Small Volume Research Articles

Sonogenetics for Monitoring and Modulating Biomolecular Function by Ultrasound.

Ultrasound technology, synergistically harnessed with genetic engineering and chemistry concepts, has started to open the gateway to the remarkable realm of sonogenetics-a pioneering paradigm for remotely orchestrating cellular functions at the molecular level. This fusion not only enables precisely targeted imaging and therapeutic interventions, but also advances our comprehension of mechanobiology to unparalleled depths. Sonogenetic tools harness mechanical force within small tissue volumes while preserving the integrity of the surrounding physiological environment, reaching depths of up to tens of centimeters with high spatiotemporal precision. These capabilities circumvent the inherent physical limitations of alternative in vivo control methods such as optogenetics and magnetogenetics. In this review, we first discuss mechanosensitive ion channels, the most commonly utilized sonogenetic mediators, in both mammalian and non-mammalian systems. Subsequently, we provide a comprehensive overview of state-of-the-art sonogenetic approaches that leverage thermal or mechanical features of ultrasonic waves. Additionally, we explore strategies centered around the design of mechanochemically reactive macromolecular systems. Furthermore, we delve into the realm of ultrasound imaging of biomolecular function, encompassing the utilization of gas vesicles and acoustic reporter genes. Finally, we shed light on limitations and challenges of sonogenetics and present a perspective on the future of this promising technology.

Up modulation of dose-averaged linear energy transfer by simultaneousintegratedboost in carbon-ion radiotherapy for pancreatic carcinoma.

Local recurrence in locally advanced pancreatic cancer (LAPC) after carbon-ion radiotherapy (CIRT) may partly attribute to low dose-averaged linear energy transfer (LETd), despite high CIRT dose. This study aimed to investigate the approaches to up-modulate the CIRT LETd and to evaluate the corresponding oxygen enhancement ratio (OER) reduction. 10 LAPCs that had been irradiated by CIRT with 67.5Gy (RBE) in 15 fractions were selected. Their original plans were taken as the control plan for the LETd and OER investigations. Our considerations for up-modulating LETd were: (1) to deliver high doses to gross tumor volume core (GTVcore), while keeping dose constraints of the gastrointestinal (GI) tract in tolerance; (2) to put more Bragg-peak (BP) within the modulated targets; (3) to increase the BP density, high doses were necessary; (4) CIRT LETd could be effectively increased to small volumes; and (5) simultaneous integrated boost technique (SIB) could achieve the aforementioned tasks.The LETd and the corresponding OER distributions of each type of SIB plan were evaluated. We delivered up to 100Gy (RBE) to GTVcore using SIB. The mean LETd of GTV increased significantly by 21.3% from 47.8 to 58.0keV/μm (p<0.05). Meanwhile, the mean OER of GTVcore decreased by 6.6%, from 1.51 to 1.41 (p<0.05). The GI LETdS in all modulated plans were not more than those in the original plans. SIB could effectively increase CIRT LETd to LAPC, thus producing reduced OER, which may effectively overcome the radioresistance of LAPCs.

Micropillar‐based channel patterning in high‐loading graphite anodes for superior Li‐ion batteries

AbstractThis study presents a low‐cost, one‐step electrode patterning method that uses a template with micropillars to indent a hexagonal array of channels in high‐loading graphite anodes for faster electrolyte infiltration and Li‐ion transport. In contrast to prior studies on using laser micro‐machining, active material losses could be completely avoided by the proposed methodology. The process can also be made roll‐to‐roll and continuous. Furthermore, the very small volume fraction of the introduced channels (&lt;1 wt.%) has little impact on practically attainable energy density or specific energy. Yet, thus introducing pore channels significantly reduces electrolyte infiltration time and improves rate performance.

