Discovery of a body-wide photosensory array that matures in an adult-like animal and mediates eye-brain-independent movement and arousal.

The ability to respond to light has profoundly shaped life. Animals with eyes overwhelmingly rely on their visual circuits for mediating light-induced coordinated movements. Building on previously reported behaviors, we report the discovery of an organized, eye-independent (extraocular), body-wide photosensory framework that allows even a head-removed animal to move like an intact animal. Despite possessing sensitive cerebral eyes and a centralized brain that controls most behaviors, head-removed planarians show acute, coordinated ultraviolet-A (UV-A) aversive phototaxis. We find this eye-brain-independent phototaxis is mediated by two noncanonical rhabdomeric opsins, the first known function for this newly classified opsin-clade. We uncover a unique array of dual-opsin-expressing photoreceptor cells that line the periphery of animal body, are proximal to a body-wide nerve net, and mediate UV-A phototaxis by engaging multiple modes of locomotion. Unlike embryonically developing cerebral eyes that are functional when animals hatch, the body-wide photosensory array matures postembryonically in "adult-like animals." Notably, apart from head-removed phototaxis, the body-wide, extraocular sensory organization also impacts physiology of intact animals. Low-dose UV-A, but not visible light (ocular-stimulus), is able to arouse intact worms that have naturally cycled to an inactive/rest-like state. This wavelength selective, low-light arousal of resting animals is noncanonical-opsin dependent but eye independent. Our discovery of an autonomous, multifunctional, late-maturing, organized body-wide photosensory system establishes a paradigm in sensory biology and evolution of light sensing.

CODEX, a neural network approach to explore signaling dynamics landscapes.

Current studies of cell signaling dynamics that use live cell fluorescent biosensors routinely yield thousands of single-cell, heterogeneous, multi-dimensional trajectories. Typically, the extraction of relevant information from time series data relies on predefined, human-interpretable features. Without a priori knowledge of the system, the predefined features may fail to cover the entire spectrum of dynamics. Here we present CODEX, a data-driven approach based on convolutional neural networks (CNNs) that identifies patterns in time series. It does not require a priori information about the biological system and the insights into the data are built through explanations of the CNNs' predictions. CODEX provides several views of the data: visualization of all the single-cell trajectories in a low-dimensional space, identification of prototypic trajectories, and extraction of distinctive motifs. We demonstrate how CODEX can provide new insights into ERK and Akt signaling in response to various growth factors, and we recapitulate findings in p53 and TGFß-SMAD2 signaling.

High-Resolution Focused Ultrasound Neuromodulation Induces Limb-Specific Motor Responses in Mice in Vivo.

Ultrasound can modulate activity in the central nervous system, including the induction of motor responses in rodents. Recent studies investigating ultrasound-induced motor movements have described mostly bilateral limb responses, but quantitative evaluations have failed to reveal lateralization or differences in response characteristics between separate limbs or how specific brain targets dictate distinct limb responses. This study uses high-resolution focused ultrasound (FUS) to elicit motor responses in anesthetized mice in vivo and four-limb electromyography (EMG) to evaluate the latency, duration and power of paired motor responses (n = 1768). The results indicate that FUS generates target-specific differences in electromyographic characteristics and that brain targets separated by as little as 1 mm can modulate the responses in individual limbs differentially. Exploiting these differences may provide a tool for quantifying the susceptibility of underlying neural volumes to FUS, understanding the functioning of the targeted neuroanatomy and aiding in mechanistic studies of this non-invasive neuromodulation technique.

The cold-induced switch in direction of chloroplast relocation occurs independently of changes in endogenous phototropin levels.

When exposed to fluctuating light intensity, chloroplasts move towards weak light (accumulation response), and away from strong light (avoidance response). In addition, cold treatment (5°C) induces the avoidance response even under weak-light conditions (cold-avoidance response). These three responses are mediated by the phototropin (phot), which is a blue-light photoreceptor and has also been reported to act as a thermosensory protein that perceives temperature variation. Our previous report indicated that cold-induced changes in phot biochemical activity initiate the cold-avoidance response. In this study, we further explored the induction mechanism of the cold-avoidance response in the liverwort Marchantia polymorpha and examined the relationship between changes in the amount of phot and the induction of the cold-avoidance response. The switch between the accumulation and avoidance responses occurs at a so-called 'transitional' light intensity. Our physiological experiments revealed that a cold-mediated decrease in the transitional light intensity leads to the induction of the cold-avoidance response. While artificial overexpression of phot decreased the transitional light intensity as much as cold treatment did, the amount of endogenous phot was not increased by cold treatment in wild-type M. polymorpha. Taken together, these findings show that the cold-avoidance response is initiated by a cold-mediated reduction of the transitional light intensity, independent of the amount of endogenous phot. This study provides a clue to understanding the mechanism underlying the switch in direction of chloroplast relocation in response to light and temperature.

