Structural disorder in molecular crystals is a fundamentallimitationfor achieving high charge carrier mobilities. Quantifying and uncoveringthe mechanistic origins of disorder are, however, extremely challenging.Here we use variable coherence transmission electron microscopy toanalyze disorder in tri-isopropyl silane pentacene films, utilizingdiffuse scattering that is present both as linear streaks and as aslowly varying, isotropic background. The former is due to thermalvibration of the pentacene molecules along their long axis, whilethe latter is due to static defects kinetically frozen during filmdeposition. The thermal vibrational amplitude is ∼0.4 Å,while the static displacement parameter in our simplified analysisis much larger (1.0 Å), because it represents the cumulativescattering of all defect configurations that are frozen in the film.Thin film fabrication therefore has an important effect on crystallinity;our technique can be readily used to compare samples prepared underdifferent conditions. Even in highly crystalline organic molecularsemiconductors, such as tri-isopropyl silane (TIPS) pentacene or rubrene,there is evidence for dynamic disorder, which arises from vibrationsof the molecular fragments about their mean lattice position.4 These vibrations are thermally driven and can have amplitudes aslarge as 0.5 Å,3 due to the relativelyweak intermolecular van der Waals forces. This dramatically altersthe orbital overlap between neighboring molecules and correspondingcharge transfer integrals,5 so that asa general rule, electrons become more localized and hopping transportdominates. Changes in orbital overlap also strongly impact the abilityof molecular pairs to undergo advanced photophysical processes suchas singlet fission, for which TIPS pentacene has emerged as an exemplarysystem.6 In addition to thermal motion,structural defects can also be kinetically frozen into the thin filmduring processing. As an example, solvents with higher boiling pointspromote better crystallinity and therefore larger mobilities in spin-coatedTIPS pentacene thin film transistors.7 Thesekinetically limited defects are described here as “static”,to distinguish them from the “dynamic” structural disordercaused by thermal vibrations.Crystallinity plays a crucialrole in the charge carrier mobility of polymer and small molecularorganic thin films.4 The electrondiffraction pattern for a given molecular crystal structure can besimulated using the multislice method,8 and the intensity profile for the diffuse streak compared with experimentto establish its accuracy and estimate disorder. Multislice is howevertime-consuming for such large supercells, especially because manyconfigurations of the dynamic disorder must be averaged to producestatistically representative results.9 Analternative method is fluctuation electron microscopy (FEM), whichis a technique used to quantify medium range order in amorphous materials.12 FEM has been applied to amorphous inorganic semiconductors13 and bulk metallic glasses,15 although there arefewer examples of quantifying disorder in crystalline materials.16 Diffuse scattering has also been extensivelyinvestigated in protein crystallography, mainly using X-rays.19Characterizing disorder at themolecular level is a highly complexand difficult challenge. Dynamic disorder gives rise to diffuse streakingin electron diffraction patterns.20 Fordiffuse scattering to occur, the coherence volume must be larger thanthe characteristic length scale of the structural disorder; smallercoherence volumes will produce less diffuse intensity in the electrondiffraction pattern. Thus, changes in the diffuse scattered intensitywith the coherence volume can be used to extract structural informationand the length scale of the disorder itself. The variable coherencemethod is demonstrated on experimental TIPS pentacene data, collectedin four-dimensional (4D) scanning transmission electron microscopy(STEM) mode, where a focused electron beam is rastered over the specimenregion of interest and a complete diffraction pattern acquired ateach scan position.21 4D STEM has had previoussuccess characterizing beam sensitive materials, such as mapping π-stackingin conjugated polymers,22 as well as extractingthe radial distribution function of amorphous organic polymer–smallmolecule blends.23 Strictly speaking, a4D STEM setup is not essential, because the same analysis can be performedusing selected area electron diffraction from a parallel electronbeam. However, 4D STEM does provide the benefit of selecting specimenregions that are oriented away from strong diffraction conditions,an important prerequisite for the variable coherence method, whichis based on kinematical scattering.Here we show that the “variable coherence” principleunderpinning FEM can also be used to directly quantify structuraldisorder in TIPS pentacene without the need for complex computer simulations.In “variable coherence”, the coherence volume over whichdiffraction takes place is varied by suitably adjusting the detectionparameters for electron scattering.TIPS pentacene thin filmswere deposited on a glass substrate witha spin-coated PEDOT:PSS layer. The TIPS pentacene was purchased fromOssila Ltd. and had a purity of >99.9%, as stated by the manufacturer.It was dissolved in chlorobenzene at a concentration of 20 mg/mL andstirred overnight. The solution was then drop casted on top of thePEDOT:PSS film, in a glovebox under a nitrogen atmosphere, followedby postdeposition annealing at 100 °C for 5 min. For the preparationof the TEM specimen, the glass substrate was immersed in deionizedwater. After a few minutes, the PEDOT:PSS layer dissolved and theTIPS pentacene film floated to the surface. The film was then pickedup with 250 mesh TEM copper grids.24 The 4D STEM step size was 20 nm, and the pixel dwell time was 250ms. The average number of electrons per scan position was 66, whichgives a fluence of ∼0.03 electrons/Å2 for a5 nm diffraction-limited probe diameter. These experimental conditionswere found to limit electron beam damage of TIPS pentacene; i.e. there was no noticeable change in the electron diffraction patternafter a 4D STEM scan. The specimen thickness is 1.3–1.5 inelasticmean free paths (∼150 nm), although the exact thickness ofthe region analyzed is unknown. The film thickness was estimated froman electron energy loss spectrum (EELS)25 acquired in a Gatan Tridiem imaging filter and a 200 kV, JEOL 2100FFEG TEM instrument at Durham University. See the Supporting Information for the EELS spectrum.4D STEM measurements wereperformed at the University of Manchester,using a 200 kV Thermo Fisher Scientific Talos field emission gun TEMinstrument, equipped with a MerlinEM direct electron detector. TheSTEM probe semiconvergence angle was 0.6 mrad, and the first condenserlens excitation was set to its highest value (i.e. “spot size”9) to improve electron beam coherence.3 and is caused by thermal vibrationsof the pentacene molecules along their long axis, which is nearlyparallel to [210]. To highlight this, 10] projection of the triclinic TIPS pentacene crystal.Out-of-plane vibrations are suppressed by large force constants dueto π-stacking, and in-plane vibrations perpendicular to thepentacene long axis are damped by enmeshing of the bulky TIPS substituents.4 Therefore, the only degree of freedom is alongthe pentacene long axis, i.e. in and out of the page in The diffuse streaking in TIPS pentacene has beendiscussed in detailpreviously20 DF images acquired further along the diffusestreak are, therefore, more incoherent.Beyondqualitative identification, dynamic disorder and associatedlinear molecular displacements can also be quantified using variablecoherence electron microscopy. This involves generating a series ofdark-field (DF) images from virtual apertures positioned along thelength of the diffuse streak. Three representative DF images are shownin 20 This is illustratedschematically in k (effectively the scatteredbeam in reverse) scattered by molecule j along wavevector k′ that falls within the STEM probe forming aperture.In the far field, the scattered wave function is fm(Δk)exp(−2πiΔk·rj), where fm(Δk) isthe scattering factor for the molecule at position vector rj and Δk = k′ – k is the scattering vector. The realspaceimage wave function ψ(r) is obtained by an inverseFourier transform:j within the illumination volume. For the sake of simplicity,we assume an infinitesimally small probe forming aperture, so that k′ is parallel to the optic axis; consequently, thereare no lens aberrations. This condition is approximately satisfiedin our measurements, because the STEM probe semiconvergence angleis only 0.6 mrad. The real space image intensity I is the square modulus of N is the number ofmolecules. The first term Nfm(Δk)2 is the incoherent intensity, while the summationis the coherence volume and represents pairwise interference betweenmolecules i and j separated by rij = ri – rj. Due to structural disorder, we assume interference is non-negligibleonly for nearest-neighbor molecules. This is because the cos(2πΔk·rij) termwill be rapidly oscillating for molecular pairs that are farther apart,i.e. large rij. Thermal vibrationof TIPS pentacene displaces the molecules along their long axis, sothat in the small displacement limitro is the equilibriumposition vector between nearest-neighbor molecular pairs, θis the change in nearest-neighbor spacing rji along the molecule long axis, and k∥ is the component of Δk parallelto the diffuse streak, i.e. perpendicular to the displacement in realspace. Assuming a Gaussian distribution of θ values with standarddeviation σ, the mean diffuse scattered intensity Idiff is given by26N′ is the numberof nearest-neighbor molecule pairs. The logarithm of diffuse intensity Idiff varies linearly with k∥2, provided that the terms within thesquare brackets in 2σ2k∥2) in 20 where suppression of thecoherence volume along the electron optic axis is due to thermal vibrationof the atoms. The gradient of a plot of ln(Idiff) versus k∥2 is −2π2σ2, which yieldsthe dynamic disorder parameter σ. Although the dynamic disorderis time-dependent, the swift passage of the STEM beam through a thinsample means that the molecules are effectively stationary duringelectron scattering.9 Therefore, σmust be interpreted as the standard deviation of the dynamic disorderat a fixed point in time.A kinematical scatteringtheory for the DF image intensity willnow be presented, where individual molecules are modeled as pointscattering objects. Following Treacy and Gibson, the direction ofelectron beam scattering by a molecule is reversed by invoking theprinciple of reciprocity.k∥ along the diffuse streak. A Friedel pair of 3 ×3 pixel virtual apertures was used for improved statistics (–1). Furthermore,only specimen regions that did not satisfy strong diffraction conditionswere analyzed, because Bragg diffraction is not included in our kinematicalmodel and can therefore result in artifacts. For example, the DF imagesin k∥ is shown in k∥ values, which is likely due to an error in estimatingthe true origin of the diffuse streak.To test this model, a series of DF images weregenerated by positioninga virtual aperture at different wavenumbers Idiff) versus k∥2 generated from the left-and right-hand side portions of the intensity profile in k∥ has been corrected to coincide withthe peak of the mean DF intensity . From Idiff arising only from dynamic disorder. Thebackground was taken to be the mean DF intensity at the largest k∥ wavenumbers, i.e. the extreme leftand extreme right data points in k∥ values,with standard deviation σ for dynamic disorder estimated tobe 0.38 ± 0.03 and 0.41 ± 0.05 Å, respectively. Incomparison, a smaller σ value of 0.13 Å is obtained bymolecular dynamics and multislice simulation of electron diffractionpatterns.3 The difference could be dueto the limitations of each technique in modeling dynamic disorder.For example, molecular dynamics can simulate only a small number ofmolecules compared to that of a real sample. On the other hand, ourmethod does not require any computer-generated molecular structures,but relies on a highly simplified kinematical scattering model todirectly quantify disorder from experimental diffraction patterns.Furthermore, some differences in σ values are to be expecteddue to variations between samples used for the two studies. Nevertheless,the agreement in the order of magnitude for σ suggests thatboth methods can provide physically meaningful results.Panels b and c of fm(Δk). The scattering vector magnitude for thediffuse streak, measured with respect to the unscattered beam, isbetween 0.6 and 1.2 Å–1. For carbon, the mainconstituent element in TIPS pentacene, the atom scattering factordecreases from 1.3 to 0.4 Å within this range. This is a relativelylarge change in absolute terms but is less of an issue when takinglogarithms for a plot of ln(Idiff) versus