Study of the efficiency of machine learning algorithms based on data of various rocks

Background: Absolute permeability plays an important role in studying the fluids flow in porous media during the development of oil and gas reservoirs, the injection of CO2 into reservoirs for storage, the monitoring of pollutants migration in underground aquifers, and the modeling of catalytic systems. Therefore, an accurate and fast evaluation of its values is an important task.&#x0D; Aim: The purpose of this article is to study the applicability of machine learning methods for predicting the absolute permeability of carbonate samples, as well as ways to improve the prediction of permeability.&#x0D; Materials and methods: The input data is 408 small volumes extracted from four cylindrical carbonate samples composed almost entirely of calcite. Input data includes total and connected porosity, specific surface area, radii of all and only connected pores, coordination number, throat radius and length, tortuosity, and absolute permeability. Permeability prediction is carried out using regression machine learning methods such as random forest, extremely random trees and extended gradient boosting. Parameters (data) of small volumes were determined using pore-scale modeling of water flow in their pore space applying a specialized Avizo software.&#x0D; Results: Data of small volumes extracted from fractured and non-fractured samples were analyzed, and the results showed that there are good relationships between many parameters of small volumes. For example, the connected and total porosity have a second-order polynomial relationship with a high correlation coefficient. Using the above-mentioned regression machine learning methods, absolute permeability values were predicted when input data divided into training and testing data in a ratio of 80/20 and 70/30.&#x0D; Conclusion: Using the logarithm of permeability instead of permeability itself and considering fractured and non-fractured samples separately, can increase the accuracy of absolute permeability prediction using the above-mentioned machine learning methods up to 90%. The extremely random trees method is the most accurate among the three machine learning methods considered for our task.

Highly-efficient full-color holographic movie based on silicon nitride metasurface

Abstract Metasurface holograms offer various advantages, including wide viewing angle, small volume, and high resolution. However, full-color animation of high-resolution images has been a challenging issue. In this study, a full-color dielectric metasurface holographic movie with a resolution of 2322 × 2322 was achieved by spatiotemporally multiplexing 30 frames with blue, green, and red color channels at the wavelengths of 445 nm, 532 nm, and 633 nm at the maximum reconstruction speed of 55.9 frames per second. The high average transmittance and diffraction efficiency of 92.0 % and 72.7 %, respectively, in the visible range, were achieved by adopting polarization-independent silicon nitride waveguide meta-atoms, resulting in high color reproducibility. The superposition of three wavelengths was achieved by adjusting the resolutions and positions of target images for each wavelength while maintaining the meta-atom pitch constant. The improvement in diffraction efficiency was brought about by the optimization of etching conditions to form high-aspect vertical nanopillar structures.

Twisted fiber microfluidics: a cutting-edge approach to 3D spiral devices.

The development of 3D spiral microfluidics has opened new avenues for leveraging inertial focusing to analyze small fluid volumes, thereby advancing research across chemical, physical, and biological disciplines. While traditional straight microchannels rely solely on inertial lift forces, the novel spiral geometry generates Dean drag forces, eliminating the necessity for external fields in fluid manipulation. Nevertheless, fabricating 3D spiral microfluidics remains a labor-intensive and costly endeavor, hindering its widespread adoption. Moreover, conventional lithographic methods primarily yield 2D planar devices, thereby limiting the selection of materials and geometrical configurations. To address these challenges, this work introduces a streamlined fabrication method for 3D spiral microfluidic devices, employing rotational force within a miniaturized thermal drawing process, termed as mini-rTDP. This innovation allows for rapid prototyping of twisted fiber-based microfluidics featuring versatility in material selection and heightened geometric intricacy. To validate the performance of these devices, we combined computational modeling with microtomographic particle image velocimetry (μTPIV) to comprehensively characterize the 3D flow dynamics. Our results corroborate the presence of a steady secondary flow, underscoring the effectiveness of our approach. Our 3D spiral microfluidics platform paves the way for exploring intricate microflow dynamics, with promising applications in areas such as drug delivery, diagnostics, and lab-on-a-chip systems.

