastic Young’s modulus, measured by Brillouin light spectroscopy, is only about 4% higher in the Silk II phase. However, differences between the two structures are pronounced regarding the photo-elastic properties due to the onset of β-sheets that dominates the Silk II structure.Silk fibroin is an important biomaterial for photonic devices in wearable systems. The functionality of such devices is inherently influenced by the stimulation from elastic deformations, which are mutually coupled through photo-elasticity. Here, we investigate the photo-elasticity of silk fibroin employing optical whispering gallery mode resonation of light at the wavelength of 1550 nm. The fabricated amorphous (Silk I) and thermally-annealed semi-crystalline structure (Silk II) silk fibroin thin film cavities display typical Q-factors of about 1.6 × 10 An important feature of several protein-based biomaterials is the metastable-stable structure transition modifying the material’s optical6 characteristics. Bombyx Mori extracted silk is a natural biomaterial renowned for its applications in clothing and surgery. At the same time, its fibroin protein, in the regenerated form, has been widely studied for its use in optical circuits, drug delivery, and sensing components7. Due to its biocompatibility, mechanical strength, high optical transmission, and tailored wettability, silk fibroin is considered a backbone for developing wearable and implantable sensing devices9, allowing the realization of skin attachable components with readily accessible optoelectronic functions10. A significant issue arising for the realization of silk-based, skin-attached11, wearable photonic devices12 is that of photo-elasticity, namely, how much the refractivity and birefringence of the silk fibroin will change under mechanical stimulation. For example, the operation characteristics of optical devices in adhesive contact with human skin tissue can depend on the mechanical forces directly exerted at the interface region. On the photo-elasticity of silk, the limited literature primarily refers to the strain-induced birefringence in pristine silkworm filaments14, whereas, to the best of our knowledge, there is no report on regenerated silk fibroin. Photo-elasticity correlates mechanical and optical properties of a material reflecting local structure onto macroscopic response. The specific local structure of regenerated silk fibroin, where protein structures such as random coils, β-sheets, and α-helixes conglomerate, render a photo-elasticity study particularly important.Optical biomaterials offer numerous physical and chemical functionalities associated with their mechanical, segmentation, and surface chemistry properties. The former functions can accelerate the implementation of such materials into innovative and functional sensing, imaging, and actuating photonic devices15 allows the detection of minimal birefringence changes yielding the stress-optical coefficient and enabling a correlation to the molecular polarizability of soft materials16. Micrometric-size silk fibroin cylindrical cavities are first formed in Silk I (metastable) and then transformed to Silk II (stable), allowing an in-situ investigation of the effect of the structure on the photoelastic behavior of silk fibroin. The formation of β-sheets in Silk II simultaneously modifies the optical and mechanical properties of the silk fibroin, dominating its photoelasticity. While silk fibroin has been used before in the fabrication of cylindrical WGM cavities onto planar substrates17, the application of WGM resonation in the study of the photo-elasticity of a protein-based biomaterial is a new approach, implemented explicitly for the case of silk fibroin. For a detailed insight into the origin of the photoelastic differences between the amorphous and semi-crystalline structures of silk fibroin, the elastic Young’s moduli and Poisson’s ratio of both phases are measured at zero strain by the non-contact, non-invasive Brillouin light spectroscopy (BLS). BLS enables the de-coupling of the mechanical properties of the two silk structures from their optical refractivity/polarizability properties18. In view of the unexpectedly very similar Young’s moduli, the photo-elasticity of silk fibroin is primarily driven by the optical properties of the β-sheet conformations formed in excess in the semi-crystalline Silk II, with the Silk I being less birefringent due to low β-sheet content and water plasticization.Herein, we report on silk fibroin's photoelastic properties, employing whispering gallery mode (WGM) light resonation in cylindrical resonators cast onto glass fiber supporting beams. Owing to the high Q-factor, the polarization-sensitive, modal dispersion of light confinement facilitated through WGM resonation19 and achieving an even shape cavity formation from a silk-water solution. The silk fibroin used was extracted from row