Waveform Plasticity under Entrainment to 12-h T-cycles in Drosophila melanogaster: Behavior, Neuronal Network, and Evolution.

A crucial property of circadian clocks is the ability to regulate the shape of an oscillation over its cycle length (waveform) appropriately, thus enhancing Darwinian fitness. Many studies over the past decade have revealed interesting ways in which the waveform of rodent behavior could be manipulated, one of which is that the activity bout bifurcates under environments that have 2 light/dark cycles within one 24-h day (LDLD). It has been observed that such unique, although unnatural, environments reveal acute changes in the circadian clock network. However, although adaptation of waveforms to different photoperiods is well studied, modulation of waveforms under LDLD has received relatively less attention in research on insect rhythms. Therefore, we undertook this study to ask the following questions: what is the extent of waveform plasticity that Drosophila melanogaster exhibits, and what are the neuronal underpinnings of such plasticity under LDLD? We found that the activity/rest rhythms of wild-type flies do not bifurcate under LDLD. Instead, they show similar but significantly different behavior from that under a long-day LD cycle. This behavior is accompanied by differences in the organization of the circadian neuronal network, which include changes in waveforms of a core clock component and an output molecule. In addition, to understand the functional significance of such variations in the waveform, we examined laboratory selected populations that exhibit divergent eclosion chronotypes (and therefore, waveforms). We found that populations selected for predominant eclosion in an evening window (late chronotypes) showed reduced amplitude plasticity and increased phase plasticity of activity/rest rhythms. This, we argue, is reflective of divergent evolution of circadian neuronal network organization in our laboratory selected flies.

Protective effects of phenformin on zebrafish embryonic neurodevelopmental toxicity induced by X-ray radiation.

Radiotherapy (RT) is a common treatment for head and neck cancers, but central nervous system function can be impaired by clinical radiation doses. This experimental study evaluated the protective efficacy of the anti-hyperglycaemic/anti-neoplastic agent phenformin against radiation-induced developmental toxicity in zebrafish embryos. Zebrafish embryos pre-treated with 25 µM phenformin 1 h before x-ray irradiation were compared to irradiation-only embryos for mortality, hatching rate, morphology, spontaneous movement, heart beat, larval swimming, activities of the antioxidant enzymes superoxide dismutase (SOD) and catalase (CAT), malondialdehyde content (MDA, a by-product of membrane lipid oxidation), and acetylcholinesterase (AChE) activity. In addition, expression levels of multiple genes related to neural development and apoptosis (sod2, bdnf, ache, p53, bax, and bcl-2) were compared by RT-PCR and associated protein expression levels by western blotting. Pre-treatment with phenformin increased hatching rate, spontaneous movement, heart beat, and larval motor activity, decreased mortality and malformation rate, increased SOD, CAT, and AChE activities, and reduced MDA compared to irradiation-only embryos. The mRNA expression levels of anti-apoptotic sod2, bdnf, ache, and bcl-2 were enhanced while mRNA expression of p53 and pro-apoptotic bax were reduced in the phenformin pre-treatment group. Further, p53, Bax, and γ-H2AX (a biomarker of DNA damage) were downregulated while Bcl-2 and BDNF were upregulated by phenformin pre-treatment. Taken together, this study supports the protective efficacy of phenformin against radiation toxicity in zebrafish embryos by suppressing oxidative stress and ensuing apoptosis.

Commissioning and Evaluation of a Third-Party 6 Degrees-of-Freedom Couch Used in Radiotherapy.

