Light-activated antimicrobial agents (photosensitisers) are promising alternatives to antibiotics for the treatment of skin infections and wounds through antimicrobial photo dynamic therapy (aPDT); utilisation of this technique is still restricted by general low efficacy requiring long exposure time (in the order of tens of minutes) that make the treatment very resource intensive. We report for the first time the possibility of harvesting the cell penetrating properties of poly-beta-amino esters (PBAEs) in combination with toluidine blue O (TBO) to shorten aPDT exposure time. Candidates capable of inactivation rates 30 times quicker than pure TBO were discovered and further improvements through PBAE backbone optimisation could be foreseen. Efficacy of the complexes was PBAE-dependent on a combination of TBO uptake and a newly discovered and unexpected role of PBAEs on reactive species production. Chemometric approach of partial least square regression was employed to assess the critical PBAE properties involved in this newly observed phenomenon in order to elicit a possible mechanism. The superior antimicrobial performance of this new approach benefits from the use of well established, low-cost and safe dye (TBO) coupled with inexpensive, widely tested and biodegradable polymers also known to be safe. Moreover, no adverse cytotoxic effects of the PBAEs adjuvated TBO delivery have been observed on a skin cells in vitro model demonstrating the safety profile of this new technology. SSTI symptoms range from mild conditions, such as pyoderma, to serious life-threatening such as necrotizing fasciitis; besides the affected area (i.e. hands or legs) may lose functionality depending on the severity of the infection2. Clearly SSTI are simultaneously a threat to life and quality of life to patients and an economic burden to health care providers4; i.e. it has been estimated that every MRSA infection costs an extra 9000 £ to the NHS5 while in the USA the total spent on the treatment of SSTIs tripled between 2000 and 2012, from $4.4 to $13.8 billion6.Skin and soft-tissue infections (SSTIs) are some of the most common infections observed in ambulatory practice and hospitals; to further concern, their rate has increased in the last decades8; nonetheless it is expected that antibiotic resistance will outpace the development of new drug thus different antimicrobial treatment is urgently needed.Antibiotics are the first line approach to treat SSTI, however their efficacy is rapidly decreasing in consequence of the growth of antibiotic resistance among pathogens. New antibiotics, such as oritavancin or delafloxacin, to treat SSTI, have emerged9, periodontitis10 and diabetic foot ulcer12. The mechanism of action of PDT derives from a light-induced excitation of a photosensitiser (PS) to singlet state and its conversion to triplet state, whose reactivity results in the generation of radicals and ROS14. These cell death inducing capabilities of ROS have found medical applications in cancer15 and infections  treatment14.Photodynamic therapy is a local, repeatable non-invasive technique which might be an effective alternative to antibiotics for the treatment of local infections17 and by biologically compatible light activated compounds PS14. Lethality mechanism of aPDT is multi-targets; hence it is not prone to induce further resistance20. A further benefit of aPDT is the possibility of inactivating not only viable cells but also virulence factors released by pathogens that can cause tissue damage after bacterial death21. This possibility makes aPDT stand alone in the variety of antimicrobial techniques that are capable of only interacting with cells leaving virulence factors untouched. Clinical trials have also investigated aPDT for the treatment of skin infections25.aPDT feasibility depends on the higher resistance of mammalian cells than pathogens to the oxidation induced by light activated compounds26 is currently the main obstacle to its application in hospital settings as this limits the number of patients that can be treated per beds available. The challenge of reducing the irradiation time necessary to achieve aPDT satisfactory results has been addressed by developing delivery systems that augment PS uptake28 or designing novel PS that either exhibit selective affinity to bacterial cells over other tissue cells or are more potent ROS producers31. Despite the positive results, such advances are not yet sufficient in terms of efficacy or economically viable; consequently, new approaches are required in order to tackle the antibiotic resistance threat in a cost-effective way that maximises the return of health providers scarce resources.Despite the numerous mentioned benefits of aPDT, the necessary exposure time (in the order of 10–15 min)32. We hypothesised that PBAEs capabilities to actively deliver molecules inside cells could be exploited as adjuvant to microbial cell uptake of toluidine blue O (TBO) and demonstrated for the first time that PBAE-TBO complexes can have higher antimicrobial activity after laser exposure than the comparable amount of free dye on various pathogens representative of SSTI  was assessed in vitro to supplement the antimicrobial efficacy data of the technology with clear safety results.Poly-beta-amino esters (PBAEs) were first described as biodegradable and biocompatible polymers with cell membrane penetrating characteristics that could be employed as gene transfer vectors in light of their positive charges and ability to form complexes with DNA moleculesSTI Fig. . TBO was34. The polymerisation, a Michael addition reaction s were synthesized by mixing 1,4-butandiole diacrylate, 1,6-hexanediol diacrylate or tetra(ethylene glycol) diacrylate with a range of amine monomers Fig.  in dichl34. End-capped PBAEs were recovered through precipitation in diethyl-ether under vigorous mixing, the unreacted amines were removed centrifuging the suspension of PBAE in diethyl-ether/DCM for 2 min at 1155g. The supernatant was removed and the PBAEs washed twice with diethyl-ether. The end-capped PBAEs were then dried under vacuum.Acrylate-terminated polymers of were dissolved in DCM at a concentration of 31.13% w/w. A range of end capping agents Fig.  were theThe PBAEs and derivatives obtained were coded through the constituents of the backbone using a capital letter to indicate the acrylate  and a number to indicate the amine as shown in Fig. Mn) and weight-averaged molecular weight (Mw) were determined against PEG standards. Molar masses were only apparent values due to the use of PEG standards and possible different hydrodynamic volumes of PEG standards and poly(beta-amino esters) of identical molar mass34.Gel permeation chromatography (GPC) was employed to determine the molecular weight of each PBAE in the library. Samples were injected in a Shimadzu-LC-20Ai system equipped with a Superdex 75 10/300 GL column; the mobile phase was 100% sodium acetate buffer pH = 5 at a constant flow of 1 mL/min. Number-averaged (g. The supernatant was removed and the PBAEs washed twice with a diethyl-ether/DCM mixture 4:1. The end-capped PBAEs were then dried under vacuum and appeared viscous fluids at room temperature.10 mg of TBO were dissolved in DCM (25 mL) along with 100 mg of end-capped polymer; the solutions were immediately covered with aluminium foil and stirred for 24 h at room temperature. The PBAEs-TBO derivatives were recovered through precipitation in diethyl-ether under vigorous mixing, the unreacted molecules were removed centrifuging the suspension of PBAE in diethyl-ether/DCM for 2 min at 115535, we hypothesized that the PBAEs-TBO complexes would be constituted by micelles containing TBO with PBAEs on the outer shell.PBAE are known to form micelles when in aqueous fluidsEscherichia coli (NCTC10418), Staphylococcus epidermidis (ATCC12228), Pseudomonas aeruginosa (NCIMB10548), Methicillin Resistant Staphylococcus aureus (NCTC12493), Streptococcus pyogenes (ATCC19615) and Acinetobacter baumannii (NCIMB9214) were stored at − 80 °C. These organisms were subcultured, when needed, on Brain Heart Infusion (BHI) Agar overnight aerobically at 37 °C. The plates were then stored at 4 °C for no more than two weeks. Bacterial suspensions used for the experiments were grown in Brain Heart Infusion (BHI) broth after inoculation with a loopful of cells from a single colony on a BHI plate and incubated aerobically for 24 h at 37 °C statically. These overnight cultures were then diluted 1 in 100 in PBS. The resulting bacterial suspension contained approximately 106 CFU/mL. Each cell suspension was used to disperse pure TBO to concentrations of 0.2 mg/mL or PBAEs-TBO derivatives to an equivalent TBO concentration.The organisms used in this study were representatives of pathogens encountered in SSTI. 200 μL aliquots of the resulting suspension were immediately poured in a GREINER 96 U-BOTTOM well plate. The well plate was then irradiated with a laser light (633 nm) using a 500 mW red laser  for different time periods (between 10 s to 3 min).After exposure the bacterial cells (L+S+) were counted through serial dilutions and plating on BHI Agar. Along this test, different experiments were performed including the testing of dark toxicity (labelled with L−S+), samples exposed uniquely to laser light (L+S−) or samples not exposed to either laser light or PBAE (L−S−).Through the experiments, all prepared samples, well plates and inoculated plates were immediately covered with aluminium foil to reduce biased measuring results due to exposure of any light besides the laser. All plates were incubated after the experiment for 24 h at 37 °C prior to colony forming units counting. All experiments were performed on three independent cultures.UV–Vis spectrum was determined using the Cary 60 UV–Vis Spectrophotometer .Absorption of pure TBO and produced TBO–polymers complexes were measured using standard cuvettes. All samples were diluted in PBS to concentrations of equivalent to 20 μg/mL of pure TBO.The generation of reactive oxygen species (ROS) was assessed using a singlet oxygen Sensor Green reagent (SOSG) . The SOSG was stored at ≤ − 20 °C and protected from light prior to use. The working solution was prepared with methanol to a final concentration of 5 mM. The prepared reagent was further diluted in methanol (1:100) before each experiment. Each sample was dispersed in PBS to a concentration equivalent to 0.2 mg/mL of pure TBO. 100 μL of diluted sample, 100 μL PBS and 20 μL reagent were immediately poured in two adjoining wells of a GREINER 96 U-BOTTOM well plate. The irradiation of one well (L+) was in each case carried out for a period of 1 min with a 500 mW red laser ; whilst the other well was covered with aluminium foil (L−). After exposure, ROS were determined using the FLUOstar OPTIMA .g, afterwards the supernatant was disposed. After one wash and centrifuge with PBS, 1 mL of sample  was added to the precipitated cells. The resulting solution was vortexed and centrifuged after 3 min exposure. The exposure of samples to cells was limited to 3 min to ensure comparability to other experiments. After two more wash- and centrifugation cycles with PBS, the cells were dissolved in 1 mL 0.1 M NaOH/1% sodium dodecyl sulphate (SDS) and incubated at 37 °C for 24 h to lysate the cells.10 mL of fresh sterile BHI broth were inoculated with a loopful of cells from a single colony on a BHI plate and incubated aerobically for 24 h at 37 °C statically. The bacterial suspension was then centrifuged with an Avanti J-20XP Centrifuge  for duration of 3 min at 2938The optical density of samples was measured at a 650 nm using a plate reader (Labtech LT5000MS) against a calibration curve prepared using the corresponding bacterial lysate.36. Partial Least Square (PLS) regression analysis between PBAEs characteristics and ROS production ratio between PBAE-TBO complexes and pure TBO was carried out using R (ver 4.0)36 and the “plsdepot” package37.Hieratical clustering (using Manhattan distance between PBAEs and complete distance between clusters) was performed R (ver 4.0) and the “stats” package34. Further experimentally determined properties of the PBAEs  were also added to the properties of PBAE included in the PLS regression.The physical and chemical parameters of each PBAE constituents (amines and acrylates) were obtained from PubChem library  while the parameters related to the basic repeated polymeric unit (amine + acrylate) were calculated through ChemDraw. These included molecular weight (MW), boiling point (BP); melting point (MP); critical volume (CV), Gibb's free