Bombyx Mori cocoons following the original Rochwood’s protocol; the extracted fibroin was dissolved into an 8% (wt/vol) water solution  from an Erbium doped fiber amplifier (EDFA) was coupled into the excitation optical fiber taper, while the WGM spectral signature was traced through the same fiber (the other end), connected to a polarizing optical fiber, while using an optical spectrum analyzer  cast on sodalime glass substrates is computed from the sound velocity measured by Brillouin light scattering (BLS), using 532 nm laser radiation see “. BLS can24 we could examine only the radial (l) and azimuthal (m) modes for the TE or TM polarization states  and the refractive index  through the relationship:TX≡ λTX(ε) − λTX(0) and ΔnTX≡ nTX(ε) − nTX(0) (index X stands for either E or M), and ε denotes the applied strain along the z-axis of the silk fibroin/silica beam system. CTX is a correction factor compensating for inherently differential slopes holding for TE and TM polarizations of equal order WGM modes15, calculated from the analytical formulation for resonation in cylindrical cavities. For the specific parameters of our experiment, CTE = 5.06 × 10–2 and CTM = 2.64 × 10–2.For the silk fibroin WGM resonator, a purely cylindrical symmetry was assumed, which is a good approximation for modal excitations within its middle section. By staying far from side-located turning pointstes Fig. a25. The 26 than the propagating wavelength. This assumption allows us to use the contracted photoelastic tensor for isotropic materials, with inverse symmetry rotation simplifications applied in the Pockel’s coefficients27. Application of longitudinal strain through the supporting optical fiber beam to the silk fibroin cylindrical micro-resonator , and transversal direction t analyzed along axes x and y (Δnt)28, as according to the relationships:In the Silk I amorphous structure, we consider silk fibroin as an isotropic, modest birefringence optical material. We also treat Silk II  as an isotropic material since the crystallinity range of the dispersed β-sheets in Silk II fibroin (~ 2.6 × 3.2 × 11.6 nm) is manifold shorter0 denotes the isotropic refractive index , εt, z are the strains, and pij are the Pockel’s elasto-optic coefficients. According to Eqs.  . The εz strain values used in those calculations were directly deduced from the experiment.Here n to Eqs. , 2) and and0 den to Eqs. , the strSeveral cylindrical cavities of silk fibroin, similar to the one shown in Fig. 89 were 1.35 × 104 for Silk I, and 1.61 × 104 for Silk II, under a Lorentzian function fitting29. It is anticipated that the annealing process improves silk fibroin homogeneity and slightly increases its refractive index30. These Q-factor values rest below those reported by other groups on Silk I WGM cavities; however, they are measured in closed-shape cavities of better radial symmetry than those casted here17. The experimental free spectral range (FSR) (31). The as-cast  birefringence Δninitial of the Silk I fibroin cavity was estimated from the wavelength positioning of the two polarization notches for a fixed azimuthal/radial order compared to simulated ones  to be ~ 0.5 × 10–3. For the annealed Silk II fibroin cavity, Δninitial≡ ΔnΤM − ΔnΤE ~ −5 × 10–3. Thus, the refractive index difference Δninitial was found to have an opposite sign and a tenfold larger absolute value in Silk II than Silk I. This difference in residual birefringence is attributed to the thermally induced β-sheet formation and their orientation inside the cavity volume of Silk II32.Experimental spectral data of WGM resonances for both polarizations over a broad wavelength range for a silk fibroin resonator as cast (Silk I) are depicted in Fig. 33 using COMSOL Multiphysics (see Supplementary part). Before employing the numerical simulations in the silk fibroin cavities under study, their accuracy was verified in an archetypical cylinder resonator geometry by comparing the simulation results against rigorous analytical solutions34 . In the simulations, the silk fibroin cavity was considered perfectly cylindrical The WGM resonation was described with full-wave eigenvalue simulationsentclass1pt{minimaentclass1pt{minimasee Fig. . The disentclass1pt{minimaFrom the above modal simulations, it was decided that all strain birefringence measurements should be focused on the spectral examination of first-order-radial modes. While higher radial order modes have also being experimentally traced (yet not presented here), their spectral behavior  rendered their interpretation laborious and of limited accuracy. The as-cast (Silk I) fibroin cavity was subjected to controllable longitudinal strain, and the spectral data of the TE and TM WGM of modal order m = 89, l = 1 are presented in Fig. 35; the WGM spectra for this cavity in the Silk II structure  and anisotropic (∝ p44) components of the segmental polarizability