The occurrence and development of coronary heart disease (CHD) are closely linked to fluctuations in blood glucose levels. While the efficacy of intensified treatment guided by HbA1c levels remains uncertain for individuals with diabetes and CHD, this review summarizes the findings and conclusions regarding HbA1c in the context of CHD. Our review showed a curvilinear correlation between regulated level of HbA1c and therapeutic effectiveness of intensified glycemic control among patients with type 2 diabetes and coronary heart disease. It is necessary to optimize the dynamic monitoring indicators of HbA1c, combine genetic profiles, haptoglobin phenotypes for example and select more suitable hypoglycemic drugs to establish more appropriate glucose-controlling guideline for patients with CHD at different stage of diabetes. Cardiovascular diseases (CVDs), encompassing ischemic heart disease, stroke, heart failure, peripheral arterial disease, and an array of other cardiac and vascular ailments, constitute the leading cause of mortality globally, greatly impairing individuals’ quality of life , 2. IschType 2 diabetes (T2DM) is the well-known economic and social burden for today’s real world . It is wTherefore, our endeavor involved summarizing the divergent perspectives on the use of HbA1c as a predictor of clinical outcomes in patients with both T2DM and CHD. We also examined the inconsistent advantages of intensified treatment targeting HbA1c reduction in this specific population. Additionally, we cited potential factors that might contribute to this lack of consistency.Our review firstly investigated the inconsistent benefits of intensive treatment for patients with both T2DM and CHD, specifically focusing on the population receiving PCI or CABG. In this section, the search terms used were “HbA1c” OR “Glycosylated Hemoglobin” AND “coronary artery disease” OR “CHD” AND “T2DM” OR “Type 2 diabetes mellitus” AND “intensified glycemic control” OR “intensive glycemic control” AND “PCI” OR “Percutaneous Coronary Intervention” AND “CABG” OR “Coronary Artery Bypass Grafting”. We then focused on the curvilinear correlation between HbA1c and CVD risk reduction. The search terms in this section included “J-shaped” OR “U-shaped” OR “curve relationship” AND “intensive glycemic control” OR “intensified glycemic control”. We further listed the factors that might impact the inconsistent benefits of intensified glycemic control. In this section, the search terms were “HbA1c variability” AND “haptoglobin phenotype” OR “HP phenotype” AND “GLP-1 RA” OR “Glucagon-like peptide 1 receptor agonist” AND “SGLT2i” OR “Sodium-glucose cotransporter 2 inhibitors” AND “MACE” OR “major adverse cardiovascular events” AND “hypoglycemia”. There were no restrictions on study type, language, or time. We utilized the electronic databases PubMed and Web of Science. Two authors (Jing-yang Chen and Dong Yin) retrieved the full texts of these studies and assessed them for eligibility. Disagreements were resolved through discussion.Typically, the HbA1c level in a human body should be maintained within the range of 4–5.7%. When healthcare professionals implement intensified glycemic control, they are referring to interventions such as lifestyle modifications and medication regimens that aim to reduce a patient’s HbA1c level to a range like that of a healthy individual, which is generally below 6.0–7.0% . Poor glIt seems that no matter for short-term (≤ 3–5 years) or long-term (≥ 5 years) benefit, lowering HbA1c level fails to bring consistent result. Generally, most of research approve the short-term benefit of intensified HbA1c control. The PROactive study found that for T2DM elder patients (≥ 70 years old) with a history of macrovascular disease, intensified glycemic control (lowering HbA1c 0.8% in average) significantly reduced the risk of MACEs and all-cause mortality . The ADVThe uncertainty of the benefits of intensified treatment is also reflected in studies of percutaneous coronary intervention (PCI) patients, with inconsistent conclusions mainly regarding the timing and intensity of glycemic control. Corpus et al. demonstrated that patients who achieved optimal glycemic control (HbA1c level ≤ 7.0%) prior to undergoing PCI experienced significantly lower risks of target vessel revascularization, cardiac rehospitalization, and recurrent angina compared to those with poor glycemic control . This fiThe discrepancy is also evident in patients who underwent coronary artery bypass grafting (CABG). It is noticeable that many studies only focused on preoperative HbA1c level, which mirrors the glycemic control before CABG. Pre-surgery HbA1c level either fails to predict in-hospital mortality or acute postoperative adverse events (within 30 days) , 47. ButIn 1998, Bonora et al. conducted a study that revealed a U-shaped or J-shaped relationship between serum insulin levels and coronary heart disease in the general population . InteresWhether it follows a U-shaped –60 or a The uncertainty surrounding the benefits of different studies makes it challenging to manage patients with T2DM in clinical settings. Factors such as the duration of diabetes, preexisting macrovascular disease, occurrence of hypoglycemic events and significant complications should be considered when deciding on the appropriate glycemic management strategy . It is eHbA1c, also known as glycated hemoglobin, has emerged as a potentially valuable biomarker of cardiovascular risk and prognosis in patients with diabetes. However, the existing body of evidence is currently insufficient to establish consistent findings on this matter. We surmise that researchers’ inability to comprehensively understand the exact glucose fluctuations of patients throughout the follow-up period leads to incomplete or inconsistent findings. We hypothesize that three potential factors may contribute to the inconsistency of the results: variability in HbA1c levels, the phenotype of haptoglobin and the selection strategy for hypoglycemic drugs.Blood glucose fluctuation is an emerging metabolic index that has gained attention in recent years. It can be categorized into two types: long-term blood glucose fluctuation and short-term blood glucose fluctuation. The latter can be evaluated by continuous blood glucose monitoring  or blood glucose levels during hospitalization to predict poor cardiovascular outcomes –73. The HbA1c is widely recognized as a reliable measure of long-term blood glucose variability. In recent years, the concept of Visit-to-visit HbA1c variability (VVV of HbA1c) has been proposed by some scholars to track HbA1c fluctuation at different treatment periods –82. “VisFor patients who undergo PCI, the post-procedural maintenance phase is an extended and protracted period. During this phase, long-term complications, such as restenosis, frequently occur after a significant duration. Therefore, we contend that the prediction of prognosis should be based on dynamic changes in HbA1c levels during the follow-up. Yang et al. discovered that the HbA1c variation rate (CV), standard deviation (SD), and VIM levels were closely related to the risk of stent restenosis in PCI and stenting patients, subjects with HbA1c ≤ 7% exhibited more severe restenosis when HbA1c-CV was high, as compared to subjects with HbA1c > 7% . This coThe dynamic observation of HbA1c levels in patients can better demonstrate the significance of HbA1c in the management of cardiovascular complications. Nonetheless, it is crucial to note that the predictive effectiveness of different indicators may differ. Critchley et al’s study revealed that HbA1c-CV had a significant dose-response relationship with all-cause mortality in patients with diabetes compared to traditional average HbA1c, whereas average HbA1c exhibited more precise predictive value in predicting the risk of mortality and hospitalization in individuals with coronary artery disease . CeriellRegrettably, at present, there is no universally recognized “golden standard” to quantify the variability of HbA1c, and there exists a lack of consensus regarding its potential clinical significance. Therefore, it is essential to determine an accurate, efficient, feasible, and widely recognized classification of the various HbA1c measurements as soon as possible to further promote its importance in prevention, prognosis, and treatment guidance. Additionally, further randomized controlled trials are required to evaluate and verify the predictive value of HbA1c variability in the risk of cardiovascular events in patients with pre-diabetes and patients without diabetes.Haptoglobin (Hp) is an acidic glycoprotein, which widely exists in human and various mammalian species and mainly synthesized in the liver. The main function of Hp is to combine with serum hemoglobin to form a stable Hp-Hb complex . BrieflyHp has two genotypes, Hp1 and Hp2, with allele frequencies of approximately 40% and 60% worldwide respectively . There aThe Hp-Hb complex is primarily captured by the scavenger receptor CD163, which is predominantly expressed on M2 macrophages, and subsequently undergoes decomposition . Though Consistent with the result of in vitro study, several studies have also shown that diabetes interacts with Hp phenotype and the association of Hp 2-2 with CVD is observed only in population with diabetes –119. In The difference of Hp phenotype may explain the differences in efficacy of intensified blood glucose control tests among subjects of different regions and races . RecentlThe heightened risk associated with intensive glycemic control, particularly in patients undergoing PCI or CABG, primarily stems from the potential occurrence of hypoglycemia. We propose that medications with minimal hypoglycemic risk should be prioritized as the preferred option for achieving intensive glycemic control in such patients. Considering the widespread utilization and low hypoglycemic risk, we particularly consider two sorts of newly developed hypoglycemic agents: Glucagon-like peptide-1 receptor agonists (GLP-1 RAs) and Sodium-glucose cotransporter 2 inhibitors (SGLT2is).GLP-1 RAs represent a novel class of hypoglycemic drugs that operate by activating the GLP-1 receptor. These agents enhance insulin secretion in response to glucose levels, suppress glucagon secretion, and delay gastric emptying , reducinUp to now, scientists have proven that some of GLP-1 RA plays a role in protecting patients with T2DM and CVD from MACEs, including liraglutide , 135, efFurthermore, a study conducted by Nathan et al. revealed that, when combined with metformin, liraglutide exhibited a significant capacity to decrease the risk of MACEs in patients with T2DM, in comparison to glargine, glimepiride, and sitagliptin . As a reSGLT2is, a novel class of medications used for diabetes management, operate by inhibiting the reabsorption of glucose in the kidneys. This mechanism results in an elevated excretion of excess glucose through the urine . SGLT2isDespite being primarily utilized for glycemic control in patients with T2DM, SGLT2is now have showed notable benefits in terms of heart protection and been recommended for patients with heart failure, irrespective of their T2DM status –151. HowInterestingly, while SGLT2is have been shown to significantly lower HbA1c levels, including HbA1c variability , it is nOverall, based on our analysis, we propose that GLP-1 RAs are considered as an optimal class of hypoglycemic drugs for implementing safer intensive glycemic treatment in patients with T2DM, while SGLT2i may be a suitable option, particularly for patients with acute or previous MI, no matter with T2DM.It is crucial to underscore the importance of continuous HbA1c measurement as it represents a dynamic and fluctuating indicator. Many studies have only conducted HbA1c measurements at baseline, which does not offer a complete monitoring of patients’ glycemic control. Long-term follow-up is necessary to fully comprehend the association between blood glucose levels, their stability, and the risk of coronary disease. Dynamic observation of HbA1c can provide a more comprehensive understanding of this relationship. However, for demonstrating the predictive value of acute or perioperative complications, short-term HbA1c measurements may be sufficient. Nonetheless, to obtain more objective data support, it is ideal to have multiple HbA1c measurements throughout the course of the study.Further verification and improvement of the predictive efficiency of HbA1c variability is necessary. Though HbA1c variability shows weak and inconsistent associations with complications in the early stages of follow-up, its significance becomes more apparent over time . We propAn alternative approach to improve the HbA1c prediction model is to use the hemoglobin glycosylation index (HGI), defined as the measured HbA1c minus predicted HbA1c, which combines fasting blood glucose (FBG) and HbA1c to overcome the incompatibility between HbA1c and average blood glucose . The preTo improve the predictive efficacy of coronary heart disease events, individual HbA1c needs to be combined with genes. A Mendelian randomized analysis study on HbA1c genetically confirmed a causal relationship between HbA1c and CAD risk, driven not only by blood glucose but also by factors unrelated to blood glucose, such as age, sex, race, and red blood cell content , 167. ThThis review highlights the uncertain benefits of intensified treatment based on HbA1c levels for diabetes patients with CVDs, indicating a curvilinear trend in the correlation between HbA1c level and therapeutic benefit. Promising advancements in glycemic management can be achieved by incorporating factors such as HbA1c variability, individual characteristics such as Hp phenotype, and the appropriate selection of hypoglycemic drugs. This comprehensive approach holds the potential to optimize treatment outcomes and improve overall patient care.