energy (GFE), logP, solubility (logS), pKA, molar refractivity (CMR), heat of formation (HtF), TPSA and the ratio between the MW of amine and acrylate. TPSA  represents the total area of all polar atoms (mainly oxygen and nitrogen) including their affixed hydrogen atoms while logP is the partition-coefficient between two immiscible phases at equilibrium which is proportional to hydrophobicity of the compound of interest39.The minimum number of PLS components to describe the role of different PBAEs chemical–physical properties on the ROS production was determined as the minimum number of PLS components that return at least 80% of the initial input data and an inflection in the scree plot representing the root mean squared error (RMSE) of the predictions vs. number of PLS components was40 from Cardiff University and grown in Minimum Essential Medium Eagle (MEM) supplemented with 10% heat-inactivated foetal bovine serum (FBS). Both media were supplemented with 1% penicillin–streptomycin (PS). Both cells lines were incubated at 37 °C in a humidified air atmosphere with 5% CO2; media were changed twice per week. 96 well plates were seed with 150 μL containing 10,000 viable cells as determined through trypan blue staining and incubated for 5 days at 37 °C in a humidified air atmosphere with 5% CO2; media were changed after 2 days.Human keratinocytes cells (ATCC PCS-200-011) were grown in Dermal Cell Basal Medium supplemented with Keratinocyte Growth Kit (ATCC); whilst human dermal fibroblast cells were kindly supplied by Prof. StephensCells were washed three times with PBS and incubated with 100 μL of either TBO 0.2 mg/mL or TBO–PBAE containing TBO 0.2 mg/mL After 2 h, wells were washed once with 100 µL of treatment media and maintained in this media thereafter. The well plate was then irradiated with a laser light (633 nm) using a 500 mW red laser  for 3 min.Control experiments included testing of dark toxicity (labelled with L−S+), samples not exposed to TBO and uniquely to laser light (L+S−) or samples not exposed to either laser light or PBAE (L−S−).2. After 4 h, the supernatant was then removed and 100 μL of dimethyl sulfoxide were added. The absorbance was measured by a spectrophotometer (Lab Tech LT5000MS) at 560 nm; mitochondrial activity was normalised against cell exposed to fresh medium only.20 μL of MTT stock solution (5 mg/mL) were added to each well and incubated in a humidified atmosphere at 37 °C with 5% COtotal and LDHreleased respectively) were determined as OD, at 490 nm, after correcting for the reading from the negative control. Cell viability was calculated according to the following equation:41LDH was quantified in the media (LDH released) and after adding the cell lysis solution  according to manufacturer’s protocols. Total and released LDH  in the MTT and LDH assays were tested through ANOVA with a significant level p = 0.05.p = 0.05.Multivariate analysis of variance (MANOVA) was carried out to test impact of the different end-capping agents on the antimicrobial activity, TBO cell up-take and ROS production of PBAE-TBO against pure TBO, using the Pillai test with a significant level UV–vis spectra of pure TBO showed a maximum of absorbance at about 633 nm; spectra profiles did not vary when TBO was complexed with PBAE Fig. .Figure 4S. epidermidis, MRSA and S. pyogenes) than the Gram- .In all six species tested Fig. , viable In all cases, samples exposed to the polymer-TBO complex or to pure TBO without irradiation (L−) did not return any cell reduction over the time considered. In comparison, irradiated (L+) samples show tendencies of progressive reduction. The comparison with pure TBO showed an obvious improvement in the antimicrobial effect of laser exposure in all cases when the same amount of photosensitiser was employed along with A3-e1. Furthermore, when cells were exposed to laser light without the presence of photosensitiser (L+S−) did not reveal any antimicrobial activity. Furthermore, PBAE alone (without TBO) did not exhibit antimicrobial activity towards any of the pathogens tested either in the dark or after laser irradiation.E. coli) and an example of Gram+ (S. epidermidis) is shown in Fig. N,N-bis[(3-methylamino)propyl]methylamine (amine 20) along with 3-(dimethylamino)-1-propilamine (amine 5) and cyclopentylamine (amine 15) resulted in similar PBAE than any of the other amines.The comparison of the efficacy of other PBAE as delivery system of TBO for antimicrobial PDT against an example of Gram− (p < 0.05).Using similar amine and end-capping agent for the preparation of PBAE, 1,6-Hexanediol diacrylate (acrylate B) was usually more effective than 1,4-Butanediol diacrylate (acrylate A) with Poly(ethylene glycol) diacrylate (acrylate C) returning the worst antimicrobial activity of the three. Most of the PBAE-TBO complexes improve the antimicrobial activity compared to the same amount of pure TBO. PBAEs end-capped with e2 had better antimicrobial activity that the respective counter-part end-capped with e1  diacrylate generally did not enhance TBO cell uptake. PBAEs end-capped with e2 resulted in higher TBO cellular uptake that the respective counter-part end-capped with e1 (p < 0.05).All PBAE tested increased the uptake of TBO in both dis Fig. , polymerp < 0.05).The amount of ROS generated by pure TBO only PBAE-TBO complexes is shown in Fig. No ROS were detected when only PBAE were exposed to laser light, hence PBAE are not photosensitisers. This demonstrate that PBAE do not only act in enhancing TBO uptake by cells but also have a direct role in the mechanism of radicals formation by PDT.S. epidermidis or E. coli was not solely depended on TBO uptake but was a combination of both uptake and ROS as not linear correlation was noticeable  of the amine component of the polymer was positively correlated to ROS formation Fig. .Figure 1Mitochondrial activity, estimated through MTT assay, and cell viability determined through LDH assay of both keratinocytes and fibroblasts were not impacted (p > 0.05) by exposure to a selection of the TBO–PBAE prepared, additionally not dark toxicity was detected in either cell lines Fig. .Figure 142; however a critical drawback is the still relative long treatment time required (in the order of tens of minutes) that impacts the largescale application of this technology26. The design of novel and more efficient photosensitisers is generally accompanied by elevated drug costs; similarly, the use of drug delivery systems brings additional costs that must be offset by the enhanced efficacy. We have brought a new approach to boost aPDT efficacy through the use of polymers enhancing intracellular drug uptake; our starting point was the use of a photosensitiser commercially available and approved for use in humans, specifically TBO 43, and polymers proven biocompatible and low cost such as PBAE. The safety profile of the compounds used in this technology offers both a shorter route to market and a high likelihood of future in vivo trials of this approach to prove safety while the use of low-cost chemicals also improves opportunities to be cost-effective.aPDT has numerous benefits as potential alternative to antibiotics for the treatment of infections such as skin and tissues that can be accessed with light sources45; subsequently the role of polymer backbone constituents was assessed through a broad library of PBAE and a new discovery, the impact on ROS, beside TBO uptake was also observed. PBAE features critical in this newly noticed phenomenon were assessed through PLS, one of chemometrics data analytic techniques that have been employed in drug design and analytical chemistry47, to also elicit possible mechanism. Initial in-vitro tests on keratinocytes cells and fibroblasts were also carried out exposing the cells to a selection of the PBAE-TBO complexes that exhibited the greatest enhancement of antimicrobial activity; in order to gather more robust safety data, two independent assays (MTT and LDH) were utilised with both showing the safety of the complexes PBAE-TBO on skin tissue cells.We firstly validated the hypothesis of using PBAE to augment PDT on a variety of pathogens representing those most commonly encountered in SSTI50 and is widely considered to be explained by the relatively porous layer of peptidoglycan and lipoteichoic acid constituting the cell wall of Gram+ that allows the photosensitiser to cross the cell wall compared to the cell envelope of Gram- bacteria that exhibit higher resistance to photosensitiser uptake48.The higher vulnerability of Gram+ over Gram− observed in this study Fig.  was expe32; subsequent evidence has validated such properties and identified key features and optimised the polymer chain backbone in order to maximise the opportunities offered by such polymers to transfer genes into target cells51. Further applications of PBAEs have been proposed for cancer drug delivery55, in layer-by-layer deposition of drug releasing coatings61 and drug localisation in cartilage34; currently no application of these polymers in antimicrobial technologies has been reported. Depending on the application, different PBAE features have been shown to pivot the outcome; for example PBAEs belonging to the family of B3 and A16 are known cell uptake enhancer in DNA delivery51 while A5 and B5 are better at driving drug uptake into cartilage34; additionally it has also been proven that end-capping has a further critical role35. Because of the essential role on TBO uptake into bacterial cells in controlling aPDT effect, it was not unexpected that polymer synthesised with acrylates such as A and B with amines such as 3 and 16 had the greatest impact on TBO uptake  is necessary for the augmented ROS formation by TBO when in presence of PBAEs. Moreover, the high correlation between TSPA (the level of polar surface) characteristics of PBAE components further support this hypothesis; under this hypothesis, it was expected that the correlation between TPSA of acrylates and amine had opposite signs as amine have positive charges whilst acrylates only exhibit oxygen atoms that results in negative charges.All three constituents of PBAEs, namely diacrylate, amine and end-capping, influence all the outcomes investigated here Figs. , 7, 8 inble Fig. , one is ble Fig. , in ordePBAEs are biodegradable, therefore, it is expected that storage of aqueous suspensions of PBAE-TBO would exhibit progressively diminishing improvements on aPDT. This obstacle could easily be overcome as we envisage the storage of the prepared PBAE-TBO dried in airtight containers with PBS added just before use.63, antimicrobial polypeptides66 or a bacterial wall disruption agent (i.e. gentamicin67 or vancomycin70). Another type of approach is the encapsulation of the photosensitiser in liposomes or micelle that drive the light activate compound inside the bacterial cell72. Compared to these approaches, the simple use of PBAE complexation with TBO that we developed removes the need of the conjugation or the encapsulation step thus reducing costs and maximising yield.The duration of the exposure time necessary to reach satisfactory antimicrobial effects is key in controlling the number of patients that can be treated given the available beds/treatment bays capacity; thus, increasing inactivation rates and, consequently, shortening treatment times, reduces the indirect costs of the treatment or the required investments to set-up facilities with a set turn-over of patients. Most of the technologies developed to enhance photosensitiser uptake rely on the conjugation of the light activated compound to a bacterial targeting polymerAntimicrobial PDT has the potential to replace antibiotics in the treatment of skin and tissue infections helping the fight against antibiotic resistance; current applications are still significantly restricted by long exposure time necessary.4, bleach or silver are the safety, low cost and biological tolerance . Additionally, our technology has the advantage that the PBAE-TBO is already a gel and such its topical application is very easy and does not require further formulation into a gel.We developed a new approach to improving aPDT efficacy thought the use of PBAE to improve bacterial uptake of photosensitisers resulting in shorter exposure times that, in turn, men higher treatment bays turnover. Reducing treatment times means that more patients can be treated with the currently available resources maximising their return. Further benefits of this technology over the current treatments of chronic wounds including the use of Iodine (Povidone), KMnOSupplementary Information.