tensor, respectively37. Unexpectedly, the depolarized BLS intensities imply that the optical anisotropy per optical axis in the amorphous Silk I is about 20% higher than in its semi-crystalline form II. Notably, the WGM experiment gives The polarized and depolarized BLS intensities obtained, respectively, from the longitudinal and transverse acoustic phonons in Silk I and II  and the stress optical coefficient, K (by more than 50%), but only marginally (by 4%) the elastic material response. These findings demonstrate that segmental orientation is a fundamental quantity for photo-elasticity. This notion is corroborated by the different The amorphous Silk I and semi-crystalline Silk II have the same chemical stoichiometry but different refractive and photoelastic properties, as revealed from the WGM of thin film cavities. Silk II reveals a pronounced optical birefringence, with the higher refractive index component measured along the longitudinal axis of the WGM cavities. However, Silk II is characterized by Young’s modulus only slightly higher than Silk I. Hence the transformation of Silk fibroin from the amorphous to the semi-crystalline structure affects predominantly the optical birefringence, Δn20. The basically amorphous structure of Silk I is also confirmed by its low initial  birefringence  for tuning the formation and orientation of β-sheets emerges as a promising approach for developing optical biomaterials with high polarizability properties, for linear or non-linearSilk Fibroin (SF) was extracted from row Bombyx Mori cocoons following the original Rochwood’s protocol. Cocoons were sliced, then boiled in a 0.02 M sodium carbonate (Alfa Aesar) solution for 30 min in order to remove the glue-like cladding made of sericin. Degummed fibroin obtained upon separation from sericin was then washed three times in highly pure water for 20 min for removing eventual sericin excess; then, left to dry overnight under hood. Degummed fibroin then dissolved in 9.3 M lithium bromide (Alfa Aeser) solution for 4 h in a conventional oven at 60 °C, forming an amber-like solution. After cooling down, this solution was dialyzed for 48 h in a dialyzer cassette  against ultra-pure water to remove salts. After dialysis, the SF aqueous solution was centrifuged twice at 9000 rpm for 20 min at 4 °C to remove the unsolved portion of fibroin; a ~ 8%( wt/vol) solution has been accordingly produced and storage at 4 °C in the fridge prior to use.−2. With the help of a micro-syringe silk fibroin droplets were dispersed onto the hydrophilic optical fiber tapers, on a vertical placement mode, for forming elongated cylindrical shape cavities of typical length of ~ 100 μm and diameters of ~ 33 μm; namely, a thin silk fibroin overlayer of ~ 1.5 μm thickness was casted on the glass surface. These silk fibroin cylindrical cavities were left to fully dry in ambient atmosphere, so the soft material reaching the Silk I amorphous state. Accordingly, for transforming the cavities to the crystalline Silk II structure an annealing procedure took place at 180 °C for 10 min in ambient air, resulting a nominal diameter shrinkage with shrinkage ratio 0.34%. The total tile of the annealing process for reaching the temperature of 180 °C lasted for ~ 20 min, with a typical ramping rate of 8 °C min−1.Optical fibers were thermally tapered down to ~ 30 μm diameters with the use of a GPX-3000 Vytran glass fiber processor. Then, these SMF-28 optical fiber tapers were irradiated using 193 nm excimer laser radiation for improving their surface hydrophilicity. This irradiation process took place with the supporting SMF-28 tapered optical fiber being immersed into deionized water, through a tungsten amplitude mask for defining hydrophilic areas of typical lengths of ~ 100 μm; the total energy dose dissipated was 13 J cmThe experimental setup for spectrally characterizing whispering gallery mode resonation inside silk fibroin cavities the investigation of the photo-elastic behavior, is depicted in the Supplementary Part, Fig. The tapered optical fiber-silk fibroin cavity components were mounted on a bronze fork using UV glue see Fig. , with thWe used Brillouin light scattering (BLS) to determine the mechanical properties of Silk I and II by measuring the velocity of longitudinal (2 substrates, which resemble more the samples used in the WGM experiment. The thinner silk films could be measured with BLS in backscattering geometry (Fig. In 90A with VV polarization Fig. a the freion Fig. b we probtry Fig. d from whIn addition to the mechanical properties, BLS can be used to derive the relative values of the photo-elastic constants in Silk I and II. Although the mechanical properties are identical, the optical anisotropy per optical axis of Silk I is significantly higher than Silk II (see the intensities in Fig. The BLS intensity for transverse waves and VH polarization is:Supplementary Information.