Optimization of pump–probe signal requires a completeunderstandingof how signal scales with experimental factors. In simple systems,signal scales quadratically with molar absorptivity, and linearlywith fluence, concentration, and path length. In practice, scalingfactors weaken beyond certain thresholds  due toasymptoticlimits related to optical density, fluence and path length. Whilecomputational models can accurately account for subdued scaling, quantitativeexplanations often appear quite technical in the literature. ThisPerspective aims to present a simpler understanding of the subjectwith concise formulas for estimating absolute magnitudes of signalunder both ordinary and asymptotic scaling conditions. This formulationmay be more appealing for spectroscopists seeking rough estimatesof signal or relative comparisons. We identify scaling dependenciesof signal with respect to experimental parameters and discuss applicationsfor improving signal under broad conditions. We also review othersignal enhancement methods, such as local-oscillator attenuation andplasmonic enhancement, and discuss respective benefits and challengesregarding asymptotic limits that signal cannot exceed. Variations of the techniquecan differ by the number of independent variables resolved in a spectrum.In the simplest case, transient absorption spectroscopy resolves aspectrum as a function of the probe frequency and time delay betweenthe pump and probe, enabling observation of how certain chemical speciesor populations change as a function of time. Transient absorptionhas seen a variety of applications, with examples ranging from photoinducedprocesses in molecular biology,6 polaritons in waveguides,7 and energy or charge transfer in nanostructures9 and photovoltaics.10Ultrafast pump–probe spectroscopyis a powerful tool formeasuring the structural dynamics and functions of molecules rangingbetween the millisecond and attosecond time scales.11 Applications of 2D spectroscopy are far reaching, withexamples ranging from investigating protein folding motions,14 protein structure and dynamics,16 chemical reactions,18 solvent dynamics,20 mechanisms of charge separationand energy transfer in photosynthetic systems,25 and excitons in semiconductors.26Time-resolvedtwo-dimensional (2D) spectroscopy is another variationof the pump–probe technique that contains all the same informationas transient absorption, while also adding more by resolving the spectrumas a function of both pump and probe frequencies. This enables disambiguationof combination bands, overtones, and Fermi resonances and allows forobserving how populations evolve over time through correlations infrequency. Polarization schemes offer further insight into molecularstructure through the relative orientation of modes.27 This requiresa complete understanding of how signal scales with experimental factors.In the simplest case, signal scales quadratically with molar absorptivityand linearly with fluence, concentration, and path length. But conventionalscaling laws do not account for asymptotic limits related to opticaldensity, fluence, and path length. For example, the upper limit ofsignal for a two-level system under incoherent excitation is |ΔOD|≤ OD, with equality corresponding to 100% transmission of theprobe beam through the pumped sample. Furthermore, this upper limitis only valid for small optical densities (<0.1), but as we discusslater, increasing the optical density of a sample leads to higherattenuation of the pump beam and therefore weakens signal scalingfactors. For example, increasing the optical density of a sample from0.05 OD to 0.1 OD leads to a 1.9× increase in signal, but anincrease from 0.5 OD to 1.0 OD merely leads to a 1.3× increasein signal. Accounting for solvent absorption and fluence further complicatesscaling factors. Hence, conventional scaling laws are inadequate beyonda very limiting case of experimental conditions, and a more generalizedformulation is necessary for accurately understanding how signal scaleswith experimental factors.The improvement of signal-to-noise in pump–probespectroscopyremains an ongoing challenge in order to expand the range of systemsaccessible by the technique.29 Decades later, Jonas and co-workers explored the effect of opticaldensity on signal both experimentally and theoretically through theuse of beam propagation models based upon Maxwell’s equations32 or a modified form of Beer’s law to account for third-orderresponse.34 In addition to high optical density, earlystudies of photon echoes also identified a nonlinear relationshipbetween signal and intensity (or fluence).35 For a two-level system, the ratio of ground state to excited stateoscillators cannot exceed unity due to competing processes of absorptionand stimulated emission, which is the basis for fluence saturation.Early studies of photon echoes identifieda nonlinear relationshipbetween signal intensity and optical density due to accumulated attenuationof the excitation beams passing through the sample.36 including an approximation to another solution publishedby Cho et al.33 A number of publicationshave reported formulas for estimating relative quantities of signal,concentration, and cross sections but require a secondary measurementor calibrant to compute absolute quantities.40 Here we identify concise formulas for calculating absolute valuesof signal in cases of high optical density and/or high fluence givenknown quantities of molar absorptivity, concentration, path length,pulse energy, spectral bandwidth, line width, beam waist at focus,and optical density of the solvent. Furthermore, we analyze theseformulas in the context of experimental strategies to enhance signaland identify asymptotic limits that signal cannot exceeded.Many of the formulas here build on previous work,Following is the layout of the manuscript: 1spot size41beam waistRayleigh lengthaperture diameterThe Gaussian beam isa solution to the scalar wave equation thatassumes a laser beam primarily propagating in one direction (2Fεa2caF is the fluence(energy/area) of the pump beam, εa and ca are the molar absorptivity and concentration of theanalyte, and 33 Here we present a shortened version of theirmodel based on Beer’s law that utilizes integral approximationsto generate a concise formula. For simplicity’s sake, our derivationassumes a simple two-level system. We also note that similar shortenedformulas have recently appeared in literature.40The simplest modelfor pump–probe signal assumes scalingof ΔOD ∝ 38We define signal, ΔOD, as the change in opticaldensity ofthe probe pulse between pump “on” and “off”,defined in ca in Nabs = Nres(1 – 10–OD), where Nres is the number of incidentphotons resonant (overlapping) with the oscillator’sline shape. For an absorbing solvent, ΔNabs is reduced by a factor of a and ODs are optical densities of the analyte and solvent and ODT is the sum total of the two. Hence, the number of photons absorbedby the analyte is Nabs and Vobs to estimate the average concentration ofanalyte excited by the pump beam. Division by Avogadro’s number NA in ca carries unitsof molarity.The factor of ΔNres,we assume the pumpspectrum and oscillator line shape are both Gaussian, with FWHM denotedby Δbw and Δlw, respectively. Furthermore,we assume that Δbw ≫ Δlw,which enables the approximation Ntot is the total number of pump photons. Ntot is related to the pump pulse energy by Ntot = Epump/hcν̃,where h is Planck’s constant, c is the speed of light and ν̃ is the center frequencyof the pump spectrum. These expressions combine in Nres.To calculate ca from Nres from T → 0. The term“competitive” refers to any scenario in which ODT is significant due to competing absorption by the solvent(ODs), the analyte (ODa), or both. The secondnumber, F̃/Fsat, is a measure of saturation induced by the pump beam. In fact, thequantity approximates twice the relative change in population, F̃/Fsat ≈ 2Δca/ca, in the lowfluence and low OD limit.We recast ΔODinto FεaOD, as“linear” becauseit assumes ΔOD is bilinear in optical density and fluence forconstant εa. ca, a. As shown in panelA, with respect to changes in concentration or path length, the twomodels are similar below OD < 0.1 and then diverge with increasingOD due to the asymptote imposed by competitive absorption of the pumplight. This stems from the factor of (1 – 10–OD) in 42 The dependency on εa is similar to a higher order.Because a–ε)εa, ΔOD(εa) transitions between quadratic scaling below ODa < 0.1 to linear scaling above ODa > 0.5. Due totheimpact of OD on signal scaling, we divide our discussion in We refer to the conventional scalinglaw, ΔOD ∝ 33 These results assume a two-level system in the low fluence limit,pumped and probed by identically sized Gaussian beams. We find strongagreement between the competitive absorber model and the Jonas model(>90%) for systems with ODa < 0.3 measured by largespectral bandwidths (2× line width or greater), and we stillfind good agreement (>60%) in more extreme cases. The agreementbetweensolutions is explainable through integral approximations. The exactsolution to Beer’s law is ΔOD = −log10(Pron/Proff) where Pron and Proff correspond toeqs 9 and 10 in ref . Then we approximate the integral in eq A7, ref (puα0 exp(−α0Z)2σΔbw evaluatedat the line shape peak, and a factor of 2 accounts for GSB + SE. And,finally, the substitutions Fsat = hcν̃/2σ and integration over Z yields the same expression as a and a and overestimate when the bandwidth of the pump spectrum is comparableto the line width of the line shape.3The competitiveabsorber model is valid at low fluence for anyoptical density. Now we discuss the saturable absorber model, whichis valid at low optical density for any incoherent excitation fluence.At low intensities, chromophores act as “linear” absorberswhich means that, given an infinitesimally thin sample, the numberof photons absorbed by the sample is linearly proportional to thenumber of photons incident upon it, i.e., the Beer–Lambertlaw. The linear absorber model works well at low intensities, typicalof an FTIR light source, where just a small fraction of oscillators(<1%) absorb light. But given a sufficiently intense beam of light,the Beer–Lambert law predicts a sample could absorb more photonsthan are oscillators in solution. Therefore, the linear absorber modelis unphysical