k∥2. Similarly, the cos(2πΔk·ro) term in 27 However, thecharacteristic scattering vector magnitude for the (π+σ)plasmon in TIPS pentacene is only 2.2 × 10–3 Å–1 (see the EELS spectrum in the Supporting Information) and is considerably narrowerthan the width of diffuse scattering  termin J0 is thezero-order Bessel function of the first kind and Rij is the magnitude of rij in the plane of the specimen. In k ispredominantly perpendicular to the optic axis. Note that the integrationover azimuthal angle ϕ ignores any variation in fm(Δk), although it will be shown belowthat this has a negligible effect on the final analysis. The in-planecoherence “volume” for hollow cone illumination is thereforea Bessel function.20 Due to static disorder, Rij will change by a small amountθ from the equilibrium in-plane nearest-neighbor molecule spacing R0. The Bessel addition theorem gives26m = 0. From Idiff for a Gaussiandistribution of static disorder (standard deviation σ) is givenby26I0 is thezero-order modified Bessel function of the first kind. k generated from virtualannular apertures of 3 pixels (0.09 Å–1) inwidth. The smallest Δk was limited to 0.27Å–1 to avoid overlap with any strong Braggreflections. The mean DF intensity was calculated from the same specimenregion used for analyzing dynamic disorder. In k to normalize for the virtual annular aperture size. Thefollowing function is then least-squares fitted to the data points:A represents the incoherent background and the expression within thesquare brackets is the normalized static disorder diffuse intensity and J0(2πΔkR0), althoughas we shall see, any changes in these variables are suppressed bythe dominant exponential term (C is equal to π2σ2 and yields the static disorder parameter.The data points in k values between 0.6 and 0.7 Å–1,which coincides with the most intense part of the dynamic disorderdiffuse streak. Overall, however, the experimental data are well representedby 7A scattering model forstatic disorder can also be developed alongsimilar lines. The diffuse scattering here is due to imperfect molecularconfigurations “frozen” into the structure during thinfilm fabrication. If the atoms in the molecule vibrate independently,the resulting thermal diffuse scattered intensity can overlap withthe static diffuse intensity background. However, thermal vibrationsin TIPS pentacene are largely correlated,ntensity7, with Bial term 7. Coeffi4 and is known to significantly alter the charge transferintegrals. Static disorder can also be frozen into the solid duringprocessing and can arise from many different defect configurations.A variable coherence method is developed to quantify the dynamic andstatic disorder from the diffuse scattering observed in electron diffractionpatterns. While the static disorder parameter in our model may notresemble the molecular displacement due to an individual defect configurationand must therefore be treated with caution, we anticipate that thismethod could nevertheless be used to compare disorder in thin filmsprepared under different conditions, such as solvents with differentboiling points, postdeposition annealing, etc.In summary, variable coherence transmission electronmicroscopyis a promising technique for probing disorder in crystalline molecularsolids. In addition to TIPS pentacene, dynamic disorder is presentin many other organic materials, such as rubrene,


The prospect of using the concept of auniversal reduced potential energy curve (RPC) for a broader classof radial molecular functions is explored by performing appropriatemodel calculations for the electric dipole moment functions of thehydrogen halides HF, HCl, and HBr. The reduced radial functions ofthe model systems, constructed from their best available theoreticalapproximants, coincide so closely that they can be used as few-parameteruniversal representations of functions available in the literature.Given the mathematical nature of the problem addressed here, the resultsare not limited to the functions studied but can be applied equallywell to all radial molecular functions that have similar shapes, suchas electric quadrupole moment and dipole polarizability functions. For instance, for a 1Σ+ diatomic molecule in weak interaction with fixedexternal charges, one obtains:2Tkin isthe kinetic energy operator of the free molecule, r is the internuclear distance between two clamped nuclei, VBO(r) is the field-free BOpotential energy curve, μ(r) and Θ(r) are the BO electric dipole and quadrupole moment curves,respectively, Z1 and Z2 are the atomic numbers of the two atoms, Ne is the number of molecular electrons,and F and G are the electric fieldand the field gradient due to the external fields, respectively. Thesame averaging can obviously be performed over any conceivable term(HX) of the general molecularHamiltonian to obtain the corresponding “radial” function X and subsequently a purely “radial” spin–rotation–vibrationHamiltonian H for each electronic molecular state.Ne, Z1, Z2) only. Unfortunately,however, there is no method in contemporary mathematics for explicitlyconstructing such functions.5 In practice,such functions can be evaluated straightforwardly using the first-principlesquantum-chemical approaches  |v′>.Formally,from a mathematical point of view, the radial functionsof a diatomic system are based on the three fundamental parameters or, ag., refs ). Still,also ref ), an unaa priori and because the elementary mathematical functions available maynot be flexible enough , they easily becomerather too awkward to allow “smooth” fitting. For instance,to describe quantitatively an ab initio potentialenergy function of He2 supporting only one bound state,Janzen and Aziz20 had to use 45 fittingparameters, and even the best approximants directly fitted to experimentaldata . Unfg., refs −18 and r.g., ref ) may stiab initio approximants by their fitting to accurate experimental data. Wherepotential energy functions of diatomic molecules are concerned, thisprocedure certainly appears to be a suitable tool when it is basedon the RPC method of Jenč. For instance, ithas enabled the proper spectral assignment of highly excited vibrationalstates using the experimental s-wave scattering length24 and has helped to reveal the existence of the12th vibrational state of the beryllium dimer and its two rotationalstates.25 The same approach can obviouslybe used for modeling any pair potential, including interaction potentialsmeasured by atomic force spectroscopy;26 note that potentials modeled this way significantly deviate fromthose produced by the usually used Lennard–Jones model.27One of the waysof overcoming the above problems may consist ofmorphing approximate, but topologically correct, r)), quadrupole moment(Θ(r)), and dipole polarizability (αzz(r)) functions. Like thepotential energy function, μ(r), Θ(r), and αzz(r) are fundamental molecular characteristics  and αzz(r), most of these studies focus on the region around the equilibriuminternuclear distance. To allow for the probing of higher-lying vibrationalstates, some such studies have attempted to construct appropriatedipole and quadrupole moment functions over a broad range of internucleardistances r by combining limited experimental datawith reliable ab initio approximants. The resultingfunctions, obtained either as Padé approximants32 or as piecewise-continuous functions,39 exhibit physically correct shapes at small and large internuclearseparations and closely coincide with genuine dipole and quadrupolemoment and polarizability functions in the equilibrium region. However,although contemporary theory provides the sought-after radial functionsquite accurately , it g., refs −45). Mor2Xref(r)  is used to generate its reduced form x(ρ) , whichis defined as follows:Deref is the depth of Xref(r) and the reduced variable ρis related to r via the expressionreref is the distance for which Xref(r) acquires its minimumand ρij satisfies the transcendentalequationij).The reduced radial propertycurve (RRC) approach based on the RPCof Jen consists of two steps: First, a given single-minimum (reference)radial function X(r) as a function of x(ρ), namelya priori unknown parameters De, re, ρij, α, β,and δ, which are to be determined by fitting the experimentaldata available .In the second step, the reducing procedure is reverted byexpressing vJ|X(r)|v′J′⟩,which are usually represented as power series in the vibrational v and rotational J quantum numbers, respectively.vJ >are obtainedby solving the Schrödinger equation for the following effectivero-vibrational Hamiltonian for an isolated 1Σ+ state47VBO is the“mass-independent” part of the molecular potential energycurve (assumed to include the Born–Oppenheimer and relativisticterms) and the terms V′(r), gr(r), and gv(r) account for QED, residual retardation, adiabatic, and nonadiabaticeffects. The sum Veff = VBO(r) + V′(r) is assumed to be determinable by fitting to the experimentaldata available; relying on the results obtained in refs (gr(r) factor function is tentativelyexpressed as gr(r) = g0 + g1(r – re)/(r + re)2, where g0 and g1 are fitting parameters and thevibrational g-factor gv(r) is neglected.The ro-vibrational wave functions | in refs , the rot3Tables S1–S5, the schemeenables the construction of highly accurate RPC curves not only bymorphing highly accurate empirical potential energy functions butalso when using their less accurate ab initio approximantsor even potential energy functions of chemically similar moleculeswhile relaxing the “correction” parameters α,β, and δ .To illustrateits properties, we first used the probed RRC schemeto construct the potential energy functions needed for generatingthe appropriate vibrational basis sets. As can be seen in the toppanels of zz) . However, because the level of accuracy achievable by bothexperiments and theory is only rarely comparable to that of data determiningpotential energy functions , the did in ref  for HF azz) see S1. HowevTables S6 and S8–S12). Subsequently, these data werecompared to data calculated by using empirical and theoretical functionsfrom the literature and their reduced counterparts constructed inthis study. Regarding HF, in 29 reproduces thereference off-diagonal <v|μ(r)|v′> data far better than its “best”theoretical counterparts, the theoretical functions are similarlybetter at describing the diagonal <vJ|μ(r)|vJ> data. Interestingly (see thebottompanels), when morphing the “best” empirical EDM of (Table S7), whereas “accurate” empiricalEDM functions usually require the inclusion of more than twice asmany fitting parameters.Obviously, a simple visual comparison of the probe curvescan bemisleading. To provide better insight into their usage as fittingfunctions for physically correct interpolation and extrapolation,it is more appropriate to compare their ability to reproduce availabledata. Taking into account the amount of literature that has been publishedon relevant experimental measurements and theoretical calculations,it seems particularly appropriate and beneficial to perform thesemodel calculations for the electrical dipole moment functions of theground electronic states of hydrogen halides. To arrange for it, referencedata sets have been generated mostly from the experimental data recommendedin ref  and fromTables S8–S12), one can arrive at similarconclusions as in the case of HF. Thefollowing two facts are worth mentioning explicitly: (a) Morphingthe “best” CCSD(T) reduced EDM functions of ref |4> and <0|μ(r)|5> of HCl and (<0|μ(r)|6>, <0|μ(r)|7> and <0|μ(r)|8>) of HBr with the rest of available data, questioningthus the physical adequacy of corresponding empirical functions fromthe literature. Interestingly, in full agreement with the results,a very recent experimental study51 on H35Cl and H37Cl reports line intensities for the5–0 bands, which are about 23% greater than the reference valuesof ref (As concerns the remaining probed models (see s of ref  while res of ref . (b) Morsof ref 4The concept of a universalreduced radial curve (RRC) allowingthe systematic study of radial functions of diatomic molecules ina unified scheme has been probed by performing actual numerical calculationsfor ground electronic states of the halide hydrides HF(DF), HCl, andHBr. Within the framework of this scheme, the radial functions ofdifferent molecules and different molecular states, both empiricaland theoretical, may be directly compared, and their regularities/irregularitiesmay be conveniently visualized. Being “physics-guided”,the RRC scheme enables the construction of reliable radial functionsover a large range of interatomic distances using a much smaller numberof fitting parameters than the number of fitting parameters neededwhen using usual polynomials or splines. The approach appears to beespecially advantageous for “noisy” transition intensitydata exhibiting high uncertainties, which cannot be fitted safelyusing low-order polynomials.