Volumetric 3D‐Printed Piezoelectric Polymer Films

AbstractA novel additive manufacturing route using a tailored resin containing Poly(vinylidene fluoride) Trifluoroethylene (PVDF‐TrFE) to 3D print piezoelectric films is demonstrated. Piezoelectric films are printed within 2 seconds in a single step by simultaneously focusing initiating and inhibiting excitations within the liquid resin to locally confine the photochemical reaction. The printed films are patterned with an array of holes with a diameter of 30 µm and a pitch of 55 µm. The piezoelectric response is homogeneous across the film, indicating that the print pattern does not impact the PVDF‐TrFE microstructure. Although the printed films contain only a small volume fraction of PVDF‐TrFE (3 wt.%), their piezoelectric response (d33 = 20.3 pC/N) is comparable to the highest literature values reported for PVDF‐TrFE films. The printed PVDF‐TrFE films are predominantly β‐phase, and no electrical poling, post‐processing, piezoelectric or inorganic additives are used in the fabrication. Analysis using piezoresponse force microscopy (PFM) and scanning electron microscopy (SEM) reveals that the enhanced piezoelectric response is due to the preferential formation of oriented PVDF‐TrFE phases during printing. These results demonstrate how the dedicated design of photoactive resins in combination with volumetric additive manufacturing can be applied to rapidly fabricate functional 3D structures.

Active mid-infrared ring resonators

High-quality optical ring resonators can confine light in a small volume and store it for millions of roundtrips. They have enabled the dramatic size reduction from laboratory scale to chip level of optical filters, modulators, frequency converters, and frequency comb generators in the visible and the near-infrared. The mid-infrared spectral region (3−12 μm), as important as it is for molecular gas sensing and spectroscopy, lags behind in development of integrated photonic components. Here we demonstrate the integration of mid-infrared ring resonators and directional couplers, incorporating a quantum cascade active region in the waveguide core. It enables electrical control of the resonant frequency, its quality factor, the coupling regime and the coupling coefficient. We show that one device, depending on its operating point, can act as a tunable filter, a nonlinear frequency converter, or a frequency comb generator. These concepts extend to the integration of multiple active resonators and waveguides in arbitrary configurations, thus allowing the implementation of purpose-specific mid-infrared active photonic integrated circuits for spectroscopy, communication, and microwave generation.

Emergency radiation therapy in modern-day practice: Now or never, or never again ?

Radiation therapy plays a fundamental role in oncological emergencies such as superior vena cava syndrome (SVCS) and metastatic epidural spinal cord compression (MESCC). These are two examples of critical complications of metastatic cancer in terms of pain and functional impact (respiratory and/or neurological). The aim of this review is to explore the current indications, treatment optionsand outcomes for emergency radiotherapy regarding to these complications.Regarding SVCS, studies are mostly retrospectiveand unanimously demonstrated a beneficial effect of radiotherapy on symptom relief. Spinal cord compression remains an indication for urgent radiotherapy, and should be combined with surgery when possible. The innovative stereotactic body radiotherapy (SBRT) showed promising results, however this technique requires small volumes and more time preparation and therefore is often unsuitable for SVCS and MESCC emergencies.This review concluded that radiotherapy has a central role to play within a multimodal approach for SVCS and MESCC treatment. Further prospective studies are needed to confirm the effectiveness of radiation and establish the criteria for selecting patients to benefit from this treatment option.

Comparing novel sperm extenders for the internally-fertilizing tiger salamander (Ambystoma tigrinum)