Fluorescence techniques dominate the field of live-cell microscopy, but bleaching and motion blur from too long integration times limit dynamic investigations of small objects. High contrast, label-free life-cell imaging of thousands of acquisitions at 160 nm resolution and 100 Hz is possible by Rotating Coherent Scattering (ROCS) microscopy, where intensity speckle patterns from all azimuthal illumination directions are added up within 10 ms. In combination with fluorescence, we demonstrate the performance of improved Total Internal Reflection (TIR)-ROCS with variable illumination including timescale decomposition and activity mapping at five different examples: millisecond reorganization of macrophage actin cortex structures, fast degranulation and pore opening in mast cells, nanotube dynamics between cardiomyocytes and fibroblasts, thermal noise driven binding behavior of virus-sized particles at cells, and, bacterial lectin dynamics at the cortex of lung cells. Using analysis methods we present here, we decipher how motion blur hides cellular structures and how slow structure motions cover decisive fast motions. In live-cell microscopy, motion blur limits resolution and contrast. Here the authors use 100 Hz super-resolving Rotating Coherent Scattering (ROCS) microscopy on various dynamic biological systems, and time-window analysis to understand biological effects. But the problem is even more severe, since the signals of moving structures can disappear completely in the background noise, if the signal intensity is spread out over too many pixels in a given time window. Hence, a meaningful image requires enough photons detected per pixel and time window, which are usually not available for fast and small structures.Living cells consist of a large variety of proteins and other molecules, undergoing thermal and transported motions as monomers, filaments, membranes, and globular structures. All these cellular structures move the faster, the smaller they are. However, the smaller the cross-section of a structure, the less light will it typically re-emit by fluorescence or by direct (coherent) light scattering after illumination. And this is a significant problem in life cell microscopy: recording the motion of a dynamic structure requires a sufficiently high number of photons on the camera to enable short integration timesrms = 200 nm within only 10 ms (in a medium 5x more viscous than water). Within 100 ms camera integration time, it diffuses already xrms ≈ 600 nm, making a particle practically invisible because of significant motion blur. And in water the situation would be 5x more severe.For example, a 100 nm spherical particle diffuses over an average distance of xAnother problem in life cell microscopy is that often the decisive moments for the biological interactions cannot be predicted. Sometimes thousands of images need to be recorded, if the decisive interactions take place on the millisecond timescale.1 is multiplied with another, stronger electric field E2, such that the amplification is E1·E2. Interferometric enhancement was often difficult to apply in cell biology, because of too many unwanted interference patterns (laser speckles) arising from the strongly structured surfaces of cells.Despite the well-accepted strengths of fluorescence imaging techniques, fluorophore bleaching and the limited output of fluorescence photons restrict many investigations in dynamic living cells. Laser light, which is known for its strong  coherence and interference ability, shows a way how to achieve both high temporal and high spatial image resolution. Interference means that a photon scattered at a structure can be amplified in its number (or intensity), when its electric field E6. Oblique illumination allows to access information from light diffracted under large angles effectively increasing the detection numerical aperture NAdet.Increasing spatial resolution requires more complex illumination patterns and more illumination time. Resolution doubling can be achieved by oblique illumination, leading to either a modulation of the electric field in the coherent case or a modulation of the intensity in the fluorescence case. In the latter case, two oblique laser beams must interfere as in the case of structured illumination microscopy8.Rotating coherent scattering (ROCS) microscopy is an emerging technique, able to overcome the above-mentioned drawbacks: first, by increasing spatial resolution nearly twofold through highly oblique sample illumination, and second, by rotating a collimated laser beam over a 2π azimuthal angle leading to nearly speckle- free images. These static and deterministic speckles are nothing else but deformed images of object structures, which locally shift the phase and result in multiple interferences on the camera. Speckles are induced by the small objects structures and therefore must not be averaged out, but assigned correctly to the object, which is achieved by the ROCS rotation. Hence, by the 2π angular integration, the deformed multiple interferences (speckles) reform into meaningful, clear images of the objectsSimilar to fluorescence techniques, evanescent sample illumination in total internal reflection (TIR) mode removes unwanted background signals and increases image contrast also in ROCS microscopy.9. Conventional resolution at λ/2 is achieved by gradient light interference microscopy (GLIM), which allows 3D imaging of refractive index changes10. Optical diffraction tomography12, which is able to reconstruct phase objects in 3D, uses an oblique rotating beam as well, which is typical for tomography applications, but does not rely on speckle interferences to maximize the contrast of small structures and has not yet achieved spatial resolutions below 200 nm13.Other label-free contrast-enhancing techniques, such as differential interference (DIC) microscopy, phase contrast microscopy or reflective wave interference microscopy, are limited in spatial resolution by the minimal spot distance of half the used light wavelength λ. This limits resolution also in iSCAT microscopy, which can measure the smallest refractive index changes, but is not well suited for cell imaging14, we have improved the ROCS technology by nearly doubling the mean acquisition rate, by varying the polar illumination angle and the laser wavelength, thereby adapting the illumination scheme to the individual cells and adhesion properties, which leads to significantly increased image quality. Furthermore, we are now able to record ROCS signals and fluorescence signals at the same time, thereby combining the specificity of fluorescence imaging with the millisecond dynamics of label-free ROCS imaging. Although structure-specific light emission is currently not possible with ROCS microscopy, we extract relevant biological information even without specificity that cannot be obtained by any other imaging technology.In comparison to our earlier study2–104 images obtained by 100 Hz ROCS (and 1–10 Hz fluorescence) imaging, such as frequency decomposition, where cellular activity can be analyzed and compared on different timescales. This methodological combination provides a clearer picture of how virus-sized particles bind tighter to cellular binding sites (receptors) over time, how filopodia tips undergo a so far undetected search-and-tumble mechanism, how actin backbones undergo ultrafast retrograde flow upon addition of Latrunculin, how extracellular tunneling nanotubes of cardiac fibroblasts (connecting to cardiomyocytes) vibrate at 50–100 Hz and become stiffer over time, how mast cells open degranulation pores in times <10 ms  and how bacterial lectin A patches move with enormous velocities within and underneath the cell membrane, requiring unexpected actin densities and myosin transport mechanisms.In the current study, we investigate the relevance of spatiotemporal resolution, where we demonstrate that limited temporal resolution strongly affects the spatial resolution due to motion blur. We introduce several methods to analyse stacks of 10−1. For more technical details see Supplementary Note We have developed an improved ROCS microscope, which is able to use three different laser beams  at different polar angles for variable ROCS imaging depending on the cells properties and their adhesion to the coverslips. ROCS can be acquired in parallel to fluorescence imaging with either the same laser wavelength or a different laser line. In addition, incoherent images can be taken with a blue LED (at 435 nm) for control and comparisons. As sketched in Fig. 0 = 0°, 90°, 180°, 270°) is sketched, together with the coherent wave trains propagating towards the camera. The exemplary intensity images I(ϕ) formed by each wave train (see blue distorted wavefronts) are visible as speckle patterns below, which do not resemble the image of a cell. However, after adding up e.g., 72 images from 72 equally distributed illumination directions, a high contrast, superiour-resolved image of a J774 mouse macrophage becomes visible  at all interfaces of lenses and filters, since this light can become easily brighter than the light backscattered from small sample structures.Figure ill and the detection NAdet. For cases NAdet < 0.85·NAill, ROCS can be used in darkfield (DF) mode, leading to high image contrast, when the illumination light is blocked by a diaphragm. The case NAdet > 0.85·NAill corresponds to brightfield (BF) mode. If in addition NAill > nmed, (refractive index nmed of cell medium), the TIR mode is possible both with darkfield and brightfield . The effective maximum optical transfer is 6.2 µm−1, corresponding to ΔxROCS = 160 nm resolution in ROCS microscopy, but is only 4.1 µm−1 in fluorescence microscopy corresponding to ΔxFLUO = 240 nm (0.61·λ/NAdet = 220 nm < ΔxFLUO). It is well known that the (incoherent) resolution estimate 0.61·λ/NA for a high-aperture lens is a strong idealization. Although the maximal bandwidth transferred remains the same, the object spectrum is shifted in ROCS, such that significantly more information is transferred close to the cutoff frequency. In Supplementary Fig. Whereas spatial resolution is decreased by 20% in TIR-F imaging by the inherent Stokes shift , TIR-ROCS imaging is decreased in resolution by around 20% because of the darkfield mode, leading to an effective NAs in ROCS image above) describes the speckle dependence on the azimuthal angle ϕ or as I on the rotation time t. The length of the specular distributions along the horizontal axis is an estimation about the angular memory effect of ROCS imaging. Six exemplary vertical linescans display six intensity speckle distributions, which form the final (black) image profile after 72 line profiles have been added up. At this point it should be emphasized again, that the speckles are not averaged out, but represent a deformed interference image of an object structure. Only by superposition of all deformed interference images, the final superiour-resolution image of the structure is obtained. It was shown recently8 that a reduction of the degree of spatial coherence led to a decrease in image resolution because of less interferences, i.e. less pronounced speckles. The role of spatial and temporal coherence is further illustrated in Supplementary Note How fast do the speckles change with the azimuthal illumination directions? The intensity distribution I in kymograph Fig. 15, which is obtained by dividing spatial resolution through temporal resolution. In the case of ROCS we find a STBW = 100 Hz/(0.16 μm) = 625 Hz/μm, being e.g. 4x higher than the STBW = 20 Hz/(0.13 μm) of fluorescence SIM (at NA = 1.46).The degree of spatial and temporal resolution can be described by the spatiotemporal bandwidth STBWFigure 16 (shown in gray colors). Although these cells were not activated by any stimuli, we recorded high intracellular dynamics, which are displayed in the kymograph , Δω1 = 0–10 Hz for slow structure motions, Δω2 = 10–33 Hz for fast and Δω3 = 33–100 Hz for very fast structure motions. So-called activity maps σ  of these difference images δIjn for each pixel , one obtains the space resolved average intensity changes within a certain time window . Assuming lateral