We then investigatethe interplay between intracellular forces and the cytoplasmic rheologyin live cells. Finally, we identify different rheological regimesand reveal evidence of active trafficking and details of the nanoscaleviscoelasticity of the cytoplasm.The viscoelasticityof the cytoplasm plays a critical role in cellmorphology, cell division, and intracellular transport. Viscoelasticityis also interconnected with other biophysical properties, such astemperature, which is known to influence cellular bioenergetics. Probingthe connections between intracellular temperature and cytoplasmicviscoelasticity provides an exciting opportunity for the study ofbiological phenomena, such as metabolism and disease progression.The small length scales and transient nature of changes in these parameterscombined with their complex interdependencies pose a challenge forbiosensing tools, which are often limited to a single readout modality.Here, we present a dual-mode quantum sensor capable of performingsimultaneous nanoscale thermometry and rheometry in dynamic cellularenvironments. We use nitrogen-vacancy centers in diamond nanocrystalsas biocompatible sensors for  The interdependence of physical properties in biologicalsystems can also be obfuscated by local inhomogeneity. Further, achange in one property, for example, temperature, can often affectothers such as viscosity, the speed of chemical reactions, or therate of cell division. The relationship between two properties isthus hard to capture effectively if the level of an external perturbationcannot be measured accurately and independently. Multimodal sensorsoffer the opportunity to reveal such interdependence.Optical techniques canprovide means for investigating intracellularphenomena at the nanoscale in a noninvasive way. These methods areoften susceptible to variations in autofluorescence,23 Nanodiamonds containing nitrogen-vacancy centers(NV) are one of the leading candidates: their properties include stablephotoluminescence (PL), minimal cytotoxicity at high concentrations,25 amenability to surface functionalization,26 and robustness against changes in pH.27 The ground-state spin transition that is utilized for sensing canbe effectively uncoupled from background fluorescence fluctuations,enabling NV measurements to be unaffected by local changes in theoptical environment. The NV has the capability to measure severaldifferent quantities, as demonstrated separately for temperature,28 magnetic field,29 electric field,30 pressure,31 reactive oxygen species,32 and through targeted surface functionalization, pH.33 These demonstrations position the NV as a promisingcandidate for multimodal sensing implementations.Amongthe many approaches to nanoscale sensing in biological systemsthat are currently being explored, nanoparticles provide a platformwhich enables robust optical intracellular sensing.In this work,we perform nanothermometry and nanorheology usingoptically detected magnetic resonance (ODMR) and particle trackingof NV-containing nanodiamonds. We first demonstrate the operationalprotocol and achieve 3.7 nm spatial resolution with 9.6 ms updaterate and a temperature sensitivity of Supporting Information Section 1). 4 nm2 s–1 in cells34 (Supporting Information Section 2). These dynamic environments requirethe nanodiamond to be tracked throughout the optical spin readoutmeasurements. We achieve this through a double-plane orbital trackingmethod,35 which provides real-time feedbackcontrol of the nanodiamond’s location, as illustrated in Supporting Information Section 3). In To achieve opticalreadout of the NV spin, which underlies the sensingconcept of nanodiamonds, we use a home-built confocal microscope .While tracking the nanodiamond in real time, we simultaneouslyperform continuous-wave ODMR for temperature sensing. The ground statezero-field splitting of the NV can be optically read out by drivingthe NV spin from the Supporting Information Section 5) andmicrowave delivery for the manipulation of NV spins, we developeda custom fabricated chip. All measurements are performed using a gold-patternedglass coverslip comprising a coplanar waveguide, two resistive heatersand a resistive temperature detector (RTD), as highlighted in The ODMR-based thermometrytechnique requires the delivery of microwavesto the region of interest. This typically leads to heating of thesubstrate and intracellular medium. These effects can be a challengeto control and may vary from sample to sample. To achieve reproducibletemperature control, sample heating with minute-scale temporal resolution(Supporting Information Section 7).To benchmark the thermometry modality, we firstquantify the temperaturesensitivity of a stationary nanodiamond drop-cast on the quantum sensingchip in the absence of any fluidic environment. As seen in Supporting Information Section 8). r(t + τ) – r(t)| 2⟩, where r is the position vectorin the transverse plane and τ is the time interval. The MSDdepends linearly on the time interval for a particle undergoing Brownianmotion, as MSD = 4Dτ, where D is the diffusion coefficient. The measured diffusion coefficientagrees with the input diffusion coefficient as highlighted in D = 5 × 104 nm2 s–1, exceeding the typical intracellulardiffusion coefficients observed with similarly sized nanodiamonds(Supporting Information Section 2). Whenthe particle is stationary, we measure a resolution of 3.7 nm witha 9.6 ms update rate, as shown in e2 point-spreadfunction. Our particle tracking is capable of following nanodiamondsin a range of dynamic environments with a high enough velocity andspatial resolution to allow the extraction of viscoelastic moduli.The nanodiamonds simultaneously operate as quantum sensors for temperaturewithout the need for measurement deadtime in either modality.To benchmark the rheometry modality, we start by verifying thedynamic tracking accuracy of the single-particle tracking method.The scanning mirrors and the objective lens are moved such that astationary nanodiamond on the substrate exhibits a predefined trajectorythat mimics Brownian motion  is the temperature dependent viscosity. We study thetemperature dependence of the diffusion coefficient over a 17.5 °Crange by increasing and decreasing the temperature in steps of 3.5°C every 5 min. In the case of glycerol, η(T) is linearly dependent on temperature in the range probed, η(T) = η0 + μ(T – T0), with μ = 0.0208 Pa s/°C, T0 = 35 °C and η0 = 0.301Pa s.36 From the experimental measurementsof the diffusion coefficient in T) (black solid curve),using only the radius of the particle as a fitting constant. The estimatedhydrodynamic radius of the nanodiamond is 28 ± 1 nm, which agreeswith the nominal distribution provided by the supplier, 25 nm. Asone would intuitively expect, a proportion of the diffusion coefficient’stemperature dependence can be attributed to increased thermal energyas seen in In glycerol 37 andthe actin cytoskeleton.38 To model theseenvironments, we use the synthetic viscoelastic polymer network glycerolcross-linked xanthan (GCX). The complex modulus, G*(f) = G′(f) + iG′′(f), is usedto characterize viscoelastic materials and can be calculated fromthe MSD,40 where f is the frequencyof external perturbations at which G is measured. The real part ofthe complex modulus, G′(f), is a measure of the elasticity of the material and the imaginarypart, G′′(f), is ameasure of the viscous component. The ratio of the real and imaginaryparts of the complex modulus establishes whether an environment isdominated by viscosity or elasticity. This capability can be usedto capture how the rheological properties of the medium react to externalperturbations.In addition to sensing predominantly viscous rheologicalbehavior, our probe can reveal the viscoelastic properties of biologicalenvironments such as DNA hydrogelsG*|, as well as its real and imaginary components decrease with temperature.This is the expected behavior for viscoelastic materials from thetime–temperature superposition principle,41 as previously observed in other materials like hydrogels.42 We achieve sufficient sensitivity to distinguishbetween the two viscoelastic states with a 30 s averaging intervalwhen we cycle the temperature by 10.6 °C. Supporting Information Section 9). We expose the nanodiamond-containingcells to temperature cycles of 5.0 °C in steps of 2.5 °Clasting 5 min each. Supporting Information Section 10).Having benchmarked our dual-modal sensing approachin controlledenvironments, we next investigated the intracellular response to externaltemperature changes and the motion of nanodiamonds inside cells. Weincubate HeLa cells with nanodiamonds and confirm internalizationusing 3D confocal microscopy  of a particle’s location, ⟨x2(ω)⟩, which is the Fourier transform ofthe MSD, can be used to model this behavior. For media where the force–displacementrelation is linear, this PSD is related to the power spectra of thermalstochastic forces, ⟨ξ2(ω)⟩ ∝ kBT,43 andactive forces due to cell agitation, ⟨Fext2⟩, byHooke’s law,Unlike glycerol and GCX, the cytoplasm of a cell is an activemediumthat is neither spatially homogeneous nor in thermal equilibrium.Molecular motors cause collective agitation of the cytoplasm.K(ω) = (6πr) G*(ω) is the (complex)spring constant characterizingthe property of the medium, G*(ω) is the complexmodulus introduced in the previous section and ω is the angularfrequency corresponding to the linear frequency, f. f = 40 Hz. The PSD increases dramatically approximately15 min into the measurement, for a duration of around 10 min. Variationsin the PSD can be explained by a combination of changes in |K(ω)| and ⟨Fext2(ω)⟩. Particularbiological events such as cell division can result in large changesin viscoelasticity5 and thus changes in |K(ω)|. In the absence of such events, active microrheology47 and whole-cell AFM48 experiments suggestthat cell viscoelasticity remains constant over the time scale ofhours. The changes that we observe are therefore likely dominatedby the active forces.Here, 49 and therefore single-particletrajectories areexpected to show temperature-dependent active trafficking50 together with the Brownian motion of the particle.As the nanodiamond spends the majority of the time in Brownian motion,the time-averaging used in the MSD and PSD analysis can hide transientfeatures in the trajectory. d/l, where d and l are the displacement and distance of a trajectory portionrespectively (Supporting Information Section 11). α, the displacementbehavior is typically classified into subdiffusive (α < 1),diffusive (α = 1) and superdiffusive (α > 1) states.Separatingthe trajectories into segments reveals that when the nanodiamondsare not in the directed motion state, they on average exhibit Brownian-likebehavior, as can be seen from the MSDs in 50 Under this treatment, nanodiamond trajectories exhibited no directedmotion. Further, the average power-law exponent of 0.3 ± 0.1indicates subdiffusive motion as presented in 51 (Supporting Information Section 12).Nanodiamond internalization involves theendocytic pathwayMultimodal quantum sensing provides opportunitiesfor investigatingperturbation and response accurately and independently on the nanoscalein active biological environments. We identify the directed motionof the nanodiamonds as a possible indicator of active traffickingin the cell. Further, by removing the action of the molecular motorsassociated