Due to the concentrated nature and small volume of spermic milt that is typically produced from internally-fertilizing salamanders, an extender is needed to analyze the sperm for quality and quantity metrics. Techniques for diluting salamander sperm for analysis have thus far utilized low osmolality media, which are likely physiologically distinct from the salamander female reproductive tract. Application of mammalian sperm extenders could maintain salamander sperm motility and morphology in storage longer due to the similar nature of internal fertilization in both taxa. Several sperm extenders were tested including: Multipurpose Handling Medium, Sperm Washing Medium, Modified Ham’s F-10 Basal Medium (HAM’s), Refrigeration Medium, and 10% Holtfreter’s solution as a control. The experimental treatments were divided into two groups: diluted (90 mOsm, the approximate osmolality of salamander sperm) and undiluted (280-320 mOsm). Milt samples were collected from male tiger salamanders (N=11) and diluted 1:50 with each of the treatments and measured for sperm progressive motility, total motility, and normal morphology every 10 minutes for one hour. When comparing the diluted versus undiluted treatments of the same extender, there was no difference (p&amp;gt;0.05) for any of the response variables. In addition, we found that 10% Holtfreter’s solution is best for maintaining progressive motility, while diluted HAM’s is best for retaining total motility and normal morphology over time. Overall, we found that osmolality is not a key driver influencing tiger salamander sperm motility, but rather other factors such as ionic composition or pH could be affecting sperm form and function.

Field stiffening: A way to break the bounds on the properties of composites

It is common practice for humans to enhance the stiffness of a material by adding stiffer ingredients into it, which leads to the development of various composites of wide applications. However, irrespective of the configuration of the constituents of a multiphase composite, its compression elastic moduli are always between some bounds which are determined by the volume fractions and elastic moduli of the constituents inherently. Here, we report a magnetic composite material that is composed of a soft matrix material and two magnetic thin plates with small volume fraction. The compressive elastic modulus of the composite material is 25 times higher than the Halpin-Tsai upper bound due to the effect of the internal magnetic field. The measured maximum initial tangent modulus of the magnetic composite is ca. 40 times and 54 times higher than that of the nonmagnetic composite and PDMS matrix, respectively. This work provides a new direction for improving the performance of materials by fields.

Measuring diameters and velocities of artificial raindrops with a neuromorphic event camera

Abstract. Hydrometers that measure size and velocity distributions of precipitation are needed for research and corrections of rainfall estimates from weather radars and microwave links. Existing optical disdrometers measure droplet size distributions, but underestimate small raindrops and are impractical for widespread always-on IoT deployment. We study the feasibility of measuring droplet size and velocity using a neuromorphic event camera. These dynamic vision sensors asynchronously output a sparse stream of pixel brightness changes. Droplets falling through the plane of focus of a steeply down-looking camera create events generated by the motion of the droplet across the field of view. Droplet size and speed are inferred from the hourglass-shaped stream of events. Using an improved hard disk arm actuator to reliably generate artificial raindrops with a range of small sizes, our experiments show maximum errors of 7 % (mean absolute percentage error) for droplet sizes from 0.3 to 2.5 mm and speeds from 1.3 to 8.0 m s−1. Measurements with the same setup from a commercial PARSIVEL disdrometer show similar results. Both devices slightly overestimate the small droplet volume with a volume overestimation of 25 % from the event camera measurements and 50 % from the PARSIVEL instrument. Each droplet requires processing of 5000 to 50 000 brightness change events, potentially enabling low-power always-on disdrometers that consume power proportional to the rainfall rate. Data and code are available at the paper website https://sites.google.com/view/dvs-disdrometer/home (Micev et al., 2023).

Exploring localized ENZ resonances and their role in superscattering, wideband invisibility, and tunable scattering