at high intensities. Alternatively, the saturable absorbermodel accounts for saturation effects due to finite population andconverges to the linear absorber model in the low intensity limit.43 The model yields slightly different formulasin each limit and work in different units: intensity (energy/area/time)for the continuous wave case and fluence (energy/area) for the ultrafastcase. Assuming a femtosecond light source, we restrict our analysisto the ultrafast limit.The saturable absorber model has two limiting cases. One for continuouswave, where the lifetime of the oscillator mode is much longer thanthe duration of the light source, and the other for ultrashort pulseswith durations much shorter than the lifetime of oscillator mode.43 As with the competitive absorber modelbefore, only the resonant fluence, F̃, definedin F̃) versus F̃/Fsat in F̃/Fsat ≲ 10–2), OD isconstant, consistent with linear absorption, and then precipitouslydrops after F̃/Fsat ≈ 10–1 and approaches an asymptotic limitof OD = 0 above F̃/Fsat ≳ 5.The saturable absorption model assertsthat optical density decaysexponentially with fluence, as defined by F̃/Fsat)OD. These formulassuggest a strong connection between saturable absorption and pump–probesignal. Indeed, for a two-level system, the change in population followsa saturation curve. Therefore, the two subjects are closely related.While this point appears to have been understood several decades ago,44 the connection between the two subjects hasrarely surfaced within the ultrafast community since. We hope to revivethis perspective given the remarkably simple formulation it providesfor computing ΔOD. One limitation with this formulation is itdoes not account for subdued scaling effects in high OD samples dueto competitive absorption.F̃/Fsat. Not surprisingly, |ΔOD|/OD(0) islinear in the weak fluence limit but appears increasingly nonlinearat higher fluences. Generally speaking, 45 However, one way to avoid up-pumpingis positively chirping the pump pulse.46 Alternatively, when a pulse shaper is available, applying a maskthat removes pump light coincident with the 1–2 transition prevents up-pumpingby disabling excitation to ν = 2 and higher.According to 6 in a regular pump–probeexperiment45 and Morichika et al. for plasmonicenhancement of W(CO)6 in reflection mode,47 both equate to a saturable limit of 45 Another interesting example isazide in FDH, where 50 While the effects of saturationon measuring spectral diffusion have not been studied, for measurementsin the  domain, it is unlikely that a 10% nonlinearity is goingto change spectral diffusion in any meaningful way.Sampling a few examples from literature, data reportedby bothWitte et al. for W(CO)–1 cm–1) have ratios of 52 which leaves room for ∼20× and ∼4× enhancementby increasing the pump fluence.These examplesshow that strong chromophores such as azide andmetal carbonyls have either already or nearly reached a saturationpoint on standard 2D IR apparatuses and further increases in pumpfluence are unlikely to improve R, is proportional to the product of the electric field amplitudeand the transition dipole moment. Although the saturable absorbermodel assumes an incoherent pump source, the model is still appropriatefor coherent light sources when ωR is smaller thanthe homogeneous line width because dephasing destroys the Rabi oscillationbefore it can significantly manifest.41Finally, it is worth notingthe existence of Rabi oscillations.Upon excitation by a coherent light source, a two-level system well-isolatedfrom dephasing and relaxation processes will oscillate in populationbetween the ground and excited state as a function of fluence. Thefrequency of oscillation, known as the Rabi frequency ω4z in the sample is Δca/ca = (1/2)(F̃/Fsat)10–OD where F̃ is the resonant fluence prior toentering the sample. This fails at high fluence because (F̃/Fsat)10–OD > 1impliespopulation inversion. To account for saturation, we replace the quantities F̃/Fsat and OD with theirsaturable counterparts from ca/ca = (1/2)(1 – f)10f ·OD–, where f = exp(−F̃/Fsat). This is consistent with the competitive absorber model in thelow fluence limit, converging to (1/2)(F̃/Fsat)10–OD, and the saturable absorber model in the high fluence limit, convergingto 1/2. Then, following ca = (ca/2)(1 – f)10fcaεaz– over z and multiplyingby −2εa, which gives a for F̃/Fsat ≫1, F̃/Fsat ≪ 1, T ≪ 1, and F̃/Fsat ≪ 1 andODT ≪ 1.Themodels reviewed up to this point are suitable for either highoptical density or high fluence, but neither model is appropriateunder both conditions. Here we derive a model suitable under bothconditions which we call the saturable competitive absorber. The trickhere is finding an expression that computes the change in populationaccounting for both saturation and high optical density. For the competitiveabsorber model, the change in ground state analyte from excitationby the pump beam at point 325Analysis of the competitive absorber model in 5.1f is the focal length, λ isthe center wavelength, and D is the diameter of apertureat which 99% of light transmits just prior to reaching the focusingoptic.41 For example, using f = 50 mm parabolic mirrors, D = 20 mm irises, andλ = 5 μm light, f-number” of an optic.f-number(f/#), defined in D at which99% of light transmits, but the smallest possible beam waist f-number. Several vendors sell f/1 off-axisparabolic mirrors and f/0.5 ZnSe aspherical lenses.However, faster optics are unlikely due to manufacturing challenges.D with the optic’s diameter of apertureto achieve the smallest possible beam waist D smaller thanthe optic diameter, then Regardless of the optic chosen, it is important to match the aperturediameter M2).53 Spherical aberrations are eliminatedby replacing spherical optics with aspherical lenses or parabolicmirrors. Nanometer deviations in optical surfaces imprint wavefrontdistortions on laser beams. Manufacturers characterize surface flatnessusing a monochromatic reference, e.g., λ/4, where λ isusually 633 nm. Common values for surface flatness range from λ/2≈ 300 nm to λ/10 ≈ 60 nm. It is important to recognizethat wavefront errors accumulate over optics, e.g., 16 reflectionsoff λ/2 mirrors add in quadrature to 300 nm × Note that 5.2a in the weak absorption limit and linearly in the high absorptionlimit. In terms of general site-specific labels in proteins, the IRtransparency window of water effectively limits the available chromophoresto those listed in 5.33) into two lower energy photons,commonly referred to as the signal (ω2) and idler(ω1) where ω3 = ω2 + ω1.68 The photon conversionefficiency of an OPA, defined in P3(in)and input seed power much smaller than either the output signal power P2(out) or output idler power P1(out), the conversion efficiency is well-approximatedby either of the latter two equalities in 2.69 Noticethat power amplification stages, e.g., second pass in BBO or DFG inAgGaS2, tend to run upward of ∼30% while seed amplificationstages, e.g., first pass in BBO, tend to run at ∼10%. Overall,the gross output power primarily comes from the power amplificationstages (2nd pass in BBO and DFG in AgGaS2), so the totalmid-IR photon conversion efficiency relative to the input Ti:sapphirepump is ∼(0.28)2 = 8%, which is typical in our experience(∼30 mW of mid-IR from ∼2200 mW of second pass pump).Beyond Ti:sapphire, some 2D IR groups have acquired 100 kHz Yb laserswith remarkable performance and applications.75In the lowfluence limit, f noise. Overall, the two lasers should exhibit comparablevalues of SNR. In practice, the comparison is more complicated afterfactoring in specifications known for commercially available lasers,such as slightly higher average powers for 100 kHz systems, or implementationof calibrated referencing schemes known to remove virtually all shot-to-shotnoise.77 And, finally,we note that only the fraction of the frequency spectrum that is resonantwith the analyte line shape contributes to pump–probe signal,which is important when comparing two different light sources withdifferent spectral bandwidths.Note that ΔOD does not necessarily scalewith the power but,rather, the energy or fluence per pump pulse. The distinction is significantfrom an SNR standpoint when comparing lasers with equal average powerat different repetition rates. Consider, for example, a hypotheticalcomparison between a 1 kHz and a 100 kHz laser with identical averageoutput powers. The 1 kHz laser yields a 100× larger signal owingto a larger pulse energy. On the other hand, the 100 kHz laser reducesnoise first by a factor of 10× from 15 = 26% power loss for s-polarizedlight and 1 – 0.9615 = 46% power loss for p-polarizedlight. Pulse shapers are known to attenuate pump light by 65–80%,depending on the circumstances. Taken altogether, for a typical 2DIR pulse shaping apparatus, one should expect the pump power at thesample to be 5–20% of the initial power measured at the mid-IRsource.There are several sources ofattenuation following the mid-IR lightsource, some of which are manageable. First is compounded reflectionlosses. For example, assuming gold mirror reflectivities of 98% fors-polarized light and 95% for p-polarized light, 15 reflections wouldaccumulate 1 – 0.9841 Here λ is wavelength and D is the diameterof aperture for which 99% of light transmits at the two ends of thecolumnated range. For example, a 5 μm wavelength beam with diameterof aperture D = 10 mm can propagate 6 m before diverginglarger than 10 mm again.The last source of attenuation worth noting is accidentaldivergenceand/or long-range diffraction inherent to laser beams. Many 2D IRapparatuses propagate ∼5–8 m between the mid-IR sourceand sample cell. At these path lengths, small perturbations in thedivergence angle might easily lead to major aperture losses in pumplight. Furthermore, the diameter of the mode must be large enoughto ensure the collimated range (twice the Rayleigh length) of themode is long enough to reach the lens or parabolic mirror that focuseslight into the sample. 