with microtubules, we show that the cytoplasm is dominatedby its elastic properties. Our results also show that within our measurementsensitivity HeLa cells do not regulate their internal temperaturein the presence of an external thermal perturbation.52 or spin electron double resonance (SEDOR).53 Techniques such as optical tweezers54 and surface chemical functionalization55 can be combined with our dual-modal approach for preciselocalization of nanodiamonds with respect to subcellular organelles,such as mitochondria.56 The targeted deliveryof nanodiamonds to subcellular regions would enable probing of potentialhot spots, correlating local biochemical events with thermogenesis.This could be used to address the topic of nanoscale temperature gradientsin live cells57 and be further extendedto studying nonbiological soft matter. Active rheology techniquescould allow further exploration of the relationship between activeforces and the spring constants in cells. Combining nanodiamond sensorswith super-resolution imaging techniques58 in a multiscale imaging setting offers an exciting opportunity toprobe physical properties in the context of their biological environment.The orbitaltracking method we employ utilizes the stable photoluminescenceof the NV and enables us to track single nanoparticles for over 40min. This tracking method is not limited to the continuous-wave ODMRtechnique, and can be paired with more sophisticated quantum sensingprotocols, such as nuclear magnetic resonance (NMR)T, R(T), is linear inthe temperature range we probe and the measured RTD resistance isconverted into temperature usingT0 is the reference temperature at which R(T0) is measured. T0 is measured with a thermocouple before each chip isused to calibratethe sensor. An experimentally determined fitting constant η= 2.44 ± 0.12 × 10–3 /°C is usedin the conversion. A polydimethylsiloxane (PDMS) open-top well (withan approximate volume of 400 μL) is plasma-bonded to the substratewhen performing experiments involving fluids.The resistivity of gold at temperature –1 50 nm carboxylic-acid terminated nanodiamonds in water were centrifuged for 5 min at 12 000 g. Details on the nanodiamond fabrication and material propertiescan be found in ref , we transfer 9.9 g of glycerol-nanodiamondsuspension prepared as mentioned above into a beaker and begin agitatingby using a magnetic stirrer at a rate of 100 rpm. Then 0.1 g of xanthangum powder  is slowly added to the glycerol. Onceall the xanthan is fully incorporated into the glycerol, the beakeris heated to 80 °C with the stirring rate increased to 1000 rpmfor 1 h to facilitate cross-linking.2. To ensure cellsadhere to the sensing chip, the substrate is coated in Geltrex basementmembrane product . Sufficient cell coveragewas achieved by inoculating 10 μL of 1 × 106 cells mL–1 in 390 μL of Dulbecco’smodified eagle medium (DMEM) supplemented with 0.11 g L–1 sodium pyruvate and 10% fetal bovine serum (FBS) directly into thePDMS well. Cells were left to adhere to the substrate for a minimumof 12 h prior to the addition of nanodiamonds.We used the cervical cancer cell lineHeLa as a model system for studying intracellular temperature changes.Prior to the experiment, HeLa cells (ATCC CCL-2) are incubated inan incubator at 37 °C with 5% CO2 concentrationduring the ODMR experiments, the cells were inoculated in Leibovitz’sL-15 media  supplemented with 10% FBSfor all experiments.To ensure anuptake of ∼2 nanodiamonds per cell, 1 μL of the nanodiamondstock solution was inoculated with the cells in the well for a minimumof 4 h. Excess nanodiamonds were washed off with phosphate-bufferedsaline (PBS). To compensate for the reduction in COTo compensate for the temperature risedue to microwave heating,ODMR experiments were performed in an incubator maintained at 33 °C, which was given more than 12 h prior tothe experiments to thermalize.Supporting Information Section 9).The observation that there are mitochondria above, below, and surroundingthe nanodiamond observed confirms that the nanodiamond is internalized.We take particular caution in verifyingthe nanodiamonds are internalized inside the cells, to avoid measuringnanodiamonds attached to the surface of the cell. To achieve this,a series of 3D confocal scanning images on a Leica SP5 are taken toestablish that the nanodiamonds are situated among the mitochondrialnetwork which has been dyed with 100 nM MitoTracker Green FM   for fast-feedback control. The photon countsare collected via a high-bandwidth counter, which are read out bythe MCU. The fitting algorithm (implemented in Arduino IDE) uses alinear search and quadratic minimization algorithm. All computationsare performed within 6 μ s.The collection optics areseparately aligned, and the collimation lenses are displaced in oppositedirections so that the collected PL is 70% of the maximum.Supporting Information Section 8).The inferred trajectory ofa stationary particle still appears moving. This results from intrinsictracking error, setup drift, and other sources of movements. Thesenoises are quantified by tracking a stationary nanodiamond  within a 200 ms duty cycle. The APD countersare gated such that they do not collect APD counts during the 40 msof off time. The ODMR uses a global synchronization clock with 100kHz frequency and gathers one PL reading on each clock edge.During the 40 ms off time, the heater is switched on using a solid-stateswitch for 30 ms with a 5 ms buffer time before and after. The solid-stateswitch has a switching rise and fall time of approximately 0.5 ms.This avoids any magnetic field interfering with ODMR due to the currentsupplied to the on-chip heater.Supporting Information Figure S1) within 160 ms is read out , converted to counts per second (bydividing by 10 μs), summed to give a total counts from bothAPDs, and stored as 80 × 200 values. Subsequently, each 80 rowsof data  is averaged and stored as intermediateresults for postprocessing. Subsequent data processing does not usethe finely timed raw data due to the long processing time. For a typicalemitter giving 1 Mcps emission, in 10 μs read out time, eachAPD reads about 5 photon counts.The number of photon counts acquiredfrom both APD1 and APD2 (shown in Supporting Information Section 4). This method fits the entire data setusing piece-wise linear interpolation to define an interpolation functionand subsequently fits subsections of the data with this interpolationfunction to find any shifts in frequency, δf. These subsections are defined as independent bins of 400 ms ODMRdata and result in a sequence of frequency shifts. We then averageconsecutive nf frequency shifts to geta better estimate of the frequency shift. The error in the frequencyshift is estimated using the standard error of the nf frequency shifts considered. The resultant shift infrequency is converted into a temperature shift presented in the maintext. See Supporting Information Section 4 for more details and motivation.All data presented in thiswork use the interpolation method (r(n) = (x(n), y(n), z(n)), where n = 1, 2, 3, ..., N arethe time indices. To compute the 1D time-averaged mean square displacementin the x direction, we compute the time average ofthe square of the pairwise differences, ξi)τ(x = x(i + τ) – x(i), byK = N – τ. Aseach x is a stochasticvariable, the MSD is a stochastic variable with inherent variance,irrespective of measurement noise. To quantify this inherent stochasticvariance, an estimator is needed based on the measured values of locations,considering the intrinsic correlation between ξi)τ(x and ξj)τis estimated byD scaleswith We gathera sequence of 3D locations of the nanoparticle, denoted by –4 μ m2, limited bythe precision of the galvo mirror and signal sources used. The combinederror is given by the noise floor if the statistical error derivedabove or the MSD itself is less than the noise floor.We estimate the system noise floorto be at 10G*(f) = G′(f) + iG″(f) = | G*(f)| eiδ (f), is related to the MSD by,G̃(s) and ⟨Δ r̃2(s)⟩ are theLaplace transformof the complex modulus, f = 1/τ and the MSDwith respect to the Laplace variable s. Using themethod developed by Mason,40 this is relatedto the frequency dependence of G byG* are calculated by propagatingthe MSD error through the above equation, assuming a comparativelynegligible error on the fitted α value.At thermal equilibriumthe complex modulus, x2(f)⟩ of motion in oneaxis was calculated using Welch’s method , averaged over 28.8 s. The total PSDis given by the sum of PSD in the two transverse axes: PSD = ⟨Δx2(f)⟩ + ⟨Δy2(f)⟩. PSD data presentedin the main text are taken at f = 40 Hz.The PSD, ⟨Δ


The cellular reconstitution after childhood cancer therapy is associated with the risk of infection and efficacy of revaccination. Many studies have described the reconstitution after stem cell transplantation (SCT). The recovery after cancer treatment in children who have not undergone SCT has mainly been investigated in acute lymphoblastic leukemia (ALL), less for solid tumors. Here, we have examined the temporal evolution of total leukocyte, neutrophil and lymphocyte counts as surrogate parameters for the post-therapeutic immune recovery in a cohort of n = 52 patients with ALL in comparison to n = 58 patients with Hodgkin’s disease (HD) and n = 22 patients with Ewing sarcoma (ES). Patients with ALL showed an efficient increase in blood counts reaching the age-adjusted lower limits of normal between 4 and 5 months after the end of maintenance therapy. The two groups of patients with HD and ES exhibited a comparably delayed recovery of total leukocytes due to a protracted post-therapeutic lymphopenia which was most pronounced in patients with HD after irradiation. Overall, we observed a clearly more efficient resurgence of total lymphocyte counts in patients aged below 12 years compared to patients aged 12 to 18 years. Our results underline that the kinetics of cellular reconstitution after therapy for HD and ES differ significantly from ALL and depend on treatment regimens and modalities as well as on patient age. This suggests a need for disease, treatment, and age specific recommendations concerning the duration of infection prophylaxis and the timing of revaccination. Apart from the development of supportive measures and novel targeted therapies, this success is largely still based on the optimized and risk-adapted dosing and scheduling of conventional chemotherapeutic agents. These systemic therapies, however, lead to a suppression of hematopoiesis and general immune functions of varying degrees.Currently, overall cure rates in childhood cancer therapy exceed 80%3, and the current guidelines recommend the use of inactivated vaccines from 3 months and live vaccines from 6 months after completion of conventional antineoplastic therapy in patients who did not undergo autologous or allogeneic hematopoietic stem cell transplantation (HSCT)4. However, these recommendations are rather uniform and not disease-specific, and, in addition to other concomitant factors such as comorbidities, infections, and possible relapse, it can be assumed that the time interval until patients regain immunocompetence also varies between the underlying disease entities and the applied therapeutic regimen.Cellular and immunological reconstitution after cessation of antineoplastic therapy is critical in terms of the risk of infections and the efficacy