motions and considering a pixel width of dx =37 nm, one obtains standard deviations of dx per time dt, i.e. of structure velocities (either relative in % to the mean velocity or absolute in nm/s). In summary, this method describes a form of cell activity within a certain time window Δt at a specific position and on a specific timescale τ = 1/Δω.Proteins, vesicles or filaments are driven by thermal and molecular forces, hence moving differently fast on different timescales. For instance, an unknown structure moving once back and forth within 0.1 sec generates a washed-out, blurred image over 0.1 s integration time, implying a broad and static structure. At 100 Hz sampling, however, one may see up to 10 different positions indicating that the structure is dynamic and is of smaller shape. Therefore, a frequency decomposition is useful when analyzing image sequencessee Fig.  are geneτ1 = 100–1000 ms, τ2 = 30–100 ms, and τ3 = 10–30 ms, we could see activities >300% higher than the mean cell activity, indicating lateral motions of cortex structures, which were likely actin filaments. The actin network is momentarily displaced by the tweezers, but relaxes first quickly and then more slowly like most viscoelastic materials, leading to activity signals σ(Δω) on different short and long timescales Δω−1. In ROCS, flat membranes with a constant refractive index hardly scatter light14, whereas vesicles give strong signals. As soon as the laser tweezers and thereby lateral drag forces are switched off, the activity disappears.Figure Using evanescent (TIR) illumination, we imaged the cortex of two adjacent MK6 macrophages (cell 1 and cell 2) as shown in Fig. Macrophage filopodia have diameters of typically 100–200 nm enabling even faster dynamics than the protrusions in Fig. 17. We added 0.2 µl LatA (dissolved in DMSO) to the cell medium, resulting in a low Lat A concentration of 0.4 nM  in the filopodia tips, leading to retractions of actin backbones through retrograde flow, shown by adherent filopodia in the ROIs , likely from backbone retractions. A linescan (dark blue) through the whole section of the cell, which marks the average ROCS intensity decay and recovery within a 60 s time window relative to the signal decay and recovery of the single filopodium marked in red. The magnified time window around the strongest actin retraction at t0 + 1 s, reveals the 40 ms time delay between filopodia and cell cortex. As a control, we added the same amount of DMSO solution without LatA, which led to no visible effects  are key cells of our innate immune response and best known for their roles in allergy, asthma, and anaphylaxis. Activation of mast cells through IgE receptors triggers degranulation with the release of inflammatory mediators that are stored in hundreds of cytoplasmic secretory granules. MC degranulation occurs within few minutes after activation and involves the fusion of secretory granules with the plasma membrane by different modes of exocytosisTo test the power of ROCS-based imaging for visualizing the fast biological events during mast cell degranulation, we used primary mast cells that had been isolated from the peritoneum of mice and cultured for several weeks.Figure Figure v = 3.6 µm/s (step 3) and moves hardly for the remaining after release . Further experiments will be necessary in the future to investigate the spatiotemporal behavior of different granules before and during release with different biochemical conditions established.Importantly, the combination of TIR-F and TIR-ROCS also allowed the observation of the fast lateral release of granules from the cell surface to the surrounding environment Fig. . The gra22. One of the less known modes of cardiomyocyte-fibroblast communication are tunneling nanotubes (TNTs), previously shown to form between the two cell types and proposed to be important for e.g., cardioprotection during ischaemia24.Fast cell-cell communication is essential in the cardiac muscle for efficient integration of electrical, mechanical, and humoral signals. Hetero-cellular coupling between cardiac muscle cells (cardiomyocytes) and interstitial fibroblasts has been shown to be essential both in health and during pathological remodelingIn contrast to fibroblasts (FB), cardiomyocytes (CM) do not attach to the coverslip, making it difficult to observe them in TIR mode. By decreasing the incident polar angle of the rotating laser beam (the ring diameter in the objective lens BFP), transmitted light can also illuminate and image structures away from the coverslip (non-TIR mode). This principle is sketched in Fig. Figure Figure Figure 25. Diseases such as asthma, lung cancer, cardiovascular disease, and stroke26, are a likely consequence of worldwide combustion processes and air pollution. Both coronaviruses and ultrafine particulates  can diffuse down the lung to the alveols, enter and damage epithelial cells (and macrophages), hence preventing sufficient air exchange. However, because of a lack of sufficiently fast and robust microscopy techniques the dynamics of such small particles and their binding behavior to the cells are widely unexplored.Small, submicrometer sized particles, such as airborne particulates  or viruses dispersed in micro-droplets cause lung infections leading to severe respiratory diseases. The disease COVID-19 spreads because of amplified exchange of 100–120 nm small replicating coronaviruses entering lung cells29. As shown before, a too low temporal resolution leads to motion blur and complete invisibility of the highly dynamic particles with 600 ms mean free diffusion length within 100 ms and in a solution 5x more viscous than water. Figure Using 100 Hz TIR-ROCS, we have observed the dynamics of 100 nm glass beads and their binding behavior to J774 macrophages (see Supplementary Movies sca = 0.6% of the incident photons (Qsca = Csca/a²π based on Mie theory). For a NA = 1.2 lens 30% of the scattered light is captured, and with a 10% overall transmission to the camera, only 0.3·0.1·0.6% = 1/5555 of scattered photons reach the detector carrying information about particle motions. Due to coherent amplification, this was sufficient to precisely track >100 particles at 100 Hz inside region ROI 1 (e.g. by using TrackMate30) as displayed in Fig. 1 = 1 min, t2 = 3 min and t3 = 5 min indicating a tighter binding to the cell. 31.Figure To prove that we can successfully image such small particles in front of cells, we acquired images of the unlabeled cells with fluorescently labeled 120 nm PS particles with TIR-ROCS (at 100 Hz) and TIR-F (at 2 Hz) as demonstrated in Fig. TIR-ROCS imaging allows to investigate the thermally driven binding behavior of small particles to cells with standard tracking procedures and with high statistics, allowing to perform various binding tests in the future.Pseudomonas aeruginosa lectin LecA and its host cell glycosphingolipid receptor Gb3 (globotriaosylceramide) trigger the cellular uptake of the bacterium via the so-called lipid zipper mechanism33. However, the dynamics of LecA at the plasma membrane leading to membrane invaginations and the mechanisms of clathrin-independent endocytosis remain so far largely unknown.Bacteria produce lectin proteins in order to mediate adhesion and entry into host cells by binding to glycosylated plasma membrane receptors. The interactions between the We investigated the interactions of LecA with the periphery of H1299 lung epithelial cells with two cameras as shown in overlay image of Fig. 34, LecA binding induces a plasma membrane domain enriched in saturated Gb3 species and the GPI-anchored protein CD59 at the extracellular membrane leaflet, which probably is able to induce membrane curvature required for endocytic uptake. We hypothesize that trans-bilayer signal propagation could be achieved by long fatty acyl chains of Gb3 and CD59 that interdigitate into the cytosolic membrane leaflet, leading to interactions and clustering of long fatty acyl chains of the phospholipid PIP3, to which myosins as actin motor proteins can bind.Based on a current working model1 = 4.0 µm/s and v2 = 4.3 µm/s.We investigated the correlations between LecA uptake with TIR-F Fig.  and the 3 only by using a priori information . ROCS gives researchers the possibility to observe the decisive seconds or minutes of interaction, either starting spontaneously or induced, at an unknown point in time. We showed that the combination of ROCS in TIR mode and darkfield mode achieves extraordinary image contrast of smallest dynamic structures within only 10 ms - an integration time which has not been minimized yet, e.g. with better cameras.We have shown that ROCS microscopy provides thousands of images at a very high spatiotemporal bandwidth (STBW) of 100 Hz/0.16 µm = 625 Hz/µm without labeling and without loss in image quality. Comparable STBWs can be achieved with fluorescence microscopy (TIRF-SIM)em is distributed over a too large motion area A, such that the intensity Iem = Pem/A approaches the background level. Unexpectedly, we found that 100 Hz acquisition is still not enough for several experiments, . Based on the diffusion estimates presented in the introduction, a 2–5 fold faster image acquisition rate would be more beneficial, requiring faster scan mirrors and cameras. As shown in the Supplementary Note Motion blur limits resolution. We have demonstrated at several examples that limited temporal resolution can be more significant than limited spatial resolution. Motion blur can turn objects completely invisible, when their photon emission power P35 and by increasing the effective numerical aperture of currently NAdet = 1.2 by blocking the reflected rotating laser focus only locally.Coherence controls speckles and resolution. Object illumination and detection of laser light achieves about tenfold higher photon densities because of interference. Based on highly oblique illumination, the object spectrum is shifted in ROCS, such that significantly more information is transferred close to the cutoff frequency see Fig. . Further37, ROCS needs no postprocessing and illuminates the sample only at 1–2 high polar angles (laser rotation at high NA) to achieve fast image acquisition. And, by using fully coherent light, spatial resolution and contrast can be maximized8. By imaging cells in TIR mode, only the outer cortex is illuminated in a depth of 0.2–0.3 µm. As demonstrated in the experiment of Fig. What do we really see with ROCS? At the camera, we see a superposition, i.e. interferences of elementary waves, which vary in phase and amplitude according to the complex refractive index and polarizability of the cellular structures. This gives room for future improvements of interference patterns, which depend on the polar angle of incidence and the laser wavelength. In contrast to the related technique of Ptychography15, which represents the standard deviation of several difference images. These maps are advantageous, especially when decomposed into different frequency ranges . This becomes apparent, when a structure moves on both a low-frequency and a high-frequency: the slow motion can cover the fast motion, which can be uncovered by this decomposition , we investigated the behavior of elongating filopodia of J774 macrophages at 100 Hz over 5000 images. With exceptional spatiotemporal resolution we could show, how single, mainly adherent filopodia grow in length while the tip region (of ≈1 µm length) fluctuates strongly in lateral directions. These motions can be thermally driven, but also by a yet unknown mechanism. Nevertheless, the fast direction changes do not inhibit actin polymerization at the tip, but apparently impede adhesion to the surface. Activity plots reveal that fluctuations are more pronounced on a 10–30 ms scale than on 0.1–1 s timescale.BB = 5 µm/s. Since off-binding of (ADP) actin is on the order of 10 Hz at the barbed end38, this results in a maximal depolymerization speed of only 5.4 nm/2·10 Hz = 27 nm/sec ≪ vBB. Retraction velocities of actin backbones in J774 macrophages, based on myosin II motor pulling, have been reported to be 0.1-0.3 µm/s39 with LatA addition. The whole cortex retracted a few 100 ms later and over several µm within