5.4ca in the weak absorption limit orbounded-exponentially in the strong absorption limit. For proteinsincorporating a single, site-specific residue as an analyte reporter,the absolute upper limit for concentration is equal to the inversevolume of a single monomer of the protein. By estimating the volumeof a protein from its molecule mass and density,78 and ignoring excess volume occupied by water between proteinmolecules, the maximum concentration of a crystalline protein in unitsof molarity is approximately equal to 1.37 (M·kDa)/m(kDa), where m is the protein mass in units of kDa.If one then assumes the void between crystalline molecules is ∼1/3of the crystal volume, then 1.37 becomes 1 and a very handy rule ofthumb is that the maximum concentration of a crystalline protein inmolarity is equal to the inverse mass in kDa. A typical protein monomerweighs anywhere from 10 to 50 kDa, which suggests an upper limit of ca = 20–100 mM for crystalline protein.In practice, proteins start nucleating well before that point, whichmeans the upper limit for a nonaggregate, solution phase measurementis somewhere between 0.1 and 10 mM, depending on the protein and experimentalconditions.5.541 oralternatively, in terms of the focal length f andaperture diameter D assuming M2 = 1. For example, given h length 9. For coT ≪ 1 and T ≪1 and energy of the pump pulse rather than the physicaldimensions of the observation volume, which is more intuitive. Inpractice, independence of ΔOD on the beam waist is unlikelymerely because most samples have significant absorption from eitherthe analyte or solvent. In moderate to strongly absorbing samples,the dependence of ΔOD on the inverse beam waist should be sublinearassuming an optimized path length.In practice, one may find that the optimal pathlength is significantlyless than 5.6scs and linearly in the denominator, the net effect approximatinga hyperbolic decay. scs, with ΔOD normalized to the Rayleigh length value foreasier assessment. Stars denote points at which εscs.The results show that signal reaches 90% or more of the asymptoticlimit at path lengths equal to the inverse absorptivity of the solvent.More generally, extending path lengths longer than ODT =1 will yield diminishing gains because additional excitation is negligiblebeyond 90% attenuation of the pump beam. The necessity for low solventmolar absorptivity often limits the measurable frequency range fordilute analytes dissolved in water to between 2000 and 2600 cm–1. In that region the water bend+libration combinationband peaks at 2100 cm–1 with εs ≈ 3.1 M–1 cm–1,79 corresponding to εscs ≈ 170 cm–1. In thatregion the optimal path length for a dilute analyte would be ∼60μm.The numerator of 6Esig ∝ χ(3)IpumpEprobe where χ(3) isthe third-order susceptibility of the sample, Ipump is the intensity of the pump, and 80 At the detector, the nonlinear signal interfereswith the local-oscillator field ELO suchthat the total intensity is Recognizing ΔOD as a heterodyned signal enables a cleverform of signal enhancement by interference of light. From a theoreticalstandpoint, spectroscopists understand pump–probe signals moreaccurately as the third-order nonlinear response of the sample. Theelectric field of the third order response is Iprobe = ILO restrictsthat factor to after the sample wherethe probe beam then acts as the local oscillator. This strategy hasbeen implemented in a variety of ways which work by selectively attenuatingthe local-oscillator field faster than the signal field after thesample, including a simple postsample polarizer,81 a Sagnac interferometer,82 orsome form of attenuated reflection.83 Inpractice, the primary limitation with local-oscillator attenuationis the heterodyned signal reaches a value too small for the well depthof a detector which leads to increasing noise.Therefore, accountingfor local oscillator attenuation just requiresmultiplying ΔOD by 7Surface enhancedspectroscopies work by massively enhancing theelectric field around the surface of small metal structures (nm toμm). The field enhancement arises from the collective oscillationof free metal electrons in response to an incident laser beam whiletrapped in a metal structure comparable in size to the wavelengthof light. The term “plasmon”, which refers to the quantizationof the oscillating electrons, stems from the observation that conductionband electrons are well-modeled as a confined gas but act as chargedparticles in the presence of an electric field and, hence, behavesomething like plasma.11 Plasmonic enhancementof linear spectra consists of four interactions: (1) incident lightexcites a surface plasmon on the metal structure, (2) the near-fieldof the surface plasmon excites the oscillator, (3) the oscillatoremits a signal that interferes with the surface plasmon (known as“back action”), and (4) the surface plasmon, drivenpartially by the back action, emits a signal that interferes withthe incident light. Though other interaction pathways among the incidentlight, plasmon, and oscillator do occur, the magnitude of enhancedsignal from this four-step pathway is largest by at least an orderof magnitude.84 The interaction pathwayfor 2D IR similarly involves a plasmon excitation and subsequent near-fieldinteraction with the oscillator for each of the three incident fields,followed by the back action of the third-order response onto the plasmon.86 Plasmonic enhancement does come at the cost of scattering and absorptionof the incident fields, and the range of enhancement only extendsa few tens of nanometers from the surface into the sample.89 That being said, the enhancements in signal can be tremendous foranalytes near enough to the surface.Spectroscopists understand linear spectroscopyas two consecutiveinteractions between an oscillator and light: (1) incident light excitesthe oscillator, and in response, (2) the oscillator emits a “signal”that destructively interferes with the incident light, which appearsas absorption.2 enhancementof 2D IR signal from thiol-functionalized gold nanoparticles.90 In 2015, Selig, Siffels and Rezus reported a∼104 enhancement of 2D IR signal using randomlyspaced micron-long gold nanoantennas, lithographically deposited onCaF2.91 Shortly thereafter,Kraak and Hamm found that sputtering a 1–3 nm gold layer onthe surface of an ATR prism enhanced the 2D IR signal of monolayersby 50–450 fold.93 Since then, additional studieshave been reported on regularly spaced nanoantennas97 and trimer nanoantennas99 yielding 104–105 enhancement. Otherstudies have shown that combining plasmonic enhancement with an attenuatedreflection geometry (SEAR-2D IR) enables an added mechanism for enhancementby means of local oscillator attenuation.101 In fact,Morichika et al. reported a 107 enhancement of 2D IR signalby detecting the back scattering off of gold nanoantenna arrays.47 With the exception of Donaldson and Hamm’sgold nanoparticles, virtually all surface enhanced 2D IR experimentsreported to date are of thin film samples where the surface densityof chromophores is comparatively high relative to mM bulk samples.Unfortunately for bulk samples, the enhancement only extends a fewtens of nanometers from the surface into the sample,89 which effectively reduces the path length of bulk measurements bya few orders of magnitude. This questions the applicability of plasmonicsto conventional bulk systems, particularly given the lack of demonstrationthus far.In 2013, Donaldson andHamm first reported a 10102 The origin of this phenomenon is from two complementary phase delays:the first is acquired between the incident field that excites theplasmon and the near-field response that excites the oscillator; thesecond is acquired by the complement back action process.86 This effect is prominent when the size of the metal particle iscomparable to the wavelength of light, such as in micron sized nanoantennas,103 but appears negligiblein reports using 1–3 nm gold particles deposited by sputtering.101 As pointed out by Chuntonovand Rubtsov, the plasmonic Fano resonance only occurs along the probeaxis and is well-modeled by classical electromagnetic theory.85 This can be explained by the 2D IR signal carryinga ±ϕ1 ∓ ϕ2 + ϕ3 phase dependence such that the phase delay cancels alongthe pump axis but remains along the probe axis.86 In fact, Tek and Hamm have shown how the Fano interference can becorrected by simply applying a zero-order phase shift along the probeaxis.104One complication introduced by plasmonic enhancementin both linearand nonlinear measurements is a Fano-like line shape.One unanswered questionregarding plasmonic enhancement is whethersignal enhancement comes at the cost of enhanced temperature jumpof the surroundings from absorption of the pump. Since plasmonic enhancementwould apply to the pump field as well, one might expect that solventssuch as water should be subjected to enhanced absorption of pump lightper unit volume near the surface, which would cause a larger jumpin solvent temperature as discussed below in 8105Signalenhancement by higher pump fluence is an attractive strategyfor weaker chromophores. However, in systems with an absorbing solvent,this presents a dilemma where enhancing signal by increasing fluencealso increases the amount of light absorbed by the solvent and therebyleads to an abrupt change in temperature of the solvent surroundingthe analyte. The abrupt change in temperature induced by the pumppulse(s), known as the temperature jump, manifests as fluence andtime-delay dependent artifacts in pump–probe spectra as a resultof temperature dependent properties such as the index of refraction,dynamics, and other line shape features more generally.scs ≫ εaca) and εs isapproximately constant throughout the pump spectrum, e.g., water at2100 cm–1, then the heat absorbed by the solventis equal to the energy of the pump pulse times the fraction of lightabsorbed by the solvent, q = Epump(1 – T, following vibrational relaxation of the absorbed lightis related to the specific heat capacity C and mass m of the solvent by q = mCΔT. The time scale of thermal diffusion outof the excitation volume is on the order of hundreds of microsecondsto milliseconds,106 so ΔT does not relax prior to the arrival of the probe pulse. Therefore, m = ρVex where ρis the density of the solvent and T due to absorptionof the pump light by the solvent in If the absorptivity of the solvent is much larger than theanalyte(εT scaleidentically in terms of the inverse beam waist and pump energy, hence,the trade-off between ΔOD and ΔT as afunction of pump fluence. r = 0 is about ∼2× higher than the averagefluence of the averaged flat top beam. Therefore, the maximum temperaturejump within the sample, located at the front of the sample and centerof the pump beam, is Epump = 1 μJ, εs = 3.1 M–1 cm–1, cs = 55 M, 3, and C = 4.186 J/g·K yields ΔT ≈ 8 K.Comparing 9We have reviewed several differentformulas for computing the absolutemagnitude of pump–probe signal over a range of conditions knownto complicate signal scaling laws. Evaluating these models requires knowledge of atleast four experimentalquantities, each of which we have reduced to some combination of theanalyte molar absorptivity, concentration, path length, pulse energy,spectral bandwidth, line shape width, beam waist at focus, and theoptical density of the solvent. We analyzed functional relationshipsbetween the pump–probe signal and these quantities and discussedpractical limitations and trade-offs with respect to each. Severaloptical calculations were reviewed such as estimating the columnatedrange of a beam to identify the limit of propagation or the beam waistand Rayleigh length of a focused beam to estimate fluence and themaximum useful path length. We also reviewed other unconventionalmethods of increasing pump–probe signals, including variousforms of local-oscillator attenuation and, more recently, plasmonicenhancement. Estimations of solvent heat absorption by the pump pulsesuggest samples containing strongly absorbing solvents like watercould have significant temperature jumps that may limit high fluencestrategies including plasmonic enhancement.