of revaccinations. In everyday clinical practice, it is generally assumed that patients from about 3 months after termination of antineoplastic therapy can be considered immunocompetent and a low risk of infections as well as an adequate vaccination response can be expected. Accordingly, infection prophylaxis is usually terminated between 3 and 6 months post therapy6. Analogous to the prevalence, the cellular and immunologic recovery in pediatric patients who did not undergo allogeneic or autologous stem cell transplantation has been investigated mainly in acute lymphoblastic leukemia (ALL)14 and less extensively in other disease entities like solid tumors18.The highly complex and biologically distinct immunological reconstitution after HSCT has been studied extensively and described in numerous publications for adult and pediatric patients alikeTherefore, this analysis aimed to illustrate disease- and therapy-dependent differences in the cellular reconstitution after chemotherapy between children and adolescents with ALL and other large and homogenous pediatric patient groups treated for lymphoma and sarcoma, respectively. Consequently, we here retrospectively determined the continuous evolution of total leukocyte, neutrophil and lymphocyte counts as surrogate parameters of immunological recovery from 4 months before until 12 months after the end of conventional chemotherapy in a large pediatric cohort of patients with ALL, patients with Hodgkin`s disease (HD), who had received less intensive lymphotoxic therapy with or without irradiation, and patients who were treated for Ewing sarcoma (ES).In total, 132 patients fulfilled the inclusion criteria and could be analyzed in this study. Of these, n = 52 patients were treated for ALL, n = 58 patients were treated for HD and n = 22 patients were treated for ES.In the group of patients with ALL, 21 individuals (40%) belonged to the standard-risk (SR) group, 26 individuals (50%) belonged to the medium-risk (MR) group and 5 patients (10%) belonged to the high-risk (HR) group. Six patients (12%) experienced a relapse within the study period.Of the 58 patients with HD, 11 (19%) had been treated according to therapy level (TL) 1, 18 (31%) according to TL-2, and 29 (50%) according to TL-3. Twenty-eight individuals (48%) received additional radiotherapy. The distribution of irradiated patients was even among patients aged ≤ 11 years (47%) and patients aged 12–18 years (54%). Four patients (7%) relapsed within the study period.Of the 22 patients with ES, 16 individuals (73%) were treated in the R1 arm and 6 (27%) in the R2 arm. Seventeen patients (77%) received additional irradiation of various locations depending on the primary involvement. A relapse within the study period was diagnosed in 4 patients (18%).The cohort and subcohort characteristics are shown in Table As depicted in Fig. Starting from clearly reduced cell counts at the end of maintenance chemotherapy, patients with ALL showed the fastest recovery of all cell types assessed among the three subgroups from month 1 after the end of treatment  and in comparison to patients with ES between 2 and 5 months after chemotherapy (p = 0.002 at month 5). Children after treatment for ES exhibited the lowest proportion of individuals reaching the LLN for total leukocyte count until month 5 merging with the patients treated for HD thereafter .The separate subcohort analysis comparing patients with HD who had received additional radiotherapy and those who had not received additional radiotherapy showed highly significant differences concerning the recovery of absolute total lymphocyte counts and the consecutive achievement of the total lymphocyte LLN as depicted in Fig. Due to the heterogeneity of the localizations of radiotherapy and great disparities in group sizes, no subgroup analysis comparing patients with ES who had received radiotherapy with those who had not received radiotherapy was performed.Addressing age dependent patterns of cellular reconstitution, we found that patients aged ≤ 11 years showed a more efficient recovery of the total lymphocyte count regularly reaching the LLN earlier compared to patients aged 12–18 years. Figure 7. The authors found that, based on these parameters, a sufficiently functioning immune system was established 6 months after cessation of chemotherapy and concluded that prophylactic antibiotics can be withdrawn and immunizations started. In a subsequent publication, the same group later described a differential and age-dependent recovery of blood T cell subsets posttherapy9. Although their analysis showed a mean reversion to normal values by 6 months, some individual patients continued to have subnormal values for up to 1 year after therapy, some of whom exhibited increased susceptibility to infections. Such observations were largely confirmed in later studies in ALL20. Some of these analyses showed that in comparison, B-cells were reduced more significantly and for longer periods than T-cells. In a long-term follow-up, van Tilburg and colleagues showed that whereas naive B and T cells exhibited a relatively fast recovery, memory B-cells regenerated significantly slower and memory T-cells did not fully recover during the entire 5-year follow-up12. In a more recent large study including 116 participants, Williams and colleagues underlined that immune reconstitution differs between lymphocyte compartments21. Concerning the influence of treatment intensity, Ek et al. found that immune reconstitution after childhood ALL was most severely affected in the high-risk group10.Childhood cancer and its treatment compromise immune functions bearing implications for the risk of infections and effectiveness of revaccinations. A plethora of studies describe the immune reconstitution after allogeneic HSCT and provide recommendations for infection prophylaxis and revaccination strategies. According to the frequency of the disease, analyses of the immune recovery after the end of therapy in pediatric patients who did not undergo HSCT focused on ALL. In this context, an early report by Alanko et al. described the recovery of blood B-lymphocytes and serum immunoglobulinsThe immune reconstitution after therapy for solid tumors in children and adolescents has been characterized less extensively as compared to ALL.22. Later, Alanko and coworkers focused on the hematologic and immunologic recovery in a small analysis including 11 children who had been treated for HD (n = 3), Nephroblastoma (n = 4), Burkitt`s Lymphoma (n = 2), clear cell sarcoma (n = 1) and rhabdomyosarcoma (n = 1). The group described that the lymphocyte counts of most patients normalized during the first 12 months after therapy. The recovery in patients with HD or Burkitt`s lymphoma was slower than in patients with nephroblastoma and radiotherapy appeared to prolong immune reconstitution15. In a later investigation, Kovacs and colleagues assessed immune recovery in 88 children receiving chemotherapy for ALL (n = 43), lymphoma (n = 15), bone tumors (n = 20), and other solid tumors (n = 10). The group determined serum immunoglobulin levels (Ig), natural killer activity (NK), antibody-dependent cellular cytotoxicity (ADCC), and T and B cell proliferation 1 year after cessation of therapy. They found, that cytotoxic therapy can lead to long-term depression of the immune system, which was most prominent in patients with ALL.In an early orienting pilot study, Cranendonk et al. retrospectively determined the effects of various chemotherapeutic drug regimens on the numbers of different blood cell types in 131 children treated for solid tumors. Apart from the general cytoreductive effects during treatment, they observed that, in the majority of the children, the lymphocyte count became normal between 1 and 12 months after cessation of therapy17 and it had been shown that radiation therapy decreases the absolute CD4 T-cell counts23. As a consequence, antibody response to pneumococcal vaccine was profoundly impaired in patients who had received intensive treatment for HD24.For adults with HD, a treatment-associated marked long-term dysregulation of T-cell subset homeostasis has long been describedComparable larger studies in pediatric cohorts have not been performed.To illustrate varying characteristics of the cellular reconstitution after completion of chemotherapy in pediatric patients with ALL and other disease entities, we here present a comparative analysis of the course of total leukocyte, neutrophil and lymphocyte counts from 4 months before until twelve months after therapy in a large pediatric cohort of n = 132 patients who had been treated for ALL, HD and ES. Depending on the underlying disease and the resulting treatment regimens and modalities, we describe significant differences mainly affecting the total lymphocyte counts.10, we found no differences in the cellular reconstitution between patients with ALL belonging to the HR group and those belonging to the MR or SR group. This might be explained, however, by the relatively small proportion of patients with HR in this study.In contrast to earlier reports24.In particular, we saw a marked and prolonged post-therapeutic lymphocyte depression in patients with HD and ES as compared to patients with ALL. Lymphopenia was most distinct in individuals with HD who had received radiation therapy. This appears obvious because in the vast majority of cases the radiation field comprised the mediastinum and thus the thymic region with direct influence on T lymphocyte regeneration. In patients with HD, these findings are in line with earlier investigations in adult cohorts25 whereas others did not report comparable differences12. Considering the lower median age of patients with ALL compared to patients with HD and ES, this finding could contribute to the differences in lymphocyte reconstitution described between the disease entities in this study.In addition to these entity, treatment, and modality specific differences in cellular reconstitution, we observed a clear age dependency with patients aged ≤ 11 years showing an earlier recovery of the total lymphocyte count than patients aged 12–18 years. An age dependency of the post-therapeutic immune reconstitution has been proposed in different contexts in some earlier studies28, are still rather uniform, and do not consider potential differences for patients with solid tumors. In pediatric patients, who have been treated for childhood cancers without autologous or allogeneic HSCT, infection prophylaxis is generally withdrawn between 3- and 6 months3, and inactivated vaccines are administered from 3 months and live vaccines from 6 months after cessation of conventional antineoplastic therapy4.Overall, our study underlines that immune reconstitution after chemotherapy for childhood cancer highly depends on the underlying disease entity, the therapeutic regimen, and treatment modality as well as patient age and differs significantly between ALL and solid tumors. These discrepancies might at least in part be a consequence of the less intensive maintenance therapy applied in ALL but not in HD or ES. However, at the same time, current recommendations for infection prophylaxis and revaccination in childhood cancer have been mainly derived from pediatric patients with ALLThe results of our study could potentially contribute to discussions about adjusting the recommendations and establishing