While the role and manifestations of the localized surface plasmon resonances (LSPRs) in anomalous scattering, like superscattering and invisibility, are quite well explored, the existence, appearance, and possible contribution of localized epsilon-near-zero (ENZ) resonances still invoke careful exploration. In this paper, that is done along with a comparison of the resonances of two types in the case of thin-wall cylinders made of lossy and loss-compensated dispersive materials. It is shown that the localized ENZ resonances exist and appear very close to the zero-permittivity regime, i.e., at near-zero but yet negative permittivity that is similar to the ENZ modes in thin planar films. Near- and far-field characteristics of the superscattering modes are investigated. The results indicate that the scattering regimes arising due to LSPRs and localized ENZ resonances are distinguishable in terms of the basic field features inside and around the scatterer and differ in their contribution to the resulting scattering mechanism, e.g., in terms of the occupied frequency and permittivity ranges as well as the sensitivity to the wall thickness variations. When the losses are either weak or tend to zero due to the doping with gain enabling impurities, the sharp peaks of the scattering cross-section that are yielded by the resonances can be said to be embedded into the otherwise wide invisibility range. In the case of lossy material, a wide and continuous invisibility range is shown to appear not only due to a small total volume of the scatterer in the nonresonant regime, but also because high-Q superscattering modes are suppressed by the losses. For numerical demonstration, indium antimonide, a natural lossy material, and a hypothetical, properly doped material with the same real part of the permittivity but lower or zero losses are considered. In the latter case, variations of permittivity with a control parameter can be adjusted in such a way that transitions from one superscattering mode to another can be achieved. In turn, transition from the strong-scattering to the invisibility regime is possible even for the original lossy material. The basic properties of the studied superscattering modes may be replicable in artificial structures comprising natural low-loss materials.

Employing electrochemically derived pH gradients for Lab-on-PCB protein preconcentration devices

Protein preconcentration is an essential sample preparation step for analysis in which the targeted proteins exist in low concentrations, such as bodily fluids, water, or wastewater. Nonetheless, very few practical implementations of miniaturized protein preconcentration devices have been demonstrated in practice, and even fewer have been integrated with other microanalytical steps. Existing approaches rely heavily on additional chemicals and reagents and introduce complexity to the overall assay. In this paper, we propose a novel miniaturized isoelectric focusing-based protein preconcentration screening device based on electrochemically derived pH gradients rather than existing chemical reagent approaches. In this way, we reduce the need for additional chemical reagents to zero while enabling device incorporation in a seamlessly integrated full protein analysis microsystem via Lab-on-PCB technology. We apply our previously presented Lab-on-PCB approach to quantitatively control the pH of a solution in the vicinity of planar electrodes using electrochemical acid generation through redox-active self-assembled monolayers. The presented device comprises a printed circuit board with an array of gold electrodes that were functionalized with 4-aminothiophenol; this formed a self-assembled monolayer that was electropolymerized to improve its electrochemical reversibility. Protein preconcentration was performed in two configurations. The first was open and needed the use of a holder to suspend a well of fluid above the electrodes; the second used microfluidic channels to enclose small volumes of fluid. Reported here are the resulting data for protein preconcentration in both these forms, with a quantitative concentration factor shown for the open form and qualitative proof shown for the microfluidic.

Measuring Trajectory Similarity Based on the Spatio-Temporal Properties of Moving Objects in Road Networks

Advancements in navigation and tracking technologies have resulted in a significant increase in movement data within road networks. Analyzing the trajectories of network-constrained moving objects makes a profound contribution to transportation and urban planning. In this context, the trajectory similarity measure enables the discovery of inherent patterns in moving object data. Existing methods for measuring trajectory similarity in network space are relatively slow and neglect the temporal characteristics of trajectories. Moreover, these methods focus on relatively small volumes of data. This study proposes a method that maps trajectories onto a network-based space to overcome these limitations. This mapping considers geographical coordinates, travel time, and the temporal order of trajectory segments in the similarity measure. Spatial similarity is measured using the Jaccard coefficient, quantifying the overlap between trajectory segments in space. Temporal similarity, on the other hand, incorporates time differences, including common trajectory segments, start time variation and trajectory duration. The method is evaluated using real-world taxi trajectory data. The processing time is one-quarter of that required by existing methods in the literature. This improvement allows for spatio-temporal analyses of a large number of trajectories, revealing the underlying behavior of moving objects in network space.