In the published article, an error was made in identifying the relevant countries.Introduction, paragraph 1. This sentence previously stated:A correction has been made to the “Canada ranks 9 out of 36 countries”This should have been written as:“Canada ranks 9 out of 36 peer Organization for Economic Co-operation and Development countries”In the published article, an error was made in providing the average human costs per year.Results, paragraph 2. This sentence previously stated:A correction has been made to “human costs averaged $183,448,841 per year,”This should have been written as:“human costs averaged $188,416,841 per year,”The authors apologize for this error and state that this does not change the scientific conclusions of the article in any way. The original article has been updated.


By adjusting flow rates inthe microfluidic channel, we resolved the temporal evolution of thereaction system in the first few seconds, generating the time profilesfor speciation, ligand exchange, and reduction of Pt. Detailed analysisof the X-ray absorption near-edge structure and extended X-ray absorptionfine structure spectra with multivariate data analysis shows thatat least two reaction intermediates are involved in the transformationof the precursor H A common method forthe synthesis of platinum nanoparticles is the reduction of hexachloroplatinateprecursors, either as H2PtCl6 or K2PtCl4, with a mild reducing agent such as ethylene glycol.11 Two mechanistic scenarios are usually considered for platinum nanoparticlenucleation with glycol reductants:14 (i) classical nucleation theory (CNT),16 which suggests that Pt nanoparticles are formed by the aggregationof Pt(0) atoms once a critical concentration of Pt(0) atoms has formedin solution, while an alternative mechanism (ii) proposes that thenon-metallic complex ions form intermediate clusters, which are thenreduced to form Pt nanoparticle nuclei.14 Accordingto the latter mechanism, some Pt–Pt bond formation occurs inthese pre-nucleation clusters prior to the formation of the Pt nanoparticles.An unequivocal experimental demarcation between the nucleation mechanismsis challenging, due to the stochastic nature of the nucleation eventsand the low concentration of these sub-nm pre-nucleation clusters.Structurally incisive experimental in situ probes with sufficientspatial and temporal resolution are therefore needed to monitor thenucleation process.17Platinum nanoparticles exhibit catalyticproperties relevant fora multitude of economically important applications, for example, ascatalysts for fuel cells, in the industrial synthesis of nitric acid,in the reduction of exhaust gases from vehicles, and as componentsin biomedical applications.19 Steinfeldt11 monitored platinum nucleationand growth with ethylene glycol using in situ small-angle X-ray scattering(SAXS). However, SAXS also detects the scattering from already formednanoparticles and therefore while it throws light on the growth mechanism,it gives limited insights into the nucleation pathway. Studies byX-ray pair distribution function (XPDF) have suggested that underheating the octahedral Pt4+ precursor species convert tosquare planar moieties (likely as Pt2+) prior to theirreduction to metallic Pt nanoparticles.13 However, XPDF lacks oxidation state sensitivity and crucially requirestaking the accompanying changes in the solution matrix into account.In the present study, we apply X-ray absorption spectroscopy (XAS),which gives insights into the evolution of the oxidation state aswell as the coordination environment of the species formed throughoutthe reaction.The developmentof liquid cells for transmission electron microscopy(TEM) has allowed characterization of colloidal nanoparticles in thepost-nucleation stage, but this approach gives limited insights intothe processes occurring prior to nucleation.20 Their results suggested a single-stepnucleation process, in which the Pt(IV) precursor is directly reducedto Pt(0) forming the Pt nanoparticles, with no evidence for the formationof any intermediate species. The reduction of Pt(IV) to Pt(0) wasrapid, taking place during the first few seconds of the reaction.Harada et al.21 studied the nucleationof Pt nanoparticles by photoreduction in an aqueous ethanol solutionusing in situ XAS. They proposed that the photoreduction formationof nanoparticles proceeded in three stages—reduction–nucleation,autocatalytic surface growth of