differentiated disease-, therapy- and modality-specific guidelines considering the differences in cellular reconstitution and in particular the clear and long-lasting effect of irradiation on lymphocyte total counts.We are aware that our study bears some limitations. Above all, due to the retrospective design, we could not investigate any additional immunological parameters and therefore focused on absolute leukocyte, neutrophil, and lymphocyte blood counts as surrogate parameters of immunological recovery. Moreover, we could not draw any clinical correlations, especially between the evolution of cell counts and infectious complications. In addition, the recent implementation of the bispecific T cell engager blinatumomab in the treatment protocols might further influence and thus alter cellular reconstitution after therapy in patients with ALL.As a consequence, future joint large studies are essential to cover these aspects in a disease- and treatment-specific manner. For a few treatment protocols, such investigations have already been initiated.This retrospective study was conducted in a single pediatric cancer center in accordance with the declaration of Helsinki. It used routine clinical data acquired during patient care, which was pseudonymized for the collection and subsequently anonymized for the analyses.As also confirmed by the institutional ethic committee of the Friedrich-Alexander-Universität Erlangen-Nürnberg, analysis of test results performed during patient care for research, like in this study, is in accordance with the applicable German/Bavarian regulations and does not require patients’ explicit consent nor a separate ethical approval.Individuals with ALL treated according to the protocol AIEOP-BFM ALL 2009Individuals with HD treated according to the protocols EuroNet-PHL-C2, EuroNet-PHL-C1, GPOH-HD 2002, and HD 95Individuals with ES treated according to the protocols EWING 2008 and EWING 99.Patients aged 0–17 years at the time of diagnosis who were treated at our center between the years 2000 and 2019 belonging to one of the following groups were eligible for this study:29. In the protocols EuroNet-PHL-C2, EuroNet-PHL-C1, GPOH-HD 2002, and HD 95, therapy levels TL-1, TL-2 and TL-3 were assigned according to the disease stage and indication for additional radiotherapy was based on early response after two initial cycles of chemotherapy32. Risk groups R1, R2, and R3 according to the protocols EWING 2008 and EWING 99 were based on initial tumor stage, tumor volume and histologic response to neoadjuvant chemotherapy33.According to the treatment protocol AIEOP-BFM ALL 2009, stratification into the high risk group was based on prednisone poor-response on day 8, ≥ 10% blasts by flowcytometry on day 15, non-remission on day 33, positivity for MLL/AF4 gene rearrangement or translocation t, hypodiploidy (< 45 chromosomes) or inadequate MRD PCR responsePatients who underwent autologous or allogeneic hematopoietic stem cell transplantation as first-line therapy were excluded from the analyses. Patients experiencing a relapse within the study period were eligible until the date of relapse diagnosis. Blood counts after this date were not considered. Individuals who deceased during the study period were also excluded.34. Other reference intervals were used according to Soldin et al.35. Subgroup division for age dependent analyses was also based on data reported in these latter studies. For cumulative analyses, the proportion of patients who had reached LLN before or at the specific time points was calculated resulting in values from 0 (0%) to 1 (100%).The data sets were extracted from patients’ digital and paper records and the laboratory information system. Patient groups were categorized according to the underlying disease with respect to risk group, treatment protocol, therapy level, and treatment modalities including chemo- and radiotherapy. The study period was defined as 4 months before and 12 months after the end of chemotherapy, 1 month corresponding to 4 weeks. All machine and manual blood counts performed in our hospital during the study period including total leukocytes, neutrophils, lymphocytes, segmented neutrophils, and band neutrophils were exported from the central laboratory information system swisslab. Where available, manually determined blood cell counts were used for this analysis. Total neutrophil counts comprised the sum of segmented and band neutrophils. In case, no manually counted blood cell values were available, device counted values were employed. If more than one manually or device counted blood cell value was determined from a patient on the same day, the daily mean value was calculated and included. A monthly mean of leukocyte, neutrophil, and lymphocyte count was determined for each patient. These values were then merged according to the diagnostic categories and subcohorts resulting in a subgroup-specific monthly median with range. Age-dependent reference intervals including the lower limit of normal (LLN) for total leukocyte counts were adopted from Zierk et al.The groups were tested against each other using Fisher ‘s exact test. Results with a two-tailed p-value < 0.05 were considered significant. The following groups were tested against one another in each blood cell category: HR-ALL (n = 5) vs. non-HR-ALL (n = 47) (1), HD-TL1 (n = 11) vs. HD-TL2 (n = 18) (2), HD-TL1 (n = 11) vs. HD-TL3 (n = 29) (3), HD-TL2 (n = 18) vs. HD-TL3 (n = 29) (4), HD non-irradiated (n = 28) vs. HD irradiated (n = 30) (5), ES-R1 (n = 16) vs. ES-R2 (n = 6) (6), ALL total (n = 52) vs. HD total (n = 58) (7), ALL total (n = 52) vs. ES total (n = 22) (8), HD total (n = 58) vs. ES total (n = 22) (9), ALL ≤ 11 years (n = 41) vs. ALL 12–18 years (n = 11), HD ≤ 11 years (n = 17) vs. HD 12–18 years (n = 41), ES ≤ 11 years (n = 11) vs. ES 12–18 years (n = 11), total patients ≤ 11 years (n = 69) vs. total patients 12–18 years (n = 63), ALL ≤ 11 years (n = 41) vs HD ≤ 11 years (n = 17) vs. ES ≤ 11 years (n = 11), ALL 12–18 years (n = 11) vs. HD 12–18 years (n = 41) vs. ES 12–18 years (n = 11).The cumulative incidence of reaching LLN for the specific time points was calculated as the number of patients who had reached LLN divided by the total number of individuals in the group.


More importantly, our recent publication showed that gene delivery of MRCKα, the downstream effector of β1 subunit-mediated signaling towards upregulation of adhesive junctions and epithelial and endothelial barrier integrity, also provided therapeutic potential for ARDS treatment in vivo but without necessarily accelerating AFC, indicating that for ARDS treatment, improving alveolar capillary barrier function may be of more benefit than improving fluid clearance. In the present study, we investigated the therapeutical potential of β2 and β3 subunits, the other two β isoforms of Na+, K+-ATPase, for LPS‐induced ALI. We found that gene transfer of either the β1, β2, or β3 subunits significantly increased AFC compared to the basal level in naïve animals and each gave similar increased AFC to each other. However, unlike that of the β1 subunit, gene transfer of the β2 or β3 subunit into pre-injured animal lungs failed to show the beneficial effects of attenuated histological damage, neutrophil infiltration, overall lung edema, or increased lung permeability, indicating that β2 or β3 gene delivery could not treat LPS induced lung injury. Further, while β1 gene transfer increased levels of key tight junction proteins in the lungs of injured mice, that of either the β2 or β3 subunit had no effect on levels of tight junction proteins. Taken together, this strongly suggests that restoration of alveolar-capillary barrier function alone may be of equal or even more benefit than improving AFC for ALI/ARDS treatment.Acute Lung Injury/Acute Respiratory Distress Syndrome (ALI/ARDS) is characterized by diffuse alveolar damage and significant edema accumulation, which is associated with impaired alveolar fluid clearance (AFC) and alveolar‐capillary barrier disruption, leading to acute respiratory failure. Our previous data showed that electroporation‐mediated gene delivery of the Na It is characterized by the accumulation of pulmonary edema of noncardiogenic origin and diffuse alveolar damage (DAD) that is primarily caused by alveolar capillary barrier dysfunction and the resulting alveolar and interstitial flooding of protein rich fluid2. It is estimated that there are 190,000 cases of ARDS annually in the United States with a hospital mortality of up to 40%3. During the evolving COVID-19 pandemic, approximately 33% of hospitalized COVID-19 patients develop ARDS, and the incidence of ARDS among non-survivors of COVID-19 reaches up to 90%, indicating that ARDS accounts for the majority of COVID-19 deaths4. Even so, the development of pharmacological treatments for ARDS has lagged since there is no single gene mutation or mechanism responsible for ARDS. Clinical strategies so mainly rely on supportive care and ventilatory management5.Acute Respiratory Distress Syndrome (ARDS) is a devastating clinical condition of acute respiratory failure7. AFC is driven by the osmotic gradient which is created by active ion transport, primarily Na+, across the alveolar epithelium8. Consequently, fluid is moved out from the airspace into lung interstitium along with the Na+ flux. Many researchers have focused on overexpressing or upregulating ion channels or transporters in the lung to increase AFC, such as the Na+,K+-ATPase, ENaC, and CFTR11. Pharmacologically, β2 adrenergic agonists have been administered to enhance AFC in various animal models by increasing the activity or membrane abundance of ENaC or the Na+,K+-ATPase12. However, this strategy has failed in clinical trials and shown no significant improvement of mortality rate13. Gene delivery mediated direct overexpression of ENaC or CFTR into living lungs also induces the increased transepithelial movement of Na+ towards the interstitium and therefore, enhances AFC. When tested in animal models of acute lung injury, the benefit of increasing AFC could easily be seen when the target genes were delivered prior to induction of lung injury, indicating a protective effect, but had little if any effect when delivered after injury had been established, suggesting that these may not be viable treatment approaches15.Increasing alveolar fluid clearance (AFC) has been one primary therapeutic targets to treat ARDS based on the finding that there is impaired fluid clearance capacity and significant edema accumulation in the lung during early ARDS+,K+-ATPase subunits for treating ARDS, our lab and others found that overexpression of Na+,K+-ATPase β1 subunit into lungs not only augmented AFC, protecting animals against the subsequent lung injury, but also rescued mice from pre-existing established injury induced by endotoxin16. Functionally, the Na+,K+-ATPase is critical to AFC, and more importantly, it is closely involved in the regulation of the epithelial barrier integrity17. Underlying ARDS, the net fluid clearance responsible for edema clearance from the airspace is undermined not by the impaired AFC, but also by alveolar capillary barrier dysfunction6. Our previous data showed that electroporation mediated gene transfer of the Na+,K+-ATPase β1 subunit could treat previously injured lungs, showing significantly upregulated tight junction protein expression, decreased