only 1–2 s, followed by a minimum of activity after a few seconds. Then, the cell cortex activity was recovered within 60 s through fast re-polymerization of unblocked G-Actin going along with an expansion of the cell. The observed effects of actin inhibition by LatA were significantly faster than that those reported on fluorescence-labeled actin17 and open new questions about mechanical cytoskeleton retraction mechanisms and the efficiency of myosin—actin interaction .Cell response to Latrunculin A within a few seconds. Our goal was to record fast cellular responses at unknown time points upon biochemical stimuli. We demonstrated that polymerization of actin fibers in the cortex and of filopodia actin backbones was hindered, by blocking ATP-G-actin within a few seconds after adding 0.4 nM Latrunculin A. This was visualized by TIR-ROCS movies, difference images and high-frequency activity maps (Δω = 50–100 Hz). ROCS revealed visible retractions not of whole filopodia, but only of the actin backbone at high velocities of up to vn = 2–5). While granule release was frustrated at the cell bottom, we could observe the lateral release of individual granules in multiple steps, which could be analyzed quantitatively through ROCS kymographs. Further investigations with even higher acquisition rates may provide further insights into the driving mechanisms of ultrafast pore opening and granule release.Mast cell degranulation pores form within a few milliseconds. Given the lack of adequate fast imaging techniques, the spatiotemporal sequence of fast occurring events that underlie MC degranulation has not yet been resolved in detail. Using 100 Hz TIR-ROCS, we could see the formation of degranulation pores in stimulated mast cells 1 min after IgE-mediated activation. By difference images, we could see pore formation within <10 ms in exceptional temporal resolution, revealing clear shapes and boundaries, indicating the number n of granules to be released  protruding from fibroblasts, which were difficult to visualize with fluorescence. Using 100 Hz TIR-ROCS, we measured lateral TNT fluctuations likely driven by thermal noise. Ten minutes later and at only 9% reduced cell cortex activity, the TNT motions were significantly reduced in amplitude and time, likely because of the stiffening of the TNTs by yet unknown mechanisms. Changes in TNT stiffness might be an important determinant of cardiomyocytes-fibroblast cross-talk in the heart during homeo-stasis and after injuries.Thermal motions of virus-sized particles reveal binding behavior. We used 100 nm small glass particles, mimicking ultrafine particulates or viruses, to record their spatiotemporal binding behavior to macrophages. Due to the high ROCS image contrast, automated tracking of hundreds of particles allowed to analyze the fluctuation behavior at different stages prior and during binding to the filopodia-rich cell periphery. We found a fivefold increase in binding stiffness within only 10 min, indicating structural changes at the cell periphery. In the future, these processes must be investigated in more detail by adding receptor blockers or different molecular particle coatings. The extremely fast hopping of tiny particles between potential binding sites indicates the complexity of particle cell interactions.LecA is driven by unknown mechanisms. We imaged Cy5 labeled LecA diffusing at the surface of HT1299 cells and below in a combination of TIR-F and TIR-ROCs. Interestingly, no correlations between the Cy5 fluorescing LecA dynamics and highly dynamic refractive index changes within the cell cortex could be found. While LecA positions and uptake positions were clearly located by difference images, no locally enhanced actin cortex activity could be revealed by ROCS. However, it turned out that a combination of hindered and driven diffusion of membrane LecA patches with velocities up to 4 µm/s could be tracked precisely, as well as dynamic, defocused particles below the membrane (likely LecA-containing endosomes), which could be explained by a reduced density of the actin cortex and active transport by myosins binding to PIP3. For sure these observations open interesting questions about the driving mechanisms of both LecA clusters and endosomes, which should be investigated in the future by multiple-plane ROCS microscopy.40, by achieving optical sectioning through e.g., speckle correlations and addressing 3D imaging through lightsheet illumination or multiple-plane detection, we envision a bright and promising future for ROCS microscopy not only for cell biology.We could show by several examples of cellular systems that 100 Hz ROCS microscopy reveals various unexpected findings in the high-speed world of molecular cell structures, which in some cases would require even faster measurements. In combination with the specificity provided by fluorescence techniques, ROCS represents a profitable imaging approach, which can be well added to existing microscopes. By extracting molecular absorption and specificity through phase delaysWe confirm that our research complies with all relevant ethical regulations; approved by the local Institutional Animal Care and Use Committees in Germany .We declare that no wild animal were used in the study and that no field-collected samples were used in the study.www.atcc.org/cell-products) transfected with pLife Act-TagGFP2  were cultured in DMEM medium and in a 37 °C and 5% CO2 atmosphere until the desired cell density for the experiments is reached (approx. 30% confluency). This cell line was tested negative for mycoplasma. For bead experiments, beads were pipetted into the cell culture medium and the imaging process was started with a controlled time delay as stated in the manuscript and figure legends. For the experiments with Latrunculin A, LatA was dissolved in DMSO at a concentration of 200 nM. 2 µl of LatA-DMSO solution was added to the cell medium (1 ml), resulting in a low Lat A concentration of 0.4 nM. Then recordings were started immediately.J774 cells . Mouse breeding and husbandry were performed at the Max Planck Institute of Immunobiology and Epigenetics, Freiburg, in accordance with the guidelines provided by the Federation of European Laboratory Animal Science Association and as approved by German authorities . Mice were kept at a light/dark cycle of 14/10 h, 22 ± 2 °C temperature and 60 ± 5% relative humidity. C57BL/6 mice were only used for organ removal after euthanasia by carbon dioxide exposure and thus not subject to experimental procedures and ethical approval according to §4 (3) Tierschutzgesetz. Mouse neutrophils were isolated from bone marrow using autoMACS Pro Selector cell separator and MACS neutrophil negative selection kit according to the manufacturer’s protocol (MIltenyi Biotec). Mouse peritoneal mast cells were isolated by peritoneal lavage and cultured before use in OptiMEM Medium supplemented with 10% FSC, GlutaMAX, penicillin, and streptomycin4 cells per dish in 2 ml culture medium. Cells were coated with an IgE antibody directed against the 2,4-dinitrophenyl hapten  overnight, before mast cell degranulation was induced with a DNP-human serum albumin conjugate . Cells were kept in Tyrodes/FCS buffer that was supplemented with FITC-conjugated avidin , which recognizes the proteoglycan matrix released by exocytosed granules. Mast cell degranulation at the given concentration was validated by FACS measurements for avidin and CD63. Further references on manufacturer’s website: https://www.sigmaaldrich.com/DE/de/product/sigma/d8406Mast cells were seeded with approx. 35 × 10Two types of cardiac cells  were used. All investigations reported here conformed to German (TierSchG and TierSchVersV) animal welfare laws, compatible with the guidelines stated in Directive 2010/63/EU of the European Parliament on the protection of animals used for scientific purposes, and they were approved by the local Institutional Animal Care and Use Committees in Germany . Animal housing and handling were conducted in accordance with good animal practice, as defined by the Federation of European Laboratory Animal Science Association, FELASA. Rabbit tissue was obtained from New Zealand white rabbits . Human tissue samples were obtained from the right atrial appendage of the patient undergoing open-heart surgery at the University Heart Center Freiburg - Bad Krozingen. Informed consent was acquired prior to surgery. Tissue samples were processed by the Cardiovascular Biobank of the University Heart Center Freiburg - Bad Krozingen . Cells were maintained in standard culture conditions prior and throughout the experiments . Cultures containing CM were maintained for maximum of 3 days. Latrunculin B (LatB) treatment was applied 24 h prior to imaging. Control was exposed to vehicle (ethanol) alone. Actin was visualized using CellLight™ Actin-RFP, BacMam 2.0 (Thermo Fisher).www.atcc.org/cell-products) were cultured in RPMI medium and in a 37 °C and 5% CO2 atmosphere until the desired cell density for the experiments was reached (approx. 30% confluency). Cells were gently washed once in PBS prior to lectin addition. Cy5-labelled LecA was added with a concentration of 5 µg/ml. Excitation of LecA was done with a 561 nm laser (Cobolt) operating at 4 mW at the sample. The exposure time was 50 ms unless stated otherwise for the best compromise between photon signal and motion blur.Mycoplasma-free H1299 cells (ATCC CRL-5803, ROCS imaging was performed at 100 Hz under TIR condition with a laser power of < 1 mW at the sample, unless stated otherwise. Every optical surface/interface, especially between and sample and camera, must be anti-reflection-coated for the specific laser wavelength and must be completely free of dust/dirt, which introduces unnecessary image artifacts. Further details can be found in the Supplementary Note Further information on research design is available in the Supplementary InformationReporting SummaryPeer Review FileDescription of Additional Supplementary FilesSupplementary Movie 1Supplementary Movie 2Supplementary Movie 3Supplementary Movie 4Supplementary Movie 5Supplementary Movie 6Supplementary Movie 7Supplementary Movie 8Supplementary Movie 9Supplementary Movie 10Supplementary Movie 11Supplementary Movie 12Supplementary Movie 13Supplementary Movie 14Supplementary Movie 15Supplementary Movie 16Supplementary Movie 17Supplementary Movie 18Supplementary Movie 19Supplementary Movie 20Supplementary Movie 21Supplementary Movie 22Supplementary Movie 23Supplementary Movie 24Supplementary Movie 25Supplementary Movie 26Supplementary Movie 27Supplementary Movie 28Supplementary Movie 29Supplementary Movie 30Supplementary Movie 31