E. coli BL21 (DE3) Omp8 Rosetta under its growth condition and by osmoregulation. The effects of osmotic stress caused by salts, sugars, or pH values on the survival of the target Gram-negative bacterial strain of E. coli BL21 (DE3) Omp8 Rosetta and OmpN expression remain unknown. Here, tryptone yeast extract with varied salts and concentrations was initially used to generate an LB broth medium. To show how salts and concentration affect bacterial growth, the optical density at 600 nm was measured. The findings supported the hypothesis that salts and concentrations control bacterial growth. Moreover, a Western blotting study revealed that OmpN overexpression was present in all tested salts after stimulation with both glucose and fructose after being treated individually with anti-OmpN and anti-histidine tag polyclonal antibodies on transferred nitrocellulose membrane containing crude OmpN. Following the presence of the plasmid pET21b(+)/ompN-BOR into E. coli BL21 (DE3) Omp8 Rosetta, which was expressed in the recombinant OmpN protein (BOR), OmpN expression was demonstrated for all monovalent cations as well as MgCl2. All of the tested salts, except for BaCl2, NaH2PO4, and KH2PO4, showed overexpression of recombinant BOR after Isopropyl β-D-1-thiogalactopyranoside (IPTG) induction. Using CH3COONa, both with and without IPTG induction, there was very little bacterial growth and no OmpN expression. With NaCl, a pH value of 7 was suitable for bacterial development, whereas KCl required a pH value of 8. According to this research, bacterial growth in addition to salts, sugars, and pH values influences how the OmpN protein is produced.This study presented the expression of the outer membrane protein N in  Is the manuscript technically sound, and do the data support the conclusions?  The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #1: PartlyReviewer #2: PartlyReviewer #3: Partly**********  2. Has the statistical analysis been performed appropriately and rigorously?   Reviewer #1: NoReviewer #2: NoReviewer #3: N/A**********  3. Have the authors made all data underlying the findings in their manuscript fully available?PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.  The Reviewer #1: NoReviewer #2: YesReviewer #3: Yes**********  4. Is the manuscript presented in an intelligible fashion and written in standard English?  PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.Reviewer #1: YesReviewer #2: NoReviewer #3: Yes**********  5. Review Comments to the Author  Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. Reviewer #1: Q1. The data do not fully support the conclusion because the conclusion was poorly written which is more like summary of what has been done.Q2. Most of the result does not capture whether the analysis was significant or otherwise especially in tables and figures, indicating that can make the reader understand the validity of the data.Q3. The data points behind means were not provided, the attached files were just like what was presented in the tables i.e the Means and standard deviationsQ4. Yes BUT there is a need for improvement in some certain sections (Indicated in the attached comment sheet) so that the manuscript can be more technically soundReviewer #2: This manuscript reports the results of a laboratory research investigating the growth and expression of the outer membrane protein N (OmpN) in E. coli BL21 (DE3) Omp8 Rosetta under osmotic stress conditions caused by different salts, sugars, and pHs. I recommend the issues below to be tackled.1) The title uses ‘kanamycin antibiotic-resistant bacterial strains’ while in the manuscript only E. coli BL21 (DE3) Omp8 Rosetta is mentioned. This may cause confusion for readers expecting information about several strains. Therefore, for coherence, the authors should either provide information about other strains or revise the title to reflect the focus on only one strain.2) ‘EcOmpN’ is not a standard protein name and should be changed to ‘OmpN’ throughout the text, as it is the correct short form for the outer membrane protein N.3) In line 31, it should be ‘tryptone yeast extract’ instead of ‘tryptone yeast’.4) In lines 32-33, ‘the optical density at 600 nm was evaluated’ should be changed to ‘the optical density at 600 nm was measured’.5) In line 34, ‘all salts’ is ambiguous.6) In line 37-38, ‘Following the presence of the plasmid pEt21b(+)/ompN-BOR in these bacterial strains, which was expressed in the recombinant BOR protein’ it's not clear what ‘these bacterial strains’ refers to. It should be clarified.7) In line 38, ‘pEt21b(+)/ompN-BOR’ should be ‘pET21b(+)/ompN-BOR’.8) In line 41, define the abbreviation of ‘IPTG’ when first appeared in the manuscript.9) In line 44, ‘According to research, bacterial growth in addition to salts, sugars, and pHs influences how the EcOmpN protein is produced’ is confusing. It is unclear what research is being referred to.10) In line 61, the authors should specify which type of bacteria was used.11) In line 71 ‘There are variations in the genotypes’ is not necessary and can be deleted.12) In line 90,’ Klebsiella pneumonia’ should be ‘Klebsiella pneumoniae.’13) In line 91, ‘which increases the OmpK36’ should be ‘which increases the expression of OmpK36.’14) In line 97, ‘To create the tryptone-medium preparation, LiCl, NaCl, KCl, RbCl, CsCl, MgCl2, CaCl2, BaCl2, Na2HPO4, NaH2PO4, and KH2PO4 were used’ is unnecessarily long and redundant.15) In line 131, ‘BL-broth’ should be written as ‘LB broth’ as it stands for Luria Bertani broth.16) In line 133, the chemical ‘yeast’ should be ‘yeast extract.’17) In line 155, it is not clear what ‘left by assessment’ means.18) In line 158, the volume of glucose and fructose added to each tube should be mentioned.19) In line 164, instead of ‘including pH 4, 5, 6, 7, 8, 9, and 10,’ it should be changed to ‘ranging from pH 4 to 10.’20) In line 180, the use of ‘carried’ after ‘Rosetta’ is redundant.21) In line 193, the type of bacterial cells used should be mentioned.22) In line 211, it is not clear which groups were compared using the t-test. The names of the groups being compared should be spell out clearly.23) In line 214, the use of ***P <0.0001 to evaluate statistical significance is incorrect, should be ***P <0.001.24) In lines 232-247 and figures 1A, 1B, 2A and 2B, the text and figures described the growth of bacterial strains at varying salt concentrations, however no statistical analysis  is mentioned to support the conclusions.25) Line 257, seems like other strains apart from E. coli BL21 (DE3) Omp8 Rosetta were also used. Need to be clarified to avoid confusion for the readers.26) In lines 266-267, should specify what concentration and type of salts were used.27) In line 267, ‘With CH3COONa, a decrease in bacterial growth was noted’ is confusing because it does not specify what was the initial bacterial growth rate, or what was the control used for comparison.28) In line 368, ‘Additionally, we noticed that the same bacteria strains, with the exception of CH3COONa and NH4Cl, could not grow at all on LB-medium containing CH3COONH4.’ This statement is vague, in addition the correct term is ‘bacterial strains,’ not ‘bacteria strains.’29) In line 373, ‘Like the chlorides of MgCl2, CaCl2, and BaCl2 in group 2A, high bacterial growth was noted at 0.1 M, but not at 0.25 M, as was the case for the chlorides of other salts from other metals in group 1A.’ This statement needs to be clarified; the introduction of "group 2A" and "group 1A" here is very confusing.30) In line 380, the use of ‘other’ is redundant and should be removed.31) In line 386, ‘which was also seen’ is redundant and can be deleted.32) In 402, ‘Reduced bacterial growth as a result of the reduced pH led to little or no EcOmpN expression’ is unclear and should be improved.33) In lines 406-407, ‘The two of them, together with PhoE, were found in E. coli K-12’, it is not clear whether ‘them’ refers to the OmpF and OmpC genes or the OmpF and OmpC proteins.34) In line 417, it is not clear whose findings the authors being referred to because the statement is not properly supported by the cited references. The authors should specify whose findings they are comparing their results to.35) In line 456, the word ‘and’ seems to be incomplete.36) In line 464, ‘The authors finding indicated’ should be changed to ‘The authors' findings indicated’Reviewer #3: This manuscript requires more extensive revisions because it is very limited in its scope and presentation of its results. This already starts on the title page, where the reader does not see that the authors’ university is located in Thailand. While this is merely a minor carelessness error, this style of negligence continues in the manuscript. For example, the abstract and the introduction promptly begin with a highly complex topic that is not introduced in detail. Neither the work’s relevance nor motivation becomes evident as a result.Similarly, the abstract ends with a result without further statements about what conclusions follow from it. How this work, in turn, can be embedded in the state of research remains to be mentioned. In addition, there are, again, minor typographical errors, of which only a few can be listed. For example, Gram should be capitalized because this goes back to the name of a scientist. In the methods section, Rayong is mentioned without naming the country in which it is located. Also, typographically, there would be some things to improve. For example, the letter “x” should not be used as a substitute for the character identifying the cross product. In addition, variables should be italicized, as in the t-test, and numbers up to 12 should be written out as words. In the list of references, some terms in continuous capital letters crept in by mistake. In summary, this reviewer suggests a vast improvement of the manuscript to interest a readership beyond the very specialized studies.