lung permeability, edema accumulation, total protein and cellularity in bronchoalveolar lavage (BAL) fluid, and improved overall outcome of lung injury18. While these experiments showed that gene transfer of ENaC actively gave higher rates of AFC than did that of the Na+,K+-ATPase β1 subunit, only the β1 subunit had any treatment effect in previously injured lungs, indicating the significance of repairing alveolar capillary barrier integrity for ARDS resolution. Moreover, we recently demonstrated that CDC42 binding protein kinase alpha (MRCKα), a downstream mediator of β1 subunit-mediated signaling to upregulate tight junction and epithelial barrier integrity in vitro, also provided therapeutic potential for ARDS treatment in vivo19. Overexpression of MRCKα attenuated LPS increased pulmonary edema (Wet/Dry ratio), lung leakage, inflammation, and improved overall outcome of lung injury all to the same degree as seen with β1 gene transfer, but without improving AFC. These results further suggest that restoring lung barrier function alone may be of equal or even more benefit than improving AFC for treating ARDS.In studying the therapeutical potential of gene delivery of Na22. Although there is isoform selectivity, all of them are able to bind to the α subunit forming a functional pump for Na+ transport23. The α1β1 isozyme functions as a housekeeping Na+,K+-ATPase in all cells, vital for maintaining the intracellular ion homeostasis, and is highly conserved across species23. The regulatory role of the β1 subunit for epithelial barrier integrity is also conserved from insects to vertebrates24. For example, Nrv2, the vertebrate β1 homologue, is the only isoform involved in the epithelial septate junction barrier function in Drosophila25. Other β subunit isoforms are tissue-specific: the β2 isoform is expressed predominantly in brain and muscle, and the β3 isoform is found in lung, testis, skeletal muscle, and liver23. Sequence alignment shows there is only 25% homology among the three transport functional β isoforms with high diversity in their extracellular domains, including their degree of glycosylation. Our previous in vitro data showed that contrary to the gene transfer of the β1 subunit, gene delivery of the β2 or β3 subunit to cultured alveolar epithelial cells failed to induce tight junction expression in a monolayer19. Thus, in the present study, we tested whether overexpression of the β2 or β3 subunit of the Na+, K+-ATPase, like the β1 subunit, could upregulate pulmonary barrier function in vivo and be used to treat mouse lungs previously injured with LPS. Understanding this will help further evaluate the relative importance and contribution of lung barrier integrity and alveolar fluid clearance in the development of treatments for ARDS.To date, three β isoforms, β1, β2, and β3, have been identified as being expressed in mammalian cells+, K+-ATPase β1, β2, and β3 expressing Myc-DDK-tagged human Na+, K+-ATPase β1, β2, β3 subunits respectively, were purchased from Origene . Plasmids were purified using Qiagen Giga-prep kits  and suspended in 10 mM Tris–HCl (pH 8.0), 1 mM ethylenediaminetetraacetic acid (EDTA) and 140 mM NaCl.Plasmid pcDNA3 was obtained from Promega . Plasmids pCMV6-Na18. Twenty-four hours later, mice were anesthetized with isoflurane (2–4%) and 100 μg of plasmid in 50 μl of 10 mM Tris–HCl (pH 8.0), 1 mM EDTA and 140 mM NaCl was delivered to mouse lungs by oropharyngeal aspiration and electroporation with eight 10-ms square wave pulses at field strength of 200 V cm−1 using externally placed pediatric pacemaker electrodes  on both sides of the chest with an ECM830 electroporator  as described18. Each animal received one plasmid only  as opposed to combinations of the plasmids. Measures of lung injury were assessed 2 days after gene delivery (n = 6–8 mice/group). All experiments were repeated three independent times. To ensure that oropharyngeal aspiration resulted in plasmid delivery primarily to the lung, we delivered 50 µl of Evans Blue dye-labeled albumin to the lungs of mice and then isolated the lungs and the esophagus/stomach within 5 min of aspiration prior to drying and spectrophotometrically quantifying Evans Blue dye. As can be seen in the Fig. ALI was induced in male C57BL/6 mice (8–10 weeks) by oropharyngeal aspiration of 50 ul of Escherichia coli 055:B5 LPS  as described previously14. Following SDS-PAGE and transfer to PVDF membrane, blots were probed with primary antibodies against DDK tag , ZO-1 , occludin (Invitrogen), VE-cadherin , and glyceraldehyde-3-phosphate dehydrogenase GAPDH . Data were analyzed using the NIH Image J software.Western blot was performed using half of the left lung lobe that was homogenized in 300 μl of 1× Reporter lysis buffer , containing protease inhibitor  using a TissueLyser II  as previously described18. Briefly, lungs were removed from the mice and weighed immediately (wet weight) prior to drying them for 72 h at 70 °C and weighing again (dry weight).Wet to dry (W/D) ratios, as a measure of pulmonary edema, were measured 72 h after LPS instillation as previously described26. Briefly, 0.7 ml of sterile PBS was instilled into mouse lungs, lavaged twice, collected and centrifuged. Total protein concentration of the supernatant was measured using a Bradford assay (Bio-Rad), and the total number of cells was determined using a hemocytometer and then cells were stained with Diff-Quick  after cytospin. Albumin levels in BAL fluid of each group was assessed using a mouse albumin ELISA quantitation kit  as described18.Protein concentration and cellularity of BAL fluid was measured as described previously2O with buffered formalin, removed from the chest, fixed overnight at 4 °C and paraffin-embedded. Sections (5 µm) were stained with hematoxylin and eosin (H & E).Lungs were inflated to a pressure of 20 cm H18. Briefly, EBD  was injected into the tail vein 47 h after gene transfer and the lungs were perfused 1 h later with sterile PBS, excised, and dried at 60 °C for 24 h. EBD was quantified spectrophotometrically at 620 and 740 nm, and corrected with the formula E620 (EBD) = E620 − (1.426 × E740 + 0.030).Pulmonary permeability was performed as described to assess alveolar capillary barrier integrity by the leakage of Evans Blue Dye (EBD) labeled albumin from blood into airways18. Procaterol  was administered in the instillate as a positive control.AFC was evaluated by quantifying the rate of concentration of an isosmolar (324 mOsm) solution of EBD labeled albumin in the alveolar airspace as previously describedEach experiment was repeated at least three times with the number of animals per group indicated for each figure (n = 6–14). The data of each series is displayed as mean values ± standard error of the mean (SEM). Graphing and statistical comparison of the data were performed using Prism 8 . Measurements for more than two groups were analyzed by one-way ANOVA followed by multiple comparisons. P values less than 0.05 were considered to be statistically significant.All animal studies were approved by the University of Rochester Committee on Animal Resources and experimental procedures were carried out under the institutional guidelines for the care and use of laboratory animals in an American Association for the Accreditation of Laboratory Animal Care-approved facility. All animal studies complied with the ARRIVE guidelines.+ transport17. Extensive research in animal models and patients has shown that upregulating Na+,K+-ATPase activity or its abundance through β adrenergic stimulation or gene delivery of the Na+,K+-ATPase α1 or β1 subunits significantly enhances alveolar fluid clearance (AFC) from its basal level, indicating there is increased active Na+ transport activity. β2 and β3, two additional β isoforms, are expressed in a tissue- and cell-specific manner to form active Na+ pumps for ion extrusion20. To further validate that gene transfer of the β2 and β3 subunits could also accelerate alveolar fluid clearance by overexpressing Na+,K+-ATPase and thereby increasing Na+ absorption from alveoli, genes encoding the β2 and β3 subunits were delivered to mouse lungs by electroporation and 2 days later, AFC was measured in living lungs  by 55% and 63% compared with naïve animals  and mice that received empty plasmid pcDNA3  by 54% and 62%, respectively. There was no significant difference in AFC rates between naïve and pcDNA3 mice. Animals receiving either β1 gene transfer or procaterol (10−8 mol/L), a specific β2-Adrenergic receptor agonist, were used as positive controls, leading to increases of AFC by 73% and 78%, respectively, compared with naïve mice (p < 0.01). There was no significant difference in AFC between animals receiving the β1, β2, or β3 subunits, indicating that all 3 subunits can increase AFC to similar degrees. Further, the significantly increased AFC from the naive basal level caused by β2 or β3 overexpression suggests that these two β isoforms can heterodimerize with the endogenous α subunit to form functional Na+,K+-ATPases for ion transport in the lung.Fluid reabsorption from the alveolar space has been well documented based on the osmotic pressure created by transepithelial active ion transport, primarily Nangs Fig.  and S3. +,K+-ATPase can effectively attenuate LPS-induced acute lung injury in vivo with decreased total cellularity and PMNs in BAL fluid, compared with LPS injury alone14. This has been seen in both protection experiments where β1 is transferred prior to lung injury and in the more stringent and clinically relevant treatment model where DNA is delivered to lungs with established lung injury. Since gene delivery of the β2 or β3 subunit of the Na+,K+-ATPase could also enhance AFC to the same degree as that by β1 overexpression, we next determined whether gene delivery of β2 or β3 subunits could also attenuate inflammation in LPS pre-injured animal lungs. BAL fluid was harvested from mouse lungs and prepared for total cell counting and differential cell staining. Since PMNs are the predominant immune cell type in BAL fluid, PMNs were also counted for statistical analysis  and PMNs  in BAL fluid, compared with LPS injury alone. Surprisingly, gene transfer of the β2 or β3 subunit of the Na+,K+-ATPase into LPS pre-injured animal lungs did not decrease the total number of cells  or PMNs ) in BAL fluid, and there was no statistical difference between β2 or β3 overexpression mice and LPS injury group (not shown). Further, there was no difference in cell numbers between mice receiving β2 or β3 plasmid and animals receiving empty vector. Collectively, these results indicate that unlike the β1 subunit, electroporation mediated gene transfer of β2 or β3 Na+,K+-ATPase failed to attenuate LPS induced lung inflammation.We previously have reported that electroporation mediated gene transfer of β1-Na+,K+-ATPase β1 subunit showed a therapeutic effect on the injured lungs, notably reducing patchy areas of neutrophil infiltration, alveolar edema and deposited fibrin strands, and showing increased numbers of areas of open and clear alveoli. However, gene delivery of the β2 or β3 subunits failed to show noticeable attenuation of any of these hallmarks of diffuse alveolar damage, compared with the LPS injury group or pcDNA3. Taken together, all the data here indicate that gene transfer of β1-Na+,K+-ATPase provides a therapeutic benefit attenuating lung inflammation