PURPOSES: The newly released Protura 6 degrees-of-freedom couch (CIVCO) has limited quality assurance protocols and pertinent publications. Herein, we report our experiences of the Protura system acceptance, commissioning, and quality assurance. METHODS: The Protura system integration was tested with peripheral equipment on the following items: couch movement range limit, 6 degrees-of-freedom movement accuracy, weight test and couch sagging, system connection with Linac, isocentricity of couch and rotation alignment, kV and cone-beam computed tomography imaging of HexaCHECK with MIMI phantom (Standard Imaging), and an in-house custom 6 degrees-of-freedom quality assurance phantom. A couch transmission measurement was also performed. RESULTS: The vertical, longitudinal, and lateral ranges of the 6 degrees-of-freedom couch pedestal are 43.9 to 0.0 cm, 24.6 to 149.5 cm, -20.6 to 20.7 cm, respectively. The couch movement accuracy was within 1 mm in all directions. The couch sagging with a 200 lbs (∼91 kg) evenly distributed object is 1.0 cm and 0.4° pitch in the distal end of the couch. The isocentricity of the couch was about 0.5 mm in diameter of all crosshair projections on the couch isocenter level, and the largest couch rotation alignment observed was (0.3°) at the couch angle of 90°. The deviation from the reference position (zero position) of the HexaCHECK phantom, measured by matching the cone-beam computed tomography with the reference planning computed tomography, was found to be below 0.2 mm in the anterior-posterior and right-left dimensions, 0.4 mm in superior-inferior dimension, and 0.1° in roll, pitch, and yaw directions. CONCLUSIONS: A 6 degrees-of-freedom quality assurance phantom is helpful for the commissioning and routine quality assurance tests. Due to the third-party integration with Linac, the system is prone to "double-correction" errors. A rigorous quality assurance program is the key to a successful clinical implementation of the Protura system.

Circadian leaf movements facilitate overtopping of neighbors.

Many plants exhibit circadian clock-driven leaf movements whereby the leaves are raised during the day to achieve a relatively high angle during the evening, before lowering late in the night. Such leaf movements were first recorded over 2000 years ago but there is still much debate as to their purpose. We investigated whether such leaf movements within Arabidopsis, a ruderal rosette plant, can aid in overtopping leaves of neighboring plants. Wild type and circadian clock mutant plants were grown in an alternating grid system so that their leaves would meet as the plants grew. Experiments were performed using day lengths that matched the endogenous rhythm of either wild type or mutant. Plants grown in a day length shorter than their endogenous rhythm were consistently overtopped by plants which were in synchrony with the day night cycle, demonstrating a clear overtopping advantage resulting from circadian leaf movement rhythms. Furthermore, we found that this leaf overtopping as a result of correctly synchronized circadian leaf movements is additive to leaf overtopping due to shade avoidance. Curiously, this did not apply to plants grown in a day length longer than their endogenous period. Plants grown in a day length longer than their endogenous period were able to adapt their leaf rhythms and suffered no overtopping disadvantage. Crucially, our results show that, in a context-dependent manner, circadian clock-driven leaf movements in resonance with the external light/dark cycle can facilitate overtopping of the leaves of neighboring plants.

The propagation of active-passive interfaces in bacterial swarms.

Propagating interfaces are ubiquitous in nature, underlying instabilities and pattern formation in biology and material science. Physical principles governing interface growth are well understood in passive settings; however, our understanding of interfaces in active systems is still in its infancy. Here, we study the evolution of an active-passive interface using a model active matter system, bacterial swarms. We use ultra-violet light exposure to create compact domains of passive bacteria within Serratia marcescens swarms, thereby creating interfaces separating motile and immotile cells. Post-exposure, the boundary re-shapes and erodes due to self-emergent collective flows. We demonstrate that the active-passive boundary acts as a diffuse interface with mechanical properties set by the flow. Intriguingly, interfacial velocity couples to local swarm speed and interface curvature, raising the possibility that an active analogue to classic Gibbs-Thomson-Stefan conditions may control this boundary propagation.

Dynamic density shaping of photokinetic E. coli.

Many motile microorganisms react to environmental light cues with a variety of motility responses guiding cells towards better conditions for survival and growth. The use of spatial light modulators could help to elucidate the mechanisms of photo-movements while, at the same time, providing an efficient strategy to achieve spatial and temporal control of cell concentration. Here we demonstrate that millions of bacteria, genetically modified to swim smoothly with a light controllable speed, can be arranged into complex and reconfigurable density patterns using a digital light projector. We show that a homogeneous sea of freely swimming bacteria can be made to morph between complex shapes. We model non-local effects arising from memory in light response and show how these can be mitigated by a feedback control strategy resulting in the detailed reproduction of grayscale density images.