**********  6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.If you choose “no”, your identity will remain anonymous but your review may still be made public.Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.  Yes: Dr. Salim Faruk BashirReviewer #1: Yes: Musa Hassan MuhammadReviewer #2: Reviewer #3: No**********https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, AttachmentPONE-D-23-08157.docxSubmitted filename: Click here for additional data file.AttachmentEcOmpN expression in kanamycin antibiotic-resistant bacterial strains under osmoregulation by salts, sugars, and pHs.docxSubmitted filename: Click here for additional data file. 31 May 2023Please see in the attached file (Respond to Reviewers)AttachmentRespond to reviewers.docSubmitted filename: Click here for additional data file. 19 Jun 2023Outer membrane protein N expressed in Gram-negative bacterial strain of Escherichia coli BL21 (DE3) Omp8 Rosetta strains under osmoregulation by salts, sugars, and pHsPONE-D-23-08157R1Dear Dr. CHUMJAN,We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at onepress@plos.org.If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact Kind regards,Bashir Sajo Mienda, PhDAcademic EditorPLOS ONEAdditional Editor Comments :Reviewers' comments:Reviewer's Responses to Questions  Comments to the Author  1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.Reviewer #1: All comments have been addressedReviewer #2: (No Response)**********  2. Is the manuscript technically sound, and do the data support the conclusions?  The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #1: YesReviewer #2: Yes**********  3. Has the statistical analysis been performed appropriately and rigorously?   Reviewer #1: YesReviewer #2: Yes**********  4. Have the authors made all data underlying the findings in their manuscript fully available?PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.  The Reviewer #1: YesReviewer #2: Yes**********  5. Is the manuscript presented in an intelligible fashion and written in standard English?  PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.Reviewer #1: YesReviewer #2: (No Response)**********  6. Review Comments to the Author  Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. Reviewer #1: (No Response)Reviewer #2: This manuscript reports the results of a laboratory research investigating the growth and expression of the outer membrane protein N (OmpN) in E. coli BL21 (DE3) Omp8 Rosetta under osmotic stress conditions caused by different salts, sugars, and pHs. I recommend that the article can be accepted if the following minor revisions are addressed.In line 548, ‘’perceived’’ should be changed to ‘’observed’’In line 552, ‘’different pHs’’ should be changed to ‘’different pH values.’’In line 553, ‘’it was not present’’ should be changed to ‘’it was not detected.’’In line 553, ‘’pHs 4 and 10’’ should be changed to ‘’pH values of 4 and 10’’, and the term ‘’pH’’ should be replaced with ‘’pH value’’ throughout the manuscript.In line 553, ‘’included both NaCl and KCl in the LB broth medium’’ should be changed to ‘’when LB broth medium contained both NaCl and KCl.’’In line 554, ‘’while KCl showed high OmpN expression levels’’ should be replaced with ‘’while high levels of OmpN expression were observed in the presence of KCl.’’**********  7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.If you choose “no”, your identity will remain anonymous but your review may still be made public.Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.  Yes: Dr. Salim Faruk BashirReviewer #1: Yes: Musa Hassan MuhammadReviewer #2: **********AttachmentPONE-D-23-08157_R1.docxSubmitted filename: Click here for additional data file.Attachment2nd Review.docxSubmitted filename: Click here for additional data file. 26 Jul 2023PONE-D-23-08157R1 Escherichia coli BL21 (DE3) Omp8 Rosetta strains under osmoregulation by salts, sugars, and pHs Outer membrane protein N expressed in Gram-negative bacterial strain of Dear Dr. Chumjan:I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department. onepress@plos.org.If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact plosone@plos.org. If we can help with anything else, please email us at Thank you for submitting your work to PLOS ONE and supporting open access. Kind regards, PLOS ONE Editorial Office Staffon behalf ofDr. Bashir Sajo Mienda Academic EditorPLOS ONE


Understanding the structures and properties of the interfacial region, however, remains a major challenge for polymer nanocomposites. Here, we directly observe the presence of two interfacial polymer layers around a nanoparticle in polar polymers, i.e., an inner bound polar layer (~10 nm thick) with aligned dipoles and an outer polar layer (over 100 nm thick) with randomly orientated dipoles. Our results reveal that the impacts of the local nanoparticle surface potential and interparticle distance on molecular dipoles induce interfacial polymer layers with different polar molecular conformations from the bulk polymer. The bilayer interfacial features lead to an exceptional enhancement in polarity-related properties of polymer nanocomposites at ultralow nanoparticle loadings. By maximizing the contribution of inner bound polar layer via a nanolamination design, we achieve an ultrahigh dielectric energy storage density of 86 J/cm The nanoparticle-polymer interface plays a key role in nanoparticle-polymer composites but understanding the structures and properties of the interfacial region remains challenging. Here, the authors directly observe the presence of two interfacial polymer layers around a nanoparticle in polar polymers with different polar molecular conformations from the bulk polymer leading to an enhancement in polarity-related properties of polymer nanocomposites It is generally believed that the emerging nanoparticle-polymer interface is essential for achieving their property enhancements10. The conformation of the interfacial polymers  often changes near the surface of solid nanoparticles12, inducing a deviation in properties compared to the bulk polymer in terms of glass transition temperature Tg, mobility and viscosity due to the existence of nanoconfinement and surface effects16. Although the presence of interfacial polymers provides an opportunity to tailor the properties of nanocomposites, the interfacial effects in polymer nanocomposites remain unclear, as direct characterization of the nanoscale interfacial regions buried around tough nanoparticles in soft polymer matrix is a great challenge.Polymer nanocomposites dispersed with nanoparticles exhibit significantly improved mechanical, electrical and thermal properties compared to pure polymers22. These models describing interfacial polymers with assumed nanoscale interfacial layers can explain some phenomena in nanocomposites, but the current interface models are abstract assumptions where the existence of these interfacial layers has not been directly demonstrated. Experimentally, a model nanocomposite was designed, in which the polymer thin film was confined between two silica slides, to understand the effect of interfacial polymer layers23. The quantitative correlation between thin-film thickness and interparticle distance in the nanocomposite provides a better understanding of the effect of interfacial polymer layers on Tg in nanocomposites filled with silica nanoparticles24. More recently, direct investigation of the polymer-nanoparticle interfaces has been enabled by scanning probe microscopy techniques31. The surface potential mapping across the nanoparticle-polymer interfaces allows detection of the interfacial layer with higher electrical polarization compared to the bulk polymer25, while the interface chemical mapping in ferroelectric polymer nanocomposites reveals that the interfacial regions around the nanoparticles are highly inhomogeneous with conformational disorders26. These recent attempts have provided direct experimental evidence that there exists an interfacial region with distinct local polarization and conformation from the bulk polymer, but it remains puzzling whether the hypothesized different interfacial polymer layers in theoretical models actually exist and how these layers are spatially distributed in terms of size, structure and properties. Of particular importance is the fact that the interfacial region around one nanoparticle in a polymer nanocomposite will be severely affected by its neighboring particles, since the interparticle distance decreases with increasing nanoparticle loading. Uncovering the possible influence of adjacent particles on interfacial polymers is another challenge for a comprehensive understanding of the interfacial effect in polymer nanocomposites.To understand the interfacial effects on nanocomposite properties, a few theoretical models have been proposed, in which the interfacial polymer was considered to consist of different layers including the tightly bound layer and the loosely bonded layerHere, by protruding half of the nanoparticles out of the polar polymer matrix to facilitate the localization and detection of the buried interface region, we directly observe the interfacial polymer region surrounding the nanoparticle, which consists of two interfacial polymer layers  with different