and overall damage, whereas gene delivery of β2 or β3 isoforms of the Na+, K+-ATPase has no treatment effect on lung injury.We next looked at overall lung injury by histological analysis to determine whether gene delivery of β2 or β3 subunits could attenuate LPS induced injury. Figure +,K+-ATPase were delivered to mouse lungs and 2 days later, lungs were harvested for gravimetric analysis , or LPS plus empty vector(p < 0.01)27. However, the injured lungs receiving plasmids to overexpress the β2 or β3 subunit of Na+, K+-ATPase failed to show any changes in their wet to dry ratios , compared with injury groups , and there was no statistical significance between treatment groups of LPS with β2 Na+,K+-ATPase, or β3Na+, K+-ATPase and mice with LPS alone or LPS plus pcDNA3.One feature characterizing diffused alveolar damage is the alveolar flooding to form edema at the early stage of ARDS. We investigated whether gene transfer of the β2 or β3 subunit could reduce the general edema fluid retention following lung injury, since they markedly increase AFC following gene transfer in mice Fig. . As abovsis Fig. . Compare6. Since gene delivery of the β2 or β3 subunit of the Na+,K+-ATPase could accelerate AFC but failed to decrease or partially reverse the LPS increased edema accumulation, we further determined the injured animals’ lung barrier function after treatment intervention with the various β subunits. Thereby, in vivo lung permeability was determined by the leakage of EBD labeled albumin from blood into airways , mice injured with LPS alone showed an up to threefold increase of spectrophotometric absorbance of EBD (0.713 ± 0.044), indicating severe lung barrier leakage (p < 0.0001). Gene delivery of empty vector pcDNA3 (0.679 ± 0.047) showed no significant difference compared to LPS alone. As expected, gene transfer of β1-Na+, K+-ATPase, the positive control, significantly reversed the LPS increased lung permeability by more than 30% (0.462 ± 0.033), compared to LPS alone or LPS plus pcDNA3, indicating the significant rescue of lung barrier function . However, gene delivery of β2 or β3 subunits failed to attenuate LPS increased lung permeability with EBD spectrophotometric absorbance of 0.694 ± 0.060 for the LPS with β2 group and 0.657 ± 0.055 for the LPS with β3 group, indicating there was still severe lung leakage in β2 or β3 treated mice.The gravimetric analysis of wet to dry weight ratio is a gross indicator of lung edema accumulation, reflecting the result of net fluid clearance which is composed of both the osmotic driving force for alveolar fluid absorption and an intact alveolar capillary barrier which holds out against alveolar floodingays Fig. 14. Forty+, K+-ATPase failed to attenuate LPS induced lung hyperpermeability, we further examined the total protein and high molecular weight protein albumin level in BAL fluid of injured mice, which are other parameters reflecting the alteration of alveolar capillary barrier. Mice injured with LPS significantly increased the total protein  and albumin  level observed in mice with LPS injury alone or with following pcDNA3, further indicating there is no restoration or rescue of the lung barrier hyperpermeability and leakage by transferring genes of β2 or β3.Since gene delivery of β2- or β3-Na28. They seal the lateral space of adjacent cells at the apical side, forming a tight barrier. Upon LPS assault, tight junction complexes are disrupted with downregulated protein expression. Our previous data showed that gene delivery of the β1 subunit into mouse lungs attenuated LPS increased lung permeability/leakage with significantly upregulated protein levels of tight junction proteins, 14. Thus, we investigated whether delivering genes encoding the β2 or β3 subunit could restore ZO-1 and occludin protein expression in pre-injured mouse lungs. As described above, 1 day after LPS intratracheal administration, plasmids expressing β2- or β3-Na+, K+-ATPase were delivered by aspiration and electroporation. Then after 2 days, the lungs were harvested for western blots  or occludin  levels, compared with groups with LPS injury alone or LPS and pcDNA3. We also have shown previously that gene transfer of the β1 subunit to cultured microvascular endothelial cells and to the mouse lung can increase expression of the adherens junction VE-cadherin18. Since we have shown that electroporation-mediated gene transfer can deliver genes to all cell types in the lung, including both the epithelium and endothelium, this suggests that the Na+,K+-ATPase β1 subunit signals similarly in endothelial cells and epithelial cells, as suggested in isolated cell studies18. Thus, we tested whether gene transfer of the β2 or β3 subunit could also increase VE-cadherin levels in these mice  receptor on the endothelial cell surface, when bound by S1P, enhances endothelial barrier integrity through actin cytoskeleton reorganization and localization of catenin and VE-cadherin molecules to the endothelial surface, supporting this as a potential intervention for ARDS36. In this respect, we have previously demonstrated that the treatment benefits gained by gene transfer of the β1 subunit of the Na+,K+-ATPase come not only from enhancing AFC by increasing ion transport activity, but also, more importantly, from restoring lung barrier function by increasing the abundance of tight and adherens junction proteins at the membrane, thereby decreasing lung permeability14. We identified the kinase MRCKα as a protein interactor with the Na+, K+-ATPase β1 subunit that is necessary for the junctional complex upregulation seen in both cells and in mouse lungs19. Upon overexpression of the β1 subunit, MRCKα is activated and in turn activates actin-myosin, circumferential actin bundling, and junctional recruitment via MLC2 phosphorylation19. Interestingly, neither the β2 or β3 subunits of the Na+, K+-ATPase interacted with MRCKα and both failed to stimulate overexpression of tight junction proteins in cultured type I alveolar epithelial cells19. When examined as a possible treatment for lung injury in mice, MRCKα gene transfer was indeed able to upregulate barrier function as in cultured cells and decrease injury to the same degree as β1 subunit gene transfer, but did not positively affect AFC18.There are limited studies investigating the beneficial effects of re-establishing the alveolar capillary barrier for ARDS treatment, despite a clear understanding that both endothelial and epithelial barrier dysfunction play critical roles in the pathophysiology of the disease. In contrast, the breakdown of endothelial and epithelial barrier integrity clearly has been recognized as a key factor in the development of edema formation and establishment of ARDS+,K+-ATPase (and hence AFC) is not required for the β1 subunit to regulate barrier function. We have found that even in the presence of oubain at concentrations shown to inhibit Na+,K+-ATPase ion channel and ATPase activity, overexpression of the β1 subunit in cultured ATI cells can still increase tight junction protein membrane abundance and transepithelial electrical resistance19. Further, we have shown that individual overexpression in mouse lungs of all of the β subunits increases AFC to a similar level, roughly twofold over that seen with naïve mice or those receiving the parental empty plasmid 42, requiring the amino acid region 198–207 as well as N-glycosylation23. Interestingly, this region is lacking in both β2 and β3 subunits, and as such these β subunits fail to show homo- or heterodimer β-β interactions and cell–cell adhesive properties39. However, the β2 subunit has been shown to act as a cell adhesion protein, at least in glial cells43, although the mechanisms for this are unknown and may be limited to specific cell types. While β1-β1 trans-dimerization between cells has been suggested to be responsible for the formation and/or stabilization of epithelial adherens and tight junctions, clear mechanistic studies showing the relationship between the β1 trans-dimers and apical junctions are lacking45. There has been limited study investigating the non-transport function(s) of the β2 and β3 isoforms, especially in relation to intracellular junctions and barrier formation. However, our previously published data showed that overexpression of the β2 or β3 subunit failed to upregulate tight junction expression in vitro, indicating the role that is involved in epithelial barrier function is probably unique for the β1 subunit of the Na+, K+-ATPase.Three isoforms of the β subunits are expressed in mammalian cells. Although there is isoform selectivity, all of them are able to bind to the α subunit, facilitating the correct folding, membrane recruitment and insertion of the α subunit and forming a functional ion transport complex+, K+-ATPase reduced multiple measures of lung injury but not to the levels seen in naïve animals. For example, a 27% decrease in pulmonary edema was observed when the β1 subunit was delivered compared to injury only, while a ~ 60% decrease in lung permeability and BAL protein or albumin content was observed. In none of these measures did the LPS-induced injury return to the pre-injury baseline. Several reasons could account for this. It is possible that the timing of outcome measures was not optimized to see maximal benefit, or that the amount of gene transfer and overexpression of the β1 subunit was insufficient to cause a complete reversal of the injury. Thus, it is possible that either delivering greater amounts of β1-expressing DNA or by repeat dosing of the DNA by electroporation-mediated gene delivery at some intervals could increase levels of the β1 transgene and further decrease measures of injury. Another approach could be to co-deliver other genes known to decrease inflammation or stimulate alveolar epithelial cell proliferation, both of which have shown some success in animal studies. For example, delivery HO-1 which has been shown to upregulate IL-10 levels or delivery of IL-10 directly has been shown to decrease lung injury, by mechanisms unrelated to fluid clearance or barrier repair48. Similarly, delivery of keratinocyte growth factor (KGF) promotes type II cell proliferation and partially protects from lung injury49. While none of these approaches have provided complete protection from subsequent lung injury, it is possible that a combinatorial approach in which multiple distinct pathways are targeted could provide even greater benefit that we have seen here in our treatment studies.Our results demonstrate that gene transfer of the β1 subunit of the Na34. Taken together with results from multiple clinical trials demonstrating that upregulation of alveolar fluid clearance alone has no positive benefit in patients13, these results may have lasting impact on development of treatment strategies for ALI/ARDS in patients.In conclusion, our data support a model in which stimulation of alveolar fluid clearance alone is insufficient to attenuate previously existing lung injury caused by LPS aspiration in mice and point to repair or strengthening of the alveolar-capillary barrier by upregulation of intercellular junctional complexes as being needed for effective treatment. These studies support a growing body of data suggesting that repair of barrier function is necessary for any meaningful alveolar fluid clearance to occur that can lead to lessening of the diseaseSupplementary Figures.