the nuclei, followed by Ostwald ripening.Evidence of Pt–Pt bonds characteristic of Pt nanoparticle formationwas observed ∼600 s after irradiation. Yao et al.7 used in situ UV–vis and XAS to monitorthe nucleation of platinum nanoparticles when using ethylene glycolas a reducing agent and reported the formation of Cl3Pt–PtCl3 dimers, which were a precursor to the formation of Pt nanoparticles.This species was observed ∼1200 s after introduction of thereducing agent. This confirmed the first-principles molecular dynamicsstudy by Colombi Ciacchi et al.14 who demonstratedthat the reduction of K2PtCl4 in aqueous mediuminvolved the formation of a Pt–Pt bond between a Pt(I) complexand an unreduced Pt(II) complex. However, apart from the latter twostudies, bonding between oxidized Pt species during the nucleationprocess is not well documented. The more widely accepted hypothesisis that Pt nanoparticles are formed by the aggregation of monomericPt(0) atoms once a critical concentration of Pt(0) atoms is achieved.There is therefore a lack of a consensus on the mechanistic formationof Pt nanoparticles and so additional work based on sensitive, time-resolvedtechniques is needed to add clarity.Boita et al. carried out an in situ XAS studyon Pt nanoparticlenucleation and growth using sodium citrate and ascorbic acid as reducingagents.25 Most XAS studies reported so far monitored the reaction processesover a timescale of minutes and hours rather than seconds. While timeresolutions of sub-seconds can be achieved in beamlines equipped withquick extended X-ray absorption fine structure (EXAFS) monochromatorsand energy-dispersive EXAFS beamlines, acquisition of XAS data takesapproximately 10 min in a conventional scanning XAS beamline. MicrofocusXAS beamlines, which fall into the latter category, are additionallycapable of achieving beam spot sizes on the order of a few μmwhich makes them compatible with probing reactions within microfluidicchannels. Here, we have combined microfocus XAS with a microfluidicdevice, with high flow velocities facilitating spreading out of thereaction progress along the length of the device. By probing differentpoints along the channel, we can in effect follow the reaction progressas a function of time. We demonstrate the feasibility of this continuous-flowoperando XAS approach to studying nanoparticle nucleation, which providesnew mechanistic information. Using multivariate analysis techniquessuch as multivariate curve resolution–alternating least squares(MCR–ALS) analysis and principal component analysis (PCA) incombination with FEFF26 calculations ofthe X-ray absorption near-edge structure (XANES) and EXAFS fittingof spectra extracted from the MCR–ALS analysis, we identifytwo intermediates formed during the nucleation process.Operando XAS coupled withcontinuous-flow synthesis using a microfluidicdevice provides an efficient way of studying nucleation processes.11 Platinumnanoparticles were synthesized within the microfluidic device as follows:a solution containing 0.005 M H2PtCl6 in 9.6mL ethylene glycol with 0.35 mL 1 M NaOH and 30 mg PVP was taken ina 5 mL syringe. The solution was introduced into the microfluidicdevice  using asyringe pump. The reaction time was determined from running the reactionex situ. The flow rate was controlled in a way that ensured that thereaction was spread out within the microfluidic device in this time.Too fast a flow rate would mean that the reaction would not be completewithin the microfluidic device, in addition to leading to the formationof localized bubbles which would then distort the XAS spectra. Theflow rate was optimized to be between 0.0625 and 0.25 mL/h as a flowrate lower than this would lead to beam damage (as the same solutionwas exposed to the beam for too long). The device was mounted on analuminum plate and heated to ∼90 °C using an electricheater attached to the back of the aluminum plate . A Mo foil was attached to the aluminum plate tofilter X-ray scatter. The temperature of the device was kept at 90± 2 °C and monitored using a FLIR infrared camera. The initiationof the reaction is taken as the point at which the precursor entersthe device at initiation temperature.Platinum nanoparticlesfor ex situ characterization were synthesized at 90 °C followinga previously reported procedure.E/E of 1.4 × 10–4. The beam spot size in the horizontal direction was 400 μmwhile the vertical size was ∼250 μm. As the