Sodium-glucose cotransporter 2 (SGLT2) inhibitors include a relatively new class of glucose-lowering drugs that reduce plasma glucose concentrations by inhibiting proximal tubular reabsorption of glucose in the kidney, while increasing its excretion in urine. Recent large randomised controlled trials have demonstrated that many of these agents reduce the occurrence of major adverse cardiovascular events, hospitalisation for heart failure, cardiovascular death and/or chronic kidney disease progression in patients with and without type 2 diabetes mellitus (DM2). Given their unique insulin-independent mode of action and favourable efficacy and adverse-event profile, SGLT2 inhibitors are promising and they offer an interesting therapeutic approach for the cardiologist to incorporate into routine practice. However, despite accumulating data supporting this class of therapy, cardiologists infrequently prescribe SGLT2 inhibitors, potentially due to a lack of familiarity with their use and the reticence to change DM medication. Here, we provide an up-to-date practical guide highlighting important elements of treatment initiation based on real-world evidence and expert opinion. We describe how to change DM medication, including insulin dosing when appropriate, and how to anticipate any adverse events based on real-world experience in patients with DM2 in the Meander Medical Centre in Amersfoort, the Netherlands. This includes a simple algorithm showing how to initiate SGLT2 inhibitor treatment safely, while considering the consequence of the glucosuric effects of these inhibitors for the individual patient.The online version of this article (10.1007/s12471-021-01580-9) contains supplementary material, which is available to authorized users. Sodium-glucose cotransporter 2 (SGLT2) inhibitors include a relatively new class of glucose-lowering drugs.regardless of the presence or absence of diabetes mellitus.Recent large randomised controlled trials have demonstrated that many of these agents reduce the occurrence of major adverse cardiovascular events, hospitalisation for heart failure, cardiovascular death, and/or chronic kidney disease progression, We provide an up-to-date practical guide for the cardiologist, highlighting important elements for treatment initiation based on real-world evidence and expert opinion, including a simple algorithm that shows how to initiate SGLT2 inhibitor treatment safely.Sodium-glucose cotransporter 2 (SGLT2) inhibitors have emerged as an important new oral glucose-lowering class of drugs for the management of hyperglycaemia in patients with type 2 diabetes mellitus (DM2). Importantly, recent large randomised controlled trials have demonstrated that many of these agents reduce the occurrence of major adverse cardiovascular events, hospitalisation for heart failure (HF), cardiovascular death and/or progression of chronic kidney disease (CKD), regardless of the presence or absence of DM2 –9. TheseIn this article, we provide an up-to-date practical guide for the Dutch cardiologist, highlighting important elements for treatment initiation, dosing, anticipated adverse events and barriers, based on expert opinion and real-world experience in patients with DM2 in the Meander Medical Centre in Amersfoort, the Netherlands. It includes a simple algorithm showing how to initiate SGLT2 inhibitor treatment safely, while considering the consequence of the glucosuric effects of these inhibitors for the individual patient.The SGLT2 receptor is a sodium-glucose cotransporter located in the proximal tubule of the nephron that is responsible for approximately 90% of urinary glucose reabsorption. Inhibition of this receptor results in lower blood glucose levels through induction of glucosuria . This ef1c levels, and a number of putative mechanisms have been proposed [Beyond their effect on blood glucose levels, SGLT2 inhibitors also cause diuretic and possibly natriuretic effects, promote weight loss and lower systolic blood pressure  . These b2) [SGLT2 inhibitors have moderate effects (risk reduction ~14%) on atherosclerotic major adverse cardiovascular events that seem confined to patients with established atherosclerotic cardiovascular disease or CKD (eGFR < 60 mL/min per 1.73 m2) .At first, the benefit of reducing the number of HF hospitalisations in SGLT2 inhibitor trials –6 primarMore recently, the benefits of an SGLT2 inhibitor in treating established HF were demonstrated in the DAPA-HF  and the EMPEROR-Reduced  trial , 8. BothThe pooled treatment effects showed consistent benefits for subgroups based on age, sex, presence of DM, baseline eGFR or background treatment with an angiotensin receptor-neprilysin inhibitor, but suggested treatment-by-subgroup interactions for subgroups based on the New York Heart Association (NYHA) functional class and possibly race. The pooled hazard ratio for patients in NYHA class II  0.59–0.76) differed from that for patients in class III–IV . Beneficial effects on symptoms, functional status and quality of life in patients with HFrEF were also reported. Additional trials with various agents in patients with HF with preserved ejection fraction are ongoing.In addition, consistent reductions in the secondary outcome of risk of kidney disease progression were seen with all agents in the cardiovascular outcome trials, although the number of ‘hard’ renal events  was small. The CREDENCE  trial  and DAPAMechanisms to explain these observations may include activation of tubuloglomerular feedback, reduction in glomerular hypertension, containment of hyperfiltration injury, reduction of kidney hypoxia and possible effects on sodium-hydrogen exchange , 13.An increased risk of genital mycotic infections  has been seen with all SGLT2 inhibitors . These iAn increased risk of amputations and fractures was observed in one trial during treatment with canagliflozin; however, this phenomenon was not seen in other trials or in the other canagliflozin trial (CREDENCE). The clinical importance of any possible increase in amputation risk is also unknown, but caution is suggested in those with a history of peripheral artery disease and/or lower extremity diabetic ulcers. A recent meta-analysis indicated that SGLT2 inhibitors do not increase the risk of bone fracture in patients with DM2 compared with placebo . There wA recent meta-analysis showed no increased risk of DKA in patients with DM2 taking an SGLT2 inhibitor compared with placebo . In gene2.The 2019 Clinical Practice Guidelines by the European Society of Cardiology (ESC) on DM recommend either SGLT2 inhibitors or glucagon-like peptide‑1 receptor agonists (GLP‑1 RAs) in patients with DM2 and established atherosclerotic cardiovascular disease or in case of very high/high cardiovascular risk (e.g. ≥ 3 major risk factors or DM duration ≥ 10 years), to reduce cardiovascular events . In addi2 or urine albumin:creatinine ratio (UACR) > 30 mg/g, particularly when UACR ≥ 300 mg/g).The 2019 version of ‘Management of Hyperglycemia in Type 2 Diabetes’, a consensus report by the American Diabetes Association and the European Association for the Study of Diabetes, offers more direction on when to prescribe SGLT2 inhibitors or GLP‑1 RAs in patients with DM2 at high or very high cardiovascular risk [Most guidelines do not address patients without DM2. The Canadian Cardiovascular Society/Canadian Heart Failure Society’s Heart Failure Guidelines are among the first to recommend that SGLT2 inhibitors be prescribed in patients with HFrEF (LVEF < 40%) and without concomitant DM, to improve symptoms and quality of life and to reduce the risk of hospitalisation and cardiovascular mortality . The in 2 and UACR > 3 mg/mmol, and (2) eGFR ≥ 60 mL/min per 1.73 m2 and UACR > 30 mg/mmol [Dutch guidelines on DM, CKD and HF are currently being updated. The Dutch Diabetic Nephropathy guideline recommends SGLT2 inhibitor therapy in two patient groups: (1) eGFR 30–60 mL/min per 1.73 m mg/mmol .Established atherosclerotic cardiovascular disease: prior myocardial infarction, ischaemic stroke, unstable angina with electrocardiographic changes, myocardial ischaemia on imaging or stress test, or revascularisation of coronary, carotid, or peripheral arteries.2 and UACR > 3 mg/mmol or eGFR ≥ 60 mL/min per 1.73 m2 and UACR > 30 mg/mmol).Chronic kidney diseaseDM2 and a very high cardiovascular risk:HFrEF (LVEF < 40%)SGLT2 inhibitor therapy can be considered for patients with the following characteristics:Although the FDA-approved cardiovascular indications are different, SGLT2 inhibitors appear to have broadly similar cardiovascular and renal benefits . A summary of the doses, indications, contraindications and adverse effects of SGLT2 inhibitors is shown in Table S2 .The patient’s diabetes care provider  should always be notified with a letter stating the clinical importance of SGLT2 inhibitors, any changes in diabetes medication, the possibility of adverse events (with specific attention to DKA) and the method of follow-up.SGLT2 inhibitors should not be prescribed in patients with DM1. If the type of DM has not be established, we advise to consult an internist, who may suggest diabetes autoantibody tests.Prescribers should be aware of precipitating factors of ketoacidosis . They should educate patients about the signs or symptoms of ketoacidosis  and instruct patients to discontinue SGLT2 inhibitors and seek immediate medical attention in case of such complaints. If there is (a low) clinical suspicion of DKA, we recommend consulting an internist for further advice.1c ≥ 64 mmol/mol or the patient is treated with comprehensive lifestyle management only or in combination with glucose-lowering therapy with a low risk of hypoglycaemia , SGLT2 inhibitor treatment can be initiated safely without modifications.If HbA1c < 64 mmol/mol and the patient is treated with glucose-lowering therapy with an increased risk of hypoglycaemia , SGLT2 inhibitor can only be initiated after adjustment of these glucose-lowering agents:If patient is on a sulfonylurea drug but not on insulin, discontinue this therapy if patient is on:a) gliclazide ≤ 80 mg once dailyb) glimepiride ≤ 2 mg once dailyc) tolbutamide ≤ 500 mg twice dailyOtherwise, reduce sulfonylurea dose by 50%.If patient is on insulin but not on a sulfonylurea drug, reduce every insulin dose by 20%. Discontinue insulin when insulin dose ≤ 12 IU.If patient is on a combination of sulfonylurea and insulin, and:1c ≤ 53 mmol/mol: reduce or discontinue sulfonylurea drug as stated above and discontinue insulin when insulin dose ≤ 12 IU. If insulin dose > 12 IU, reduce insulin dose by 20%.a) HbA1c 54–64 mmol/mol: reduce or discontinue sulfonylurea drug as stated above or adjust insulin as stated above.b) HbAIf there is uncertainty about changing the insulin dose, we recommend consulting an internist for further advice.If HbASGLT2 inhibitors increase the risk of genital mycotic infections, polyuria, and potential volume depletion in the context of hyperglycaemia.Patients should be educated about the risk of genital mycotic infections and the importance of genital hygiene . In most cases, genital infections will resolve after standard antifungal therapy without discontinuation of the diabetes medication regimen. If necessary, the therapy can be temporarily interrupted.It is prudent to educate patients about the signs and symptoms of dehydration , which may be more of a concern outside the clinical trial setting, especially in frail patients. Clinical judgement should be used when initiating SGLT2 inhibitor treatment in patients who will be undergoing renin-aldosterone-angiotensin system inhibition therapy (or in whom this dose is up-titrated) if the patient’s renal function is impaired. SGLT2 inhibitors should be discontinued in case of acute kidney injury as with other medications such as sulfonylureas, angiotensin converting enzyme inhibitors, diuretics, metformin, angiotensin receptor antagonists and non-steroidal anti-inflammatory drugs.Initiation of SGLT2 inhibitor treatment may lead to a transient acute decline of eGFR at week 4, followed by a period of stable kidney function during long-term follow-up, as was shown in the EMPA-REG OUTCOME trial; eGFR gradually declined with placebo .Follow-up by the diabetes care provider is essential after two weeks if the patient is on insulin and after six weeks if the patient is not taking insulin.The clinical importance of any possible increase in amputation risk remains unclear. It is recommended to examine the feet of all patients on foot wounds and diabetic ulcers with even more awareness for this issue in patients with a history of peripheral artery disease and lower extremity diabetic ulcers. If there is clinical uncertainty about the extent of peripheral artery disease, consult a vascular surgeon. Do not start or discontinue SGLT2 inhibitors if foot wounds and/or ulcers are present.Mostly, SGLT2 inhibitor treatment can be initiated safely by a cardiologist  . In one of these patients, the internist further reduced the long-acting insulin dose based on the HbAIn total, 20 adverse events (24%) were reported, including 7 urogenital infections (Tab. A 63-year-old, obese male with a medical history of atrial fibrillation and coronary artery bypass grafting died in his sleep for unknown reasons and did not have any prodromal symptoms 6 months after SGLT2 inhibitor treatment initiation. Sudden death is not a known adverse effect of SGLT2 inhibitors –9.Six patients were lost to follow-up early on, because they wished to return to their general practitioner for diabetic care.Statistical analysis of our observational non-randomised data was not possible and conclusions based on statistically significant differences could therefore not be drawn. However, our results do show preliminary safety in this population, and our study can serve as a valuable experience for cardiologists wanting to prescribe SGLT2 inhibitors in patients with DM2.This article provided a practical guide for cardiologists on initiating and monitoring of SGLT2 inhibitor treatment. Although we intended to facilitate clinical decision-making, the information provided in this article should complement, rather than supersede, good clinical judgement. Study data from our hospital suggest that using the algorithm presented was safe and gave cardiologists enough confidence and the assurance they were not going to do harm. It is important to strive for good multidisciplinary cooperation, not only between the departments of Cardiology and Internal medicine, but also with general practitioners and pharmacists.We anticipate that our algorithm changes as new evidence emerges, but the goal is still to safely improve cardiovascular outcomes in patients with DM2 at very high risk of atherosclerotic disease.Table S1. SGLT2 inhibitors: Summary of the published SGLT2 inhibitor cardiovascular and renal outcomes trialsTable S2. Summary of currently available SGLT2 inhibitors in the Netherlands on indications, contra-indications, dosing, and most important side-effects