Structured-Light-Based System for Shape Measurement of the Human Body in Motion.

The existing methods for measuring the shape of the human body in motion are limited in their practical application owing to immaturity, complexity, and/or high price. Therefore, we propose a method based on structured light supported by multispectral separation to achieve multidirectional and parallel acquisition. Single-frame fringe projection is employed in this method for detailed geometry reconstruction. An extended phase unwrapping method adapted for measurement of the human body is also proposed. This method utilizes local fringe parameter information to identify the optimal unwrapping path for reconstruction. Subsequently, we present a prototype 4DBODY system with a working volume of 2.0 × 1.5 × 1.5 m³, a measurement uncertainty less than 0.5 mm and an average spatial resolution of 1.0 mm for three-dimensional (3D) points. The system consists of eight directional 3D scanners functioning synchronously with an acquisition frequency of 120 Hz. The efficacy of the proposed system is demonstrated by presenting the measurement results obtained for known geometrical objects moving at various speeds as well actual human movements.

Light-induced propulsion of a giant liposome driven by peptide nanofibre growth.

Light-driven nano/micromotors are attracting much attention, not only as molecular devices but also as components of bioinspired robots. In nature, several pathogens such as Listeria use actin polymerisation machinery for their propulsion. Despite the development of various motors, it remains challenging to mimic natural systems to create artificial motors propelled by fibre formation. Herein, we report the propulsion of giant liposomes driven by light-induced peptide nanofibre growth on their surface. Peptide-DNA conjugates connected by a photocleavage unit were asymmetrically introduced onto phase-separated giant liposomes. Ultraviolet (UV) light irradiation cleaved the conjugates and released peptide units, which self-assembled into nanofibres, driving the translational movement of the liposomes. The velocity of the liposomes reflected the rates of the photocleavage reaction and subsequent fibre formation of the peptide-DNA conjugates. These results showed that chemical design of the light-induced peptide nanofibre formation is a useful approach to fabricating bioinspired motors with controllable motility.

When uncertain, does human self-motion decision-making fully utilize complete information?

When forced to choose humans often feel uncertain. Investigations of human perceptual decision-making often employ signal detection theory, which assumes that even when uncertain all available information is fully utilized. However, other studies have suggested or assumed that, when uncertain, human subjects guess totally at random, ignoring available information. When uncertain, do humans simply guess totally at random? Or do humans fully utilize complete information? Or does behavior fall between these two extremes yielding "above chance" performance without fully utilizing complete information? While it is often assumed complete information is fully utilized, even when uncertain, to our knowledge this has never been experimentally confirmed. To answer this question, we combined numerical simulations, theoretical analyses, and human studies performed using a self-motion direction-recognition perceptual decision-making task (did I rotate left or right?). Subjects were instructed to make forced-choice binary (left/right) and trinary (left/right/uncertain) decisions when cued following each stimulus. Our results show that humans 1) do not guess at random when uncertain and 2) make binary and trinary decisions equally well. These findings show that humans fully utilize complete information when uncertain for our perceptual decision-making task. This helps unify signal detection theory and other models of forced-choice decision-making which allow for uncertain responses. NEW & NOTEWORTHY Humans make many perceptual decisions every day. But what if we are uncertain? While many studies assume that humans fully utilize complete information, other studies have suggested and/or assumed that when we're uncertain and forced to decide, information is not fully utilized. While humans tend to perform above chance when uncertain, no earlier study has tested whether available information is fully utilized. Our results show that humans make fully informed decisions even when uncertain.

Region-specific irradiation system with heavy-ion microbeam for active individuals of Caenorhabditis elegans.