flow directionwas in the vertical direction, the beam size was made smaller in thevertical direction. The beam size was optimized so as to minimizebeam-induced reduction of the precursor solution and deposition ofthe nanoparticles onto the walls of the microfluidic device. The devicewas oriented at 45° to the incident X-ray beam and spectra werecollected at the Pt L3 absorption edge at specific pointsalong the microfluidic channel  in the fluorescence yield mode using a four-element Vortex silicondrift detector.XAS measurements were carriedout at Beamline I18, Diamond Light Source, which operates at a 3 GeVphoton energy and 300 mA current. The beamline uses a Si (111) doublecrystal monochromator providing an energy resolution Δ3 Themis 300 transmission electronmicroscope at the Leeds Electron Microscopy and Microscopy Centre(LEMAS), Leeds.TEM measurements werecarried out with a FEI Titan27 Models for the Pt intermediatestructures were optimized within the range-separated hybrid-DFT formalismusing the Gaussian 16 code.28 Electronexchange–correlation interactions were treated with the HSEH1PBEfunctional,30 while we used the sdd basis set for the Pt atoms and cc-pvdz basis setfor all other atoms. The resulting optimized structures were usedas input for XANES calculations using the FEFF 9.0 code.31The XANES spectrawere analyzed usinglinear combination fitting using Athena, part of the Demeter softwaresuite.For the FEFF 9.0 calculations, the full multiple scatteringand self-consistent field cards were set to 8.0 and 7.5 Å, respectively,and the Hedin–Lundqvist exchange correlation potential wasused. All other FEFF parameters were set to default values.35 MCR–ALS was carried out using the MCR–ALS 2.0 toolbox.36The MCR–ALS technique aims to extract the pure constituentsof a mixture when no prior information on the nature or compositionof the mixture is available. It involves performing an ALS minimizationof the differences between the experimental data set and the reconstructeddata matrix equal to the product of the concentration matrix and thepure spectral matrix. This least-squares minimization is carried outtaking into account suitable chemically meaningful constraints. Here,the constraints are the non-negativity of both the spectral and concentrationprofiles.R-space using k2 weighting. Fitted k and r ranges were 3–11 Å–1 and 1–4Å, respectively. Pt–Pt, Pt–O and Pt–Cl scatteringpaths for the fitting derived from H2PtCl6,H2Pt(OH)4 and Pt(I)–Pt(II) dimer structureswere as described in ref (The EXAFS data were fitted using Artemissoftware of the Demetersoftware package. A Hanning type Fourier transform (FT) was used andthe fits were done in d in ref .Figure S2). TEM of the nanoparticles obtainedat the end of the operando XAFS  runs had a slightly wider size distribution when compared to theones obtained by benchtop synthesis in the laboratory: ∼1–7nm sized particles were obtained. The wider size distribution forthe nanoparticles obtained after the operando reaction may be a resultof small gradients in temperature on the microfluidic device.TEM of ex situ platinumnanoparticles as synthesizedthrough reductionusing ethylene glycol were shown to be monodisperse and consistedof particles that were ∼1–3 nm in size  suggestingthat all or at least a significant amount of Pt is present in an oxidizedstate.This process was followed by afurther decreasein the white lineintensity, indicating reduction of Pt37Monitoring of the experiment at finer time intervalsindicatedthat the ligand exchange process under these conditions took between19 and 28 s 2 indicat4+ to Pt2+ within ∼40 s followed by a slower transformationto Pt0 nanoparticles over a period of 40 min. This is consistentwith the platinum nanoparticle formation kinetics reported elsewhere.38 The average oxidation state wasobtained by fitting each spectrum with H2PtCl6, K2PtCl4, and Pt metal as standards. Whilethe linear combination fitting serves to give a rough idea of thechange in the average formal oxidation state as a function of reactiontime, a less than perfect fit was obtained for some of the spectra indicating that [PtCl4]2– is not the right species contributing to theintermediate spectra and that a different or greater number of intermediatesmay be present.39The change in average oxidation state as a functionof reactiontime is plotted in 39 When structural modelsfor the possible intermediate species are lacking, methods