polar molecular conformation from the bulk polymer. By combining simulations and experiments, we illustrate that both the nanoparticle-polymer interaction and the interparticle distance affect the formation of the interfacial polymer layers, which leads to an exceptional enhancement of polarity-related properties in dilute polymer nanocomposites. To maximize the effect of the inner bound polar layer, we design a nanolaminated nanocomposite with the polymer thin film confined between two oxide nanolayers, and achieve an ultrahigh dielectric energy storage density of the nanocomposite. Our observations provide an intuitive understanding of the interfacial effect in polymer nanocomposites and an effective strategy to tailor the polarity-related properties of nanocomposites via interface engineering.2 nanoparticles as the nanofillers. PVDF and its copolymers are typical polar polymers with excellent dielectric and piezoelectric properties33. To facilitate the localization and detection of the buried interface area from the nanocomposite surface, the PVDF-TiO2 nanocomposites were prepared via a carefully treated spin-coating process  of the PVDF polymer film is deliberately reduced to a value smaller than the diameter of nanoparticle (D), enabling the nanoparticles to protrude from the polymer matrix. In the experimental process, a pure PVDF polymer layer was first spin-coated onto silicon wafer substrates to eliminate any potential surface effect originating from the silicon substrate. As shown in the scanning electron microscopy (SEM) image  image and the carbon mapping results   as a matrix and TiOage Fig. , a visiblts Fig.  further trans , and three trans linked to a gauche  conformations have stronger polarity than the trans-gauche 35. The chemical mapping was conducted with an infrared laser at 840 cm−1 and 766 cm−1, which signify characteristic IR bands of the polar (TTTT/TTTG) and their counterpart non-polar (TGTG) conformation of PVDF chains, respectively36. The IR response of the interface region in the chemical maps is shown in Fig. −1 and a weaker conformation signal of 766 cm−1 at the interface region are observed , however, the nanoparticle shows no contrast. By combining L-PFM and V-PFM phase images, the interface dipoles are determined to be aligned perpendicular to the nanoparticle surface as illustrated in Fig. 2 nanoparticle surface without the PVDF bound layer , where an AFM tip was employed to locally detect the thermal expansion of the sample induced by infrared radiation Fig. . In PVDFved Fig. , which s−1/766 cm−1 and 1279 cm−1/766 cm−1 are calculated, which signify the concentration ratios between polar TTTT and non-polar TGTG conformations  reaches a peak value at ~0.35 vol.% of TiO2 nanoparticles  of the PVDF-TiO2 nanocomposites as a function of the nanoparticle volume fraction in the PVDF-TiO2 nanocomposites shows a similar trend to the polar polymer conformation content . As shown in Fig. ent Fig. , where t oC Fig. , which e oC Fig. . Above t oC Fig. , since P45, where both high electrical displacement  and high breakdown strength  are requisite to achieve a high energy density. The increase of D(εr) in polymer nanocomposites typically leads to a reduction in Eb46, while the rise in εr resulting from polar polymer conformations maintains a high level of Eb for the dilute nanocomposites. As illustrated in Supplementary Figs. 2 nanoparticles produces over 40% enhancement in D, while simultaneously preserving a high Eb of 630 kV/mm. The energy storage density and efficiency of the nanocomposites are calculated from the D-E loops . Al2O3 was chosen to fabricate the nanolaminated nanocomposite because high-quality ultra-thin Al2O3 nanolayer can be easily prepared by atomic layer deposition at a temperature of 100 oC, meanwhile, the PVDF-Al2O3 nanocomposites with Al2O3 nanoparticles also exhibit a similar overall dielectric enhancement trend to the PVDF-TiO2 nanocomposites  and bulk PVDF (εr ~ 7.8). The nanolaminated nanocomposite can be considered as a series connection of two Al2O3 films and PVDF film, therefore, the local εr of the interfacial PVDF layers can be calculated based on the series law of capacitors /PVDF (10 nm)/Al2O3 (28 nm) nanolaminated nanocomposite containing the inner bound polar PVDF layer has an energy storage density of 61 J/cm3. Notably, the Al2O3 (15 nm)/PVDF (10 nm)/Al2O3 (15 nm)/PVDF (10 nm)/Al2O3 (15 nm) nanolaminated nanocomposite containing two inner bound polar PVDF layers exhibits an ultra-high dielectric energy density of 86 J/cm3, which is far beyond the values so far reported in the dielectric polymers and nanocomposites, while possessing a satisfactory energy efficiency of 76%, being comparable or even superior to most dielectric polymers and nanocomposites51  were dissolved into N,N-Dimethylformamide (DMF) at a concentration of 2% w/v and stirred for 24 h for homogeneous mixing. Silicon wafer with platinum top layer was obtained from Hefei Kejing Materials Technology, and was cleaned by ultrasonic bath. A pure PVDF polymer layer was first spin-coated onto silicon wafer substrates to eliminate the possible surface effect from the silicon substrate. Afterward, the PVDF-TiO2 nanocomposite film was spin-coated onto the substrates with an as-coated PVDF layer at the spin speed of 8000 r/min, and then dried in a vacuum oven at 50 °C for 12 h.Polymer nanocomposite film with nanoparticles protruded out was prepared on the silicon wafer substrate for local interfacial characterization Fig. . The TiO2 nanocomposite mixture with PVDF/DMF solution concentration of 10% w/v was cast into films on glass substrates with a scraper. The films were then dried at 50 oC for 12 h in vacuum for the complete evaporation of solvents, after which, the flexible freestanding films were peeled from substrates. Free-standing nanocomposite films including PMMA-TiO2, PEI-TiO2, PVDF-Al2O3 were prepared with the same procedures, the Al2O3 nanoparticles have an average diameter of about 60 nm.Free-standing polymer nanocomposite films with a thickness of around ~10 μm were prepared for measuring their properties. The PVDF-TiO2O3/PVDF/Al2O3 nanocomposites, the bottom Al2O3 layer was deposited at 100 oC onto the silicon wafer with platinum electrode using atomic layer deposition system  with the Trimethylaluminum as precursor. The middle PVDF film was spin-coated onto the Al2O3 layer, and then dried in a vacuum oven at 50 °C for 12 h. The top Al2O3 layer was deposited onto the as-formed PVDF thin film also via atomic layer deposition at 100 oC. The thickness of both the bottom and top Al2O3 layers was controlled at about 1, 10 and 20 nm by controlling the deposition time duration, and the thickness of PVDF film was controlled at about 10, 50, 90, 150, 300, 450 and 1000 nm by controlling the spin speed of coating.For the fabrication of layered Al2 nanocomposite was characterized with scanning electron microscopy . The TEM observation and the associated EDX mapping were conducted using a FEI Titan Cubed Themis G2 300 microscope with a spherical aberration corrector. AFM-IR measurements were implemented using a NanoIR3 . Contact-mode Si tips coated with gold of spring constant 0.07-0.4 N/m  were used. The polarized IR laser was set at 840 and 766 cm−1 during the AFM-IR scanning.The morphology of PVDF-TiO3. The final structure consists of 200 random chains with the box size of 5 nm × 5 nm × 20 nm. TiO2 plate was perpendicular to z axis and the x-y dimensions were fixed to 4.8 nm × 4.8 nm.The initial single chain of PVDF was prepared by Materials studios (MS) 8.0 (20 monomers in each chain). The simulation model of PVDF was generated by the Amorphous cell module with the density of 1.77 g/cm52 force field. The UFF53 force field was applied to parameterize the TiO2 and to describe the interactions between TiO2 and PVDF. Periodic boundary conditions were applied to all directions. Long-range correction was used to calculate Columbic interactions beyond 12 Å cutoff. Van der Waals interaction was also calculated with 12 Å cutoff. The temperature, T, was kept constant through the Nose-Hoover thermostat algorithm54. A time step of 0.1 femtosecond was used in all simulations. All molecular dynamics simulations were performed by the LAMMPS software package55. VMD software was used for visualization and post processing of simulation data56.We combine two kinds of force fields to describe the simulation system. The PVDF was simulated with the CVFF39,ψ(r), q, ε, zi, ni, kB, and T represent the local electrical potential, unit charge, dielectric constant, the dipole moment of dipole specie i, the concentration of dipole specie i, the Boltzmann constant and temperature, respectively. In this simulation, the diameter of nanoparticles was set to 60 nm, the electric potential at particle surface was set to 100 mV, the concentration of dipole was set to 1013 cm−3, respectively.The potential distribution around the interfaces can be obtained by solving the Boltzmann-Poisson equation2 to 107 Hz at 1 Vrms. Unipolar displacement-electric field (D-E) hysteresis loops (using a triangle waveform with a frequency of 100 Hz) measurements were measured by a multiferroic ferroelectric test system  at room temperature. Electric breakdown tests were performed with a withstand voltage test system at a ramping rate of 200 V/s and 2 V/s for free-standing nanocomposite films and nanolaminated nanocomposite, respectively.Copper electrodes (4 mm in diameter) were coated onto the free-standing nanocomposites, and platinum electrodes (300 μm in diameter) were coated on the nanolaminated nanocomposites, for the measurements of dielectric and energy storage properties. Dielectric constants were measured with an impedance analyzer  with a broad frequency range from 10d33 of polymer nanocomposite films was obtained by d33 = Q/F, where Q is the charge measured by the electrometer and the F is the applied force detected by the force sensor. The open-circuit voltages of the dilute nanocomposites were detected by an electrometer  under the periodic pressing force of 10 N. The stress-strain curves were tested with a universal material testing machine .For the piezoelectric tests, copper electrodes (1 cm in diameter and 50 nm in thickness) were coated on both sides of nanocomposite film. The polymer nanocomposite films were first annealed at 140 °C for 24 h and then poled in silicone oil under the poling electric field of 150 kV/mm for 2 h. Then the film sample was held in place sandwiched by two rod probes (1 cm in diameter). The probes and electrodes with the same diameter were overlapped exactly. Dynamic force was applied on the upper probe during the test while the other was fixed, an electrometer  and a force sensor  were employed for the detection of charge and force on the sample, respectively. The Supplementary InformationPeer Review File