Radiation may affect essential functions and behaviors such as locomotion, feeding, learning and memory. Although whole-body irradiation has been shown to reduce motility in the nematode Caenorhabditis elegans, the detailed mechanism responsible for this effect remains unknown. Targeted irradiation of the nerve ring responsible for sensory integration and information processing would allow us to determine whether the reduction of motility following whole-body irradiation reflects effects on the central nervous system or on the muscle cells themselves. We therefore addressed this issue using a collimating microbeam system. However, radiation targeting requires the animal to be immobilized, and previous studies have anesthetized animals to prevent their movement, thus making it impossible to assess their locomotion immediately after irradiation. We developed a method in which the animal was enclosed in a straight, microfluidic channel in a polydimethylsiloxane chip to inhibit free motion during irradiation, thus allowing locomotion to be observed immediately after irradiation. The head region (including the central nervous system), mid region around the intestine and uterus, and tail region were targeted independently. Each region was irradiated with 12 000 carbon ions (12C; 18.3 MeV/u; linear energy transfer = 106.4 keV/µm), corresponding to 500 Gy at a φ20 µm region. Motility was significantly decreased by whole-body irradiation, but not by irradiation of any of the individual regions, including the central nervous system. This suggests that radiation inhibits locomotion by a whole-body mechanism, potentially involving motoneurons and/or body-wall muscle cells, rather than affecting motor control via the central nervous system and the stimulation response.

The effect of directed photic stimulation of the pineal on experimental Parkinson's disease.

The role of the circadian system in Parkinson's disease (PD) is a topic of increasing scientific interest. This has emerged from recent studies demonstrating an altered response of PD patients to treatment in relation to the phase of the light/dark cycle and from other work defining the functional significance of melanocytes in PD: a cell type that the nigro-striatal dopamine (NSD) system and circadian system both contain. The present study was undertaken to determine the sensitivity of the pineal, as the final common pathway of the circadian system, to light delivered directly to the pineal via surgical implantation of LEDs. Direct photic stimulation of the pineal altered the course of experimental PD while anatomical controls receiving stimulation of the frontal cortex exhibited a negative impact on the course of recovery of these animals. These effects were closely linked to the phase of the light/dark cycle. The present results suggest that while pineal photoreceptors are regarded as vestigial, functional photo-reactivity of the pineal remains. It is inferred that melanocytes are the active cells responsible for the observed effect since they remain functionally intact in mammalian pineal even though pineal photoreceptors are functionally inert. Although the stimuli applied in the present study may be regarded as artificial this study demonstrates that brain parenchyma remains differentially reactive to direct light exposure and presents a novel mechanism in circadian structures that needs to be explored.

Molecular machines open cell membranes.

Beyond the more common chemical delivery strategies, several physical techniques are used to open the lipid bilayers of cellular membranes. These include using electric and magnetic fields, temperature, ultrasound or light to introduce compounds into cells, to release molecular species from cells or to selectively induce programmed cell death (apoptosis) or uncontrolled cell death (necrosis). More recently, molecular motors and switches that can change their conformation in a controlled manner in response to external stimuli have been used to produce mechanical actions on tissue for biomedical applications. Here we show that molecular machines can drill through cellular bilayers using their molecular-scale actuation, specifically nanomechanical action. Upon physical adsorption of the molecular motors onto lipid bilayers and subsequent activation of the motors using ultraviolet light, holes are drilled in the cell membranes. We designed molecular motors and complementary experimental protocols that use nanomechanical action to induce the diffusion of chemical species out of synthetic vesicles, to enhance the diffusion of traceable molecular machines into and within live cells, to induce necrosis and to introduce chemical species into live cells. We also show that, by using molecular machines that bear short peptide addends, nanomechanical action can selectively target specific cell-surface recognition sites. Beyond the in vitro applications demonstrated here, we expect that molecular machines could also be used in vivo, especially as their design progresses to allow two-photon, near-infrared and radio-frequency activation.

Beam angle evaluation to improve inter-fraction motion robustness in pelvic lymph node irradiation with proton therapy.

BACKGROUND: Proton therapy dose distributions are sensitive to range variations, e.g. arising from inter-fraction organ motion. The aim of this study was to evaluate the inter-fraction motion robustness of proton beams from different beam angles in irradiation of pelvic lymph nodes (LNs). MATERIAL AND METHODS: Planning CT (pCT) and multiple repeat CT (rCT) scans of 18 prostate cancer patients were used. Considering left and right LNs separately, the average water equivalent path length (WEPL) over all ray paths in the beams eye view of the LNs were calculated for all gantry/couch angle combinations across all rCTs versus the corresponding pCT. Single beam proton plans were optimized on the pCT for all gantry angles