for spectraldeconvolution that do not require any prior knowledge of standardspectra can be used to deduce both the number of components contributingto a data set as well as the nature of the pure spectra contributingto each experimental spectra obtained at a particular time point.In a complex systemsuch as this with the absorber atom likely presenting as multiplespecies in solution and therefore present in multiple local environmentsat any time point, each XANES spectrum represents an average sampleover all species.41 to deduce the number of componentscontributing to the data set. The PCA analysis  shows that only the components 2–5, thatis four components, are associated with features that exhibit strongvariations in the region close to the absorption edge and thereforeare structurally incisive signals. PCA analysis only gives the numberof components corresponding to the number of possible species contributingto the data set, but the individual component spectra do not havea physical meaning. In order to extract the possible pure absorptionspectra of the chemical species involved in the reaction and theirconcentration profiles as a function of the progress of the reaction,the MCR–ALS approach was used.42 The results of the MCR–ALSanalysis are shown in We first use PCAanalysis S5 shows analysis S5 shows analysis S5 shows 2. The appearance of component D at 40 s does not fit its overallpattern. We believe it is a consequence of inefficient mixing nearthe channel entrance and possibly also non-uniform heating in thelower part of the device, leading to premature observation of componentD at 40 s.The EXAFS region was examinedto determine thecoordination numbersand approximate bond lengths in the first coordination sphere of thespecies formed during the nanoparticle nucleation process. FT (phaseuncorrected) of the EXAFS of the components extracted from the MCR–ALSanalysis are shown in 2PtCl6 as the structure model, whilecomponent B exhibits Pt–O and Pt–Cl scattering pathsthat are consistent with those in H2Pt(OH)4 andH2PtCl4, respectively. As standard solid-statecrystal structure data are only available for H2PtCl6 and not for the ligand substituted intermediates, scatteringpaths derived from geometry-optimized metal complex structures wereused for the EXAFS fits. The phase-corrected FT for component A givesa Pt–Cl mean distance of ∼2.31 ± 0.01 Å anda coordination number of 6. Fitting of component B with scatteringpaths taken from H2Pt(OH)4 and H2PtCl4 yielded a Pt–O distance of 1.94 ± 0.01Å and a Pt–Cl distance of 2.31 ± 0.01 Å. ThePt–O and Pt–Cl coordination numbers refined to 1.98± 0.2 and 1.96 ± 0.2 suggesting a 4-coordinate structurewith the composition [Pt(Cl)2(OH)2]2–.EXAFS fitting of thecomponents was carried outto confirm thestructures corresponding to each of the components. 14 The coordination numbers and bond distances were allowed to be refinedto get the best fit to the data. The fit yielded a Pt–O bonddistance of 1.987(2) Å and a Pt–Cl distance of ∼2.051(1)Å. The shorter distance obtained for Pt–Cl suggests thatthis correlation is also due to Pt–O bonding. The Pt–Ptbond distance was fitted to ∼2.747(1) Å with a coordinationnumber N of ∼5.6 and a second Pt–Ptbond distance of ∼2.912(2) Å with a coordination numberof ∼3.0. While the presence of the first Pt–Pt distanceof ∼2.75 Å corresponds to the Pt–Pt distance inPt metal, the longer Pt–Pt distance of ∼2.9 Å indicatesthe formation of a Pt–Pt bond within an oxidized cluster species,14 with a longer Pt–Pt distance than inPt metal. –1.Fitting of the EXAFS from the final Pt nanoparticleproduct provedproblematic due to high noise in the experimental data above 6 ÅA comparison of the XANES spectra of the componentsextracted fromthe MCR–ALS analysis with FEFF-calculated spectra of some modelcompounds was also carried out to give further insights on the natureof the species contributing to the pure spectra.2PtCl6 XANES indicates that it best matcheswith H2PtCl6 . Comparison with the FEFF-calculatedH H2PtCl6 8a and is4+ to Pt2+ and arises from the exchange of the Cl ligands by OH ligands. Thedecrease of the white line from that in component A indicates reductionof Pt4+ to Pt2+, while a decrease in the intensityof the feature at 11580 eV indicates that at least some of the Clligands have been exchanged with OH at this stage of the reaction.A FEFF calculation of the H2PtCl2(OH)2 spectrum n) containing Pt–Ptbonds . We