Physical development and bone mineral density of children with inflammatory bowel diseases

Relevance. Inflammatory bowel diseases can cause a lag in physical development and a decrease in the nutritional status of children. Therapy with glucocorticosteroids can aggravate the process of formation of the body and reduce the mineralization of bone tissue.Materials and methods. The study included 24 children aged 5 to 17 years with inflammatory bowel diseases, of which 19 patients did not use glucocorticosteroids in therapy – group 1, and group 2–5 children on hormone therapy. The groups did not differ statistically significantly in age and gender composition. In order to assess the level of physical development, an assessment of anthropometric indicators was carried out using the ANTHRO Plus program. Bone mineral density was assessed by densitometry. Statistical processing was carried out using Statistica 6.0 statistical programs and descriptive statistics methods. To compare independent groups of small volume, the method was used Mann-WhitneyResults. The evaluation of length, body weight and body mass index was carried out, as a result, it was found that the majority of patients who participated in the study had an average level of physical development in the population, no statistically significant differences were found by any criterion (p &gt; 0.05). When assessing the BMD in children with IBD who did not use hormone therapy in the treatment of diseases, it was found that the Z-Score level within the reference values was observed in 57.9% (n = 11) children, 1 (5.3%) child has below –2.49 and in 36.8% (n = 7) the Z-Score level was determined in the range from –1.00 SD to –2.49 SD. More than half of the patients in group 2 had no changes in BMD (60%; n = 3), the Z-Score value in 1 (20%) patient corresponded to the level of osteopenia and in 1 (20%) child – osteoporosis. No differences were found between the groups and depending on physical development, gender or age (p &gt; 0.05).Conclusions. It was found that children, regardless of the use of hormone therapy they have similar indicators in the levels of physical development and bone mineralization. The importance of monitoring the studied indicators for the timely prevention of changes is noted.

Anticoagulant effects of protein C, protein S, and antithrombin levels on the protein C pathway in young children.

The protein C (PC) pathway involves physiological anticoagulant factors (PC, protein S [PS], and factor V) and performs major anticoagulant functions in adults. Variations in overall PC pathway function due to dynamic changes in PC and PS in early childhood are poorly understood. We aimed to evaluate the contributions of PC pathway function during early childhood by measuring changes in plasma thrombin generation (TG) after administration of the PC activator protac. We evaluated correlations between anticoagulant factors and percentage of protac-induced coagulation inhibition (PiCi%). Before protac addition, TG in newborns (n = 35), infants (n = 42), young children (n = 35), and adults (n = 20) were 525 ± 74, 720 ± 96, 785 ± 53, and 802 ± 64mOD/min, and PiCi% were 42.1 ± 9.9, 69.8 ± 11.0, 82.9 ± 4.4, and 86.9 ± 3.4%, respectively. The distribution of PiCi% on the two axes of TG (with or without protac) changed continuously with age and differed from that of warfarin-treated plasma and adult PC- or PS-deficient plasma. PiCi% increased dynamically during infancy and correlated with PS levels in newborns and PC levels in young children. Addition of PC or fresh frozen plasma equivalent to approximately 25% PC to PC-deficient plasma improved PiCi%. This automatic measurement requires only a small sample volume and is useful for analysis of developmental hemostasis.

Research on Multi-Objective Optimization on Explosion-Suppression Structure-Nonmetallic Spherical Spacers

Intense burning phenomena (fire disasters) need to be prevented in the combustible gas utilization and transportation processes to ensure industrial safety. Nonmetallic spherical spacers (NSSs) have been investigated and applied in lots of explosive atmospheres to prevent explosion execution in a confined space. In this work, a novel fuzzy-based analytic hierarchy process (FAHP) is developed to take into account the uncertainty in decision-making and effectively solve the problem of factor weight allocation in multi-objective optimization. Optimal Latin Hypercube Design (Opt LHD), Chebyshev Orthogonal Polynomials (COP), and Adaptive Simulated Annealing (ASA) were combined. A multi-objective optimization method is proposed for the structural parameter optimization problem on NSSs in order to achieve conflicting multiple-objective optimization of low displacement rate and minimal deformation. That is to say, the small volume (low displacement rate) and high explosion-suppression performance (minimal deformation) of NSSs were optimized simultaneously. The results show that, compared with the original NSS model’s deformation (2.85 mm) and displacement rate (3.63%), the optimized NSSs with weight allocation had optimized the deformation by 12.98% and displacement rate by 6.1%. Compared with the optimized design model of NSSs without weight allocation with a deformation of 2.75 mm and a displacement rate of 3.48%, the deformation has been optimized by 9.82%, and the displacement rate has been optimized by 2.0%. It was verified that the proposed method is effective. At the same time, it was verified that the suppression effect of NSSs can be enhanced by changing the shape of the NSS spacer reasonably by experimental verification.