CDKN1A), damage response (γH2AX) and cell death pathways such as apoptosis  and ferroptosis (xCT/GPX4) was demonstrated. This study also shows the epigenetic regulation following a combined regime that alters the expression of chromatin modifiers such as DNMTs  and glycosylases (MBD4 and TDG). Furthermore, a study of the underlying cellular status six hours after the priming dose alone showed evidence of retained modifications on the molecular and epigenetic pathways suggesting that the priming dose infers a “radiation awareness phenotype” to the thymocytes, a sensitization key to the differential response seen after the second hit with the challenging dose. These data suggest that combined-dose radiation regimes could be more efficient at making cells respond to radiation and it would be interesting to further investigate how can these schemes be of use to potential new radiation therapies.In the quest for more effective radiation treatment options that can improve both cell killing and healthy tissue recovery, combined radiation therapies are lately in the spotlight. The molecular response to a combined radiation regime where exposure to an initial low dose (priming dose) of ionizing radiation is administered prior to a subsequent higher radiation dose  after a given latency period have not been thoroughly explored. In this study we report on the differential response to either a combined radiation regime or a single challenging dose both in mouse in vivo and in human ex vivo thymocytes. A differential cell cycle response including an increase in the subG1 fraction on cells exposed to the combined regime was found. Together with this, a differential protein expression profiling in several pathways including cell cycle control (ATM, TP53, p21 If fact, during the last years, many new trials have been set to find practice-changing radiation therapies5. Therefore, there is a necessity to improve the effectiveness of radiation treatments in order to make them more effective at killing tumour cells yet less harmful to the surrounding healthy tissue. A small conditioning (or priming) dose of ionizing radiation (less than 100 mGy) reduces the genotoxic effects induced by a higher dose  administered after a given latency interval6, demonstrating the potential of this combined treatment at preventing healthy tissue damage. Together with this, previous studies showed that T-cell lymphoma induction in mice was reduced by changing the single dose regime (1.75 Gy) to a combined regime (0.075 + 1.75 Gy)7 demonstrating that combined radiation schemes protect normal cells from neoplastic transformation. Genome-scale assessment of the combined response in human lymphoblastic cells demonstrated the upregulation of genes involved in DNA repair and stress response while down-regulated genes were associated with cell cycle control and apoptosis in adapting cell lines8. Using in vivo mouse models, us and others found that a combined regime of radiation, where the priming low dose was administered as an acute dose9 or chronic exposure10, could exert a differential effect on apoptosis in cells of the lymphoid line by regulating expression of genes involved in cell death control. This suggests that more information is needed on which cell death mechanisms are induced after a combined radiation treatment and how can they be key to the betterment of radiation therapies. Understanding the activation of the damage and cell cycle control pathways to either a single dose or a combined regime of radiation, would also be pivotal at getting an overall picture of the response to a combined scheme and whether or not this regime could be more useful at cell killing and maintaining healthy tissue homeostasis than fractionating the total treatment dose into challenging doses alone.More than 50% of cancer patients require radiotherapy as part of their cancer treatments and it is frequently used to treat the most common types, such as breast, cervical, colorectal, and lung cancer as stated by the World Health Organization (WHO) and the International Atomic Energy Agency (IAEA). It is sometimes combined with chemotherapy or immunotherapy to boost its results. Advances in radiation therapy in the past decade have led to significant improvements in tailoring the radiation dose distribution, however, radiation exposure still causes cytotoxic effects to healthy tissue11, therefore it seemed the way forward to study the outcome of a combined regime regarding its potential for new therapy regimes.Previous studies on low doses have demonstrated its effectiveness at enhancing cancer therapeutics12. These epigenetic modifications result in enduring cellular transformations without altering the underlying DNA nucleotide sequence. Mutations of epigenetic regulatory genes are common in thymic carcinomas13 and it is known that high doses of ionizing radiation result in epigenetic modifications in adult mice14. In fact, targeted disruption of Dnmt1 and Dnmt3a in cultured cells eliminates the transmission of genomic instability15. A high frequency of induction and persistence of IR-induced genomic instability, as well as a non-Mendelian mode of inheritance of transgenerational effects also suggests an epigenetic based mechanism key at transferring molecular information after exposure16. Understanding these epigenetic mechanisms within the combined regime will help understanding the overall response of thymocytes and the mechanism of transmission of information between the two doses in a combined radiation therapy.Some authors have hinted that epigenetics could play a major role in radiation responseFemale C57BL/6 J mice of comparable age (4–5 weeks) and weight (21–24 g) were purchased from Charles River Laboratories. They were kept one week in the local animal house for acclimatization. Animal experiments were carried out according to the European Commission Guidelines (Directive 86/609/CEE) on the use of laboratory animals.γ-rays); mice irradiated with an acute high dose of γ-rays 1.75 Gy same as the challenging dose of the combined regime, that were generated by a 137Cs gamma IBL-437C irradiator  and the study of the priming dose effects was performed on mice exposed to an acute single low dose of 0.075 Gy of X-rays. These mice were exposed to 0.075 Gy were kept alive for a latency period of 6 h to study. Each experimental group consisted of three mice. Mice were separated into four experimental conditions , 4 and 6 h were chosen to analyse the initial response to the combined regime when compared to the single dose treatment.Mice were separated into four experimental conditions to receive whole-body irradiation , the Ethic Committee of the Spanish National Research Council  and the Ethics Committee of Animal Experimentation (CEEA) of the Centre of Molecular Biology Severo Ochoa  (CEEA-CBMSO-23/191).Thymocytes were obtained by removing the thymus from the corresponding irradiated and non-irradiated C57BL/6 mice. Thymuses were washed in PBS and thymocytes were isolated via straining through a Nylon Mesh Cell Strainer of 40 µM .Human postnatal thymocytes were isolated from thymuses removed during congenital heart defects corrective surgery of 3 pediatric patients aged between 1 month and 3-year-old. Thymuses were mechanically disaggregated using a cell dissociation sieve-tissue grinder and isolated via ficoll density gradient  and its comparison to human pediatric samples (aged between 1 month and 3 years) is provided in the supporting information attached to the paper.Thymocytes were harvested and fixed in ice-cold 70% ethanol. On staining, cells were suspended in PBS containing 50 mg/ml RNase A (Sigma) for 15 min at room temperature. Last, we added propidium iodide (Sigma) staining solution at a final concentration of 50 µg/ml. cell cycle profiles were generated on a FACS-Calibur and Coulter Epics XL-MCL flow cytometer. The experiment was repeated 3 times for each cell type and condition.Isolated thymocytes were homogenized in radioimmunoprecipitation assay (RIPA) lysis buffer, supplemented with PhosStop phosphatase inhibitor and Complete EDTA free protease inhibitors (Roche) to isolate the soluble protein fraction. Protein concentrations were determined with the BCA™ protein assay kit (Pierce). Western blotting experiments were conducted at room temperature using 5 µg of cell lysate extracts, fractionated by 4–15% precast Long-life TGX (Tris–Glycine eXtended) Gels (Bio-Rad) and transferred to a polyvinylidene difluoride (PVDF) membrane using the Trans-Blot Turbo Transfer System according to the manufacturer’s protocols (Bio-Rad). Membranes were cut around the predicted molecular weight according to the manufacturer´s information  was chosen to perform pixel quantification of the images. Normalization method is used to correct for differences in protein abundance that are not relevant to the biological question being addressed. A summary of all western blot analysis is depicted on Supplementary Table vs. 5 serial dilution of control methylated DNA (0.5–10 ng). Specific Promoter methylation: Specific promoter methylation studies were performed on the same batch of samples using the OneStep qMethyl™ Kit (Zymo Research) according to manufacturer’s protocols and with the primers listed on the Supplementary Table Global methylation: A total of six human samples  had DNA extracted to quantify DNA methylation levels. Global DNA methylation status was performed using the Methylflash Methylated DNA Quantification Kit . DNA is bound to high DNA affinity strip wells. Methylated DNA is detected using capture and detection antibodies to 5-methyl cytosine (5-mC) and then colorimetrical quantified by reading absorbance at 450 nm using iMark™ Microplate Absorbance Reader (Bio-Rad Laboratories). The amount of methylated DNA is proportional to the OD intensity measured. The absolute amount of methylated DNA was quantified using a standard curve, plotting the OD values www.graphpad.com). Measurement data were expressed as mean ± SD, and were analyzed using the independent samples t-test. One-way ANOVA followed by Bonferroni's post hoc test were used for multiple pairwise comparisons, with an inspection level of α = 0.05. Significance levels such as *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001 and ****p ≤ 0.0001 were considered to indicate a statistically significant difference.Statistical analyses were performed using GraphPad Prism version 9.3.1 for Mac OS X,  with a 6-h interval between both doses. The outcome was compared to that of a control unirradiated cohort and a single dose cohort (1.75 Gy of γ-rays)  was delivered prior to challenging the cohort with a higher radiation dose of 1.75 Gy of CDKN1A axis in cell cycle arrest after the combined treatment. We measured the protein amounts in thymocytes exposed to either radiation regimen. Thirty minutes after exposure ATM expression in thymocytes exposed to the combined regime shows a modest reduction that increases with time. In fact, the expression of this protein was almost abolished 6 h after the combined regime exclusively (***p ≤ 0.001). Six hours after exposure to the single dose alone we found a decrease in ATM amounts but far from those levels seen in the combined regime (*p ≤ 0.05)  whilst the single dose did not accumulate comparable amounts of this protein until 4 h after exposure  Fig. . The expγH2AX in radio-adapted thymocytes 30 min after irradiation (****p ≤ 0.0001), and a uniform accumulation of this protein 4 and 6 h after any of the radiation treatments  and maintained its constant expression 6 h post-irradiation . Protein profiling of the 85 kDa PARP1 fragment Fig. C, the 41p ≤ 0.01  to the combined regime, whilst not varying much over time in thymocytes exposed to the single dose alone.The study of a panel of DNMTs and glycosylases known to play a crucial role at chromatin remodelling was performed. 