also looked atother possibilities such as tetrahedral or square planar Pt structureswith Pt–Pt bonds. Square planar Pt5OH4 clusters containing Pt–Pt bonds upon optimization yieldedcompressed square planar clusters  and these were found to have XANES spectra that matched betterwith the spectrum of component C 2 (38 in showing that reduction from Pt(IV) toPt(II) is a fast process occurring over a time period of ∼100s, while Pt(II) to Pt(0) reduction is a slower process occurring overa time period of ∼100–1500 s 2 7. This e0–1500 s 3. It wou51 while the latter one is attributedto the Pt–Pt complex with each Pt coordinated to ∼4–5ligands. The results of the EXAFS fitting points to a high ligand(Pt–O) to the Pt–Pt ratio that is higher than what wouldbe expected in a square planar oxidized cluster. This observationmay not be significant if there are EXAFS amplitude variations arisingfrom self-absorption effects, but it may also point to the formationof PtO2 in addition to Pt nanoparticles and oxidized Ptclusters. However, the most important result of our study is thatit indicates the presence of two distinct Pt–Pt bond distancesof ∼2.7 and ∼2.9 Å, which suggests the formationof distinct precursors to metal nanoparticle nucleation that are partiallyoxidized clusters with Pt–Pt bonds.PCA and MCR–ALS analysisshows that at least four componentsare involved in the nucleation process, with significant transientcoexistence of species. This coexistence could in part be a featureintrinsic to the microfluidic setup, in which not all the parts ofthe device are uniformly heated, resulting in the lower part of thedevice closer to the metal plate achieving higher temperatures andprogressing further in the nucleation process than the upper partsof the device, resulting in precursors nucleating at different rates.However, the results show quite clearly that from ∼110 s, athird component emerges quite distinctly from component 2. There areseveral reports of formation of metal–metal bonds between oxidizedclusters prior to nucleation of metal nanoparticles. Our simulationof the XANES spectra of oxidized species with Pt–Pt bonds showsthe emergence of a prominent feature at ∼11595 eV, which isnot seen in the other components. EXAFS fitting for component C indicatesthat it includes species with a short Pt–Pt distance of ∼2.75Å (CN—∼5.6) as well as species with a longer Pt–Ptdistance of ∼2.9 Å (CN = ∼3). The former speciesis attributed to Pt nanoparticles with a particle size of ∼0.3nm,Wehave demonstrated that distinct stagesof ligand exchange andredox processes during the synthesis of Pt nanoparticles can be determinedby a continuous microfluidic operando XAS experiment. The timescaleunder the reaction conditions and temperature studied here is ∼20–30s. Detailed analysis of the X-ray absorption data using PCA followedby MCR–ALS and EXAFS fitting of the extracted components suggeststhat nanoparticle nucleation is not just individual Pt atoms comingtogether but is a more complex process involving the formation ofintermediate oxidized clusters with long Pt–Pt bonds (∼2.9Å).


Two-photon (2p) microscopy has historically relied on titanium sapphire pulsed lasers that are expensive and have a large footprint. Recently, several manufacturers have developed less expensive compact pulsed lasers optimized for 2p excitation of green fluorophores. However, quantitative evaluation of their quality is lacking.We describe a simple approach to systematically evaluate 2p excitation efficiency, an empiric measure of the quality of a pulsed laser and its ability to elicit 2p induced fluorescence.By measuring pulse width, repetition rate, and fluorescence output, we calculated a measure of 2p excitation efficiency 2p excitation efficiency varied substantially among tested lasers. The Coherent Axon exhibited the best 2p excitation efficiency (We describe a simple method that both laser developers and end users can use to benchmark the 2p excitation efficiency of lasers used for 2p microscopy. These properties restrict fluorescence generation to a highly localized focal volume .All of the compact 2p lasers tested have a nominal peak wavelength of 920 nm, except for the Menlo Systems YLMO 930, which has a peak wavelength of 930 nm. To ensure that this difference in wavelength would not affect the amount of excited fluorescence and the calculated pulse quality, the methods in Secs. Click here for additional data file.