Brillouin lasing in microspheres coupled with silicon subwavelength grating waveguides

Brillouin lasing has been widely applied to microwave oscillators, optical gyroscopes, optical sensors, and other fields owing to its strong and flexible dynamics. Microspheres with ultrahigh quality factors and small mode volume are very suitable for generating Brillouin lasing, but it is difficult to integrate them on a chip. Here, we utilize subwavelength grating waveguide as a photonic coupler to connect the silica microsphere with a silicon chip and realize on-chip backward Brillouin lasing. It is experimentally indicated that when the on-chip pump power reaches 24.8 mW, the backward Brillouin lasing with an on-chip slope efficiency of 5% will be excited. Our method makes it easier to realize on-chip Brillouin acousto-optic interaction and greatly expands the application range of microspheres.

Enhanced RBE of Particle Radiation Depends on Beam Size in the Micrometer Range.

High-linear energy transfer (LET) radiation, such as heavy ions is associated with a higher relative biological effectiveness (RBE) than low-LET radiation, such as photons. Irradiation with low- and high-LET particles differ in the interaction with the cellular matter and therefore in the spatial dose distribution. When a single high-LET particle interacts with matter, it results in doses of up to thousands of gray (Gy) locally concentrated around the ion trajectory, whereas the mean dose averaged over the target, such as a cell nucleus is only in the range of a Gy. DNA damage therefore accumulates in this small volume. In contrast, up to hundreds of low-LET particle hits are required to achieve the same mean dose, resulting in a quasi-homogeneous damage distribution throughout the cell nucleus. In this study, we investigated the dependence of RBE from different spatial dose depositions using different focused beam spot sizes of proton radiation with respect to the induction of chromosome aberrations and clonogenic cell survival. Human-hamster hybrid (AL) as well as Chinese hamster ovary cells (CHO-K1) were irradiated with focused low LET protons of 20 MeV (LET = 2.6 keV/µm) beam energy with a mean dose of 1.7 Gy in a quadratic matrix pattern with point spacing of 5.4 × 5.4 µm2 and 117 protons per matrix point at the ion microbeam SNAKE using different beam spot sizes between 0.8 µm and 2.8 µm (full width at half maximum). The dose-response curves of X-ray reference radiation were used to determine the RBE after a 1.7 Gy dose of radiation. The RBE for the induction of dicentric chromosomes and cell inactivation was increased after irradiation with the smallest beam spot diameter (0.8 µm for chromosome aberration experiments and 1.0 µm for cell survival experiments) compared to homogeneous proton radiation but was still below the RBE of a corresponding high LET single ion hit. By increasing the spot size to 1.6-1.8 µm, the RBE decreased but was still higher than for homogeneously distributed protons. By further increasing the spot size to 2.7-2.8 µm, the RBE was no longer different from the homogeneous radiation. Our experiments demonstrate that varying spot size of low-LET radiation gradually modifies the RBE. This underlines that a substantial fraction of enhanced RBE originates from inhomogeneous energy concentrations on the µm scale (mean intertrack distances of low-LET particles below 0.1 µm) and quantifies the link between such energy concentration and RBE. The missing fraction of RBE enhancement when comparing with high-LET ions is attributed to the high inner track energy deposition on the nanometer scale. The results are compared with model results of PARTRAC and LEM for chromosomal aberration and cell survival, respectively, which suggest mechanistic interpretations of the observed radiation effects.