Molecular beams of NO2 are used to prepare a p (4 × 4)-O adlayer on the silver crystal. The translational energy distributions of O2 are shifted towards hyperthermal energies indicating desorption from an intermediate activated molecular chemisorption state.This study presents velocity-resolved desorption experiments of recombinatively-desorbing oxygen from Ag (111). We combine molecular beam techniques, ion imaging, and temperature-programmed desorption to obtain translational energy distributions of desorbing O Silver surfaces play important roles in large scale industrial heterogeneous catalytic processes such as partial oxidation of methanol to formaldehyde and ethylene to ethylene oxide . Because−6) of O2 -O domains  under UHV conditions with molecular oxygen is difficult due to the low sticking probability (ca. 1 × 106) of O2 . In earlnditions . The usenditions  or NO2  phase diagrams describing stable oxidized surface phases from UHV to high pressure conditions present at real world catalysts (The large number of reconstructed oxidized Ag (111) surfaces observed in experiments has motivated theory groups to develop models describing surface stability based on first principles theory . Using aatalysts . By comp2 dissociation process on Ag (111). These studies do not aim for clarifying the geometry of reconstructed surfaces but provide theoretical data on the atomic scale mechanism of the oxidation process itself (2/Ag (111) dissociative adsorption propagating on a six-dimensional potential energy surface (PES) obtained from density-functional theory (DFT)   . From thAg (111) . They alSuch theoretical work provides excellent data for comparison with surface dynamics experiments under well-controlled UHV conditions. A classical experimental approach probes the entrance channel of the reaction pathway by employing pulsed molecular beams of reactants to initiate the surface reaction . SeedingSurface reaction dynamics experiments on the exit channel probe degrees of freedom of the desorbing reaction products using quantum state-resolved detection methods in combination with translational energy dependent measurements . From th2 was detected using a velocity map imaging (VMI) setup after non-resonant ionization with a femtosecond pulse of 800 nm. The desorption process was initiated by linearly heating the sample in a TPD type approach. We identified hyperthermal velocity distributions for oxygen molecules desorbing from surface sites as well as for oxygen molecules originating from subsurface, indicating a common intermediate desorption state.Recently, we used this approach to investigate the recombinative desorption of oxygen from Rh (111) . O2 was 2 from Ag (111). We prepare p (4 × 4)-O Ag (111) by dosing the surface with a molecular beam of NO2 seeded in rare gases at a surface temperature of 510 K. Angular distributions are narrow and hyperthermal translational energy distributions indicate an activated desorption process.In this paper, we present angular distributions and translational energy distribution of recombinatively-desorbing O−10 mbar. We dose the surface with a pulsed supersonic molecular beam of 10% NO2  seeded in He  using a home-built pulsed solenoid nozzle  for multiple cycles. After annealing , the surface cleanliness is checked by Meitner-Auger electron spectroscopy (OCI BDL 450) and low-energy electron diffraction (LEED) spectroscopy (OCI BDL 450). The Ag (111) crystal  is mounted on a home-built sample holder and is resistively heated by Ta filaments; temperatures are monitored by a K-type thermocouple. With liquid nitrogen cooling, the accessible temperatures range from 100 K to 1,235 K. We use a home-written LabVIEW™ program for data acquisition and control of experimental parameters.For surface cleaning, the UHV apparatus is equipped with an ion gun (Staib Instruments IG-5-C), with which the surface is Arf = 300 mm) such that molecules are non-resonantly ionized after desorption. The ions are detected by an imaging setup which follows the design by Eppink and Parker (Velocity distributions of surface desorption products are obtained by combining velocity map imaging (VMI) and temperature-programmed desorption (TPD) experiments: we linearly heat up the surface while simultaneously detecting velocity map images of desorbing molecules. For that, the beam of a regenerativly amplified femtosecond laser  is focused by an optical lens -O phase on Ag (111) by dosing it with NO2 from a molecular beam at Tsurf = 350 K or 510 K for 2 min at a nozzle frequency of 200 Hz. We check the degree of oxidation by LEED and TPD (see 3) or pure oxygen layers may form (2 and O2 recorded with the VMI setup displayed in 2 (dashed curve) shows two different desorption features after dosing at 350 K: first, a broad desorption peak ranging up to 470 K followed by a less broad, lower intensity desorption between 480 K and 510 K. These features are attributed to NO3 decomposition into NO2(g) and O from two different states (2 (solid curve) desorbs at significantly higher temperatures at Tsurf of ≈590 K  around 590 K. We obtain the image by averaging all images that we record during a desorption peak in a TPD run. The raw image clearly displays the residual thermal gas background in the UHV chamber as circular spot as indicated in −1 and 1,500 m s−1. The angular tilt is due to a slightly tilted suspension of the crystal in the sample holder. From such images, we deduce velocity distributions by iterative integration over velocity increments within 10◦-broad angular slices as shown in 2 peak at 425 K and the O2 peak at 595 K. All curve integrals are normalized to unity. For comparison, thermal flux-weighted Maxwell-Boltzmann distributions of the shapeM denotes the molar mass of the compounds and R is the universal gas constant. We use the signal-weighted temperature ⟨T⟩ of 425 K for NO2 and 595 K for O2 for Tsurf.We use the VMI setup to determine velocity distributions for both, desorbing NO2 desorbs with hyperthermal velocities indicated by a shift of the curve’s maximum by more than 300 m s−1 compared to a flux-weighted thermal velocity distribution. In contrast, NO2 desorption is clearly thermal as it can be well-reproduced by a flux-weighted Maxwell-Boltzmann distribution of the surface temperature. We did not observe any significant difference between the two desorption features of NO3 decomposition (see From the figure it is obvious that Otion see . Table 12 and O2 desorption from Ag (111). For that, we move the surface parellel to the detector such that only molecules from certain desorption angles are detected as described previously (2 (squares) and O2 (circles) desorption. For comparison, a cos(θ)-distribution is shown, which would be expected for thermal desorption. We observe a narrow cos8(θ)-angular distribution for O2 desorption, whereas NO2 desorption resembles the cos(θ)-distribution indicative of a thermalized (or equilibrium) desorption process.Additionally, we record angular distributions of NOeviously . As VMI 2 desorption indicates an activated desorption process. Molecules have to overcome a barrier, on which they get accelerated into the gas phase. This is known to result in very peaked angular distributions  are obtained. The narrow angular distribution of Oibutions . In cont2 from Ag (111). The distributions are obtained from the velocity distributions displayed in 2 distribution is clearly shifted towards higher energies; NO2 resembles a thermal distribution.Also, velocities of desorbing molecules provide valuable information about the underlying PES. 2 energy distribution can be used to quantify energy-dependent sticking probabilities using the concepts of detailed balance  system reveal for instance significant enhanced sticking probabilities for vibrationally excited molecules . For these fast molecules, we assume no internal energy and that the recoil against the surface from the transition state results only in minor Ag phonon excitation. This value should therefore be the lower limit to the real energy barrier height as we do not account for excitation of the solid.We indicate this threshold as black arrow in 2 dissociative desorption on Ag (111) on a before computed PES  to O2 (v = 3). However, this reduction is still not sufficient to explain the discrepancy between the experimentally measured onset of this study of 0.57 eV and the minimum value of 0.8 eV (O2 (v = 3) for a bridge site) of the theoretical work. We therefore suspect that we do not map a direct dissociation trajectory but desorption from another intermediate surface state under the experimental conditions applied in this study.Interestingly, these values are significantly lower than calculated sticking probabilities based on first principles theory . Kunisaduted PES . Dependi2 molecular beams with Ag (111) at 150 K identifying several scattering pathways using ToF detection techniques  system, three adsorption states exist: a shallow physisorption well, a molecular chemisorption well, and a dissociative chemisorbed state  by combining ion imaging techniques with temperature programmed desorption. Desorption occurs at 590 K, is clearly hyperthermal, and exhibits a narrow angular distribution indicating an activated desorption process. Velocity distributions are similar to previously reported distributions from molecular beam surface scattering experiments. For both studies, the energetics of desorbing molecules indicate desorption from an intermediate molecular chemisorption state. Recent theoretical papers calculate significantly higher barriers for oxygen sticking on Ag (111) than we deduce from the translational energy distribution (We performed velocity resolved surface desorption experiments of recombinatively desorbing Oribution . The her