30 min after exposure, thymocytes exposed to the combined regime showed a reduction of the DNA methyltransferases DNMT1, DNMT2, DNMT3B, DNMT3L and the glycosylases MBD4 and TDG , the same as the p18Ser-TP53 (**p ≤ 0.01) and p21CDKN1A (*p ≤ 0.05)  , subsequent PARP1 proteolysis (****p ≤ 0.0001) and PKCδ-dependent H3T45 phosphorylation (***p ≤ 0.001) seem to be activated in those cells as well  Fig. B. Striki01) Fig. C. MoreovParp1 and Pkcδ in thymocytes exposed to the combined radiation scheme  and single dose (***p ≤ 0.001 and **p ≤ 0.01) regimes 30 min after exposure  but the protein levels were found increased exclusively in those thymocytes exposed to the combined regime as early as 30 min after exposure (****p ≤ 0.0001). The same behavior was found for caspase-3 (*p ≤ 0.05) and PARP1 (*p ≤ 0.05 full length and ***p ≤ 0.001 cleaved form), when compared to control cells. Please note the activation of these proteins in control cells potentially due to the handling of these ex-vivo samples and the thawing procedures18   in the amounts of this glycosylase  Fig. C. In thease Fig. C.p ≤ 0.0001)   and TDG (*p ≤ 0.05)  6 h after the priming dose alone, a baseline inferred by the priming dose to later be enhanced by the full combined regime. Other authors have shown that G2-phase arrest and an upregulation of cell cycle control pathways are detectable at low dosesCDKN1A response and did not impose a stop at the G1 checkpoint after DNA damage32. In this study, such reduction did not avoid TP53 accumulation nor its phosphorylated form, suggesting a differential activation of this pathway by the combined regime. One must bear in mind however that the ATM induction could have happened earlier than 30 min after exposure in the combined cohort. In fact, only in the combined exposed thymocytes cohort, early transcriptional induction of p21CDKN1A was found (30 min after exposure). The accumulation of p21CDKN1A was not visible in thymocytes exposed to a single dose regime until 4 h after exposure demonstrating a faster response of the ATM/TP53/ p21CDKN1A axis in mice hit with the combined-dose regime. When assessing the cellular background 6 h after the priming dose alone, we discovered no changes in ATM expression at that time point (which again does not discard an earlier activation of the protein) but rather increase in cellular amounts of its downstream effectors, TP53, phosphorylated-18-TP53 and p21CDKN1A. This suggests that the priming dose activates this pathway and the cells retain enough increased protein levels during the latency period as to having them available to faster and more efficient activation of the pathway after the challenging dose.Combined radiation scheme exposed thymocytes reduced ATM expression in a time dependent manner. Previous data reported that the decrease in ATM levels caused failure to obtain a p53 / p21γH2AX33 was induced by the priming dose. This made the γH2AX response to the challenging dose within a combined scheme faster both in mice and human thymocytes. In addition, γH2AX is not required for Caspase-3 activation but both cooperate to determine the final cellular fate34. Our analysis for Cleaved Caspase-3 demonstrates the presence of both p19 and p17 Caspase-3 subunits as early as 30 min after combined regime of irradiation in mouse thymocytes. It is known that Cleaved Caspase-3 propagates an apoptotic signal inducing proteolysis of several enzymes. Among them, the DNA repair enzyme including poly (ADP-ribose) polymerase 1 (PARP1)35 and the protein kinase C delta (PKCδ)36. PARP1 is specifically proteolysed by the p17 Caspase-3 subunit to a 24 kDa DNA-binding domain (DBD) and to a 89 kDa catalytic fragment during the execution of the apoptotic program38. PKCδ is a cytoplasmic p19 caspase-3 substrate resulting in the production of a 41 kDa constitutively proapoptotic active catalytic fragment (CFδ)41. In this study we found activation of caspase 3 leading to the processing of PARP1 in response to the combined regime as early as 30 min after exposure when compared to the single dose alone in mouse thymocytes. The study of the cellular status prior to the challenging dose revealed an exclusive accumulation of the p17 subunit of caspase3 and its downstream effector PARP1 induced by the priming dose. A comparable pathway activation is only found in single dose thymocytes 4 h after exposure demonstrating the effectiveness of a combined regime at inducing cell killing. These data demonstrate that the PARP1 pathway activation by the priming dose alone is crucial for the differential apoptotic induction found in the combined regime. It might, in fact, be a decisive event during the progression to cellular demise as other authors reported in different experimental models38. In human thymocytes however, caspase activation was specific to the subunit p17 both after the priming dose and the combined regime concomitant with a modest increase in PARP1 backing up the mouse data on the importance of the cleavage of this specific subunit of caspase 3 in the radiation response to the combined radiation regime.The damage recognition pathway represented by gamma . demonstrated that phosphorylation of H3T45 (H3T45ph), the downstream effector of PKCδ increases dramatically in apoptotic cells, around the time of DNA nicking and/or fragmentation42. The positive association of these two molecules is clear in our studies and the accumulation of H3T45ph is exclusive of the combined dose cohort 30 min after exposure. The altered chromatin structure favors DNA processing in late apoptosis, such as DNA nicking and/or fragmentation, explaining the abundance of the mark at this time when we subject animals to a combined regimen of irradiation. Strikingly, in the study of the effects of the priming dose alone, we find this pathway already activated as Cleaved Caspase-3, cleaved PARP1, PKCδ and phosphorylated H3T45 appear highly expressed in these cells 6 h after the priming dose alone. Following after the TP53 axis response, this pathway seems to be activated contributing at sensitization by the priming dose, allowing thymocytes to faster respond to the subsequent challenging dose in mice. In human thymocytes however, p19 and the PKCδ pathway does not seem to contribute to the response in the times assayed.In the case of PKCδ, its cleavage (mediated by the p19 subunit of Caspase-3) seems to be triggered by the challenging dose independently of whether it is a single or a combined dose cohort in mouse thymocytes. However, it is proteolysed from the full length to the catalytic fragment faster in the combined regime. Hurd et al43, is known to be activated in IR-induced cell death response44. Furthermore, IR leads to glutathione depletion through the inhibition of xCT  and, consequently, inactivates glutathione peroxidase 4 (GPX4), whose function is to remove lipid peroxides45. In this study we show the activation of this pathway by depletion of xCT exclusively in the combined regime. However, this pathway seems to need both radiation doses, as the expression of the inhibitor does not change with the priming dose alone and this leads to a later activation of this pathway that would match the cell cycle distribution data. We provide the first evidence that the combined radiation regime promotes ferroptosis. Regarding this pathway, it is known that using xCT-inhibitors increased the efficacy of other therapies47 or that administration of genotoxic agents to increase DNA damage has been harnessed clinically to improve radiotherapy efficacy and therefore improve the outcomes of patients with cancer. With these data one could suggest the increased efficacy of a combined regime at tumour sensitization regarding ferroptosis induction as well.There was a differential sub-G1 accumulation 4 and 6 h after the treatment between the combined and the single dose cohorts not explained by the canonical apoptotic pathway. Therefore, we wondered if other cell death mechanisms could be differentially induced in the combined regime. Ferroptosis, an iron-related cell death induced by excessive lipid peroxidation50. Most studies showed a global decrease in DNA methylation within a day of radiation exposure52 therefore it is not surprising that the combined exposure resulted in a significant decrease in global DNA methylation, data that was specifically seen in mice in almost all the promoters of those genes seen altered in this study. These epigenetic changes however were accompanied by decreased levels of DNMT1, DNMT2, DNMT3A, DNMT3B and DNMT3L. This reduction occurs gradually after the full radiation combined regime in mice suggesting the control of different pathways after exposure. Single dose irradiated thymocytes show less efficiency at reducing the expression of these proteins and even in some of them no changes were found. Is it known, for example, that the reduction of DNMT3B sensitized prostate cancer cells to radiation53, therefore supporting the theory of sensitized cells by the combined scheme also at an epigenetic level. As previously stated in the introduction, targeted disruption of Dnmt1 and Dnmt3a in cultured cells eliminates the transmission of genomic instability15 so one could argue about the preventing potential of the combined regime at buffering the transmission of instability54. Furthermore, inhibition of DNMTs has demonstrated reduction in tumour formation, supporting the anti-tumour potential of this combined radiation regime.Global DNA hypomethylation is a potential biomarker for cancer risk associated with genomic instability, which is an important factor in radiation-induced cancer56 and as such, the reduction reported here might be key to the induction of cell death pathways. It was previously reported that TDG knockdown strongly suppresses the tumour-forming capabilities of melanoma suggesting that one or more of the TDG activities are critical for tumour induction, maintenance, and progression57 and as shown here, it is also key to the way thymocytes respond to the combined regime which supports the theory of the anti-tumour effectiveness of the combined scheme. All these results were corroborated in human, where the combined radiation regime induced changes in the expression of epigenetic markers although differential regulation levels might be present in human cells.Furthermore, the reduction of the glycosylase MBD4 has been functionally linked to apoptosis and plays a role in the effective response to a variety of chemotherapeutic and other cytotoxic drugs26. Therefore, these findings could be of great value at shaping future radiation treatments by enhancing radiation power with small radiation doses rather than using other aggressive alternatives such as the chemo or immunotherapy, as used nowadays, with the same idea of increased sensitization of tumour cells and healthy tissue recovery, in this case led by the hormesis11 known to be induced by the low dose (priming dose) triggering a differential, more effective response in the combined regime.All these results revealed here could be key responses behind cellular mechanisms such as radio-adaptation by low radiation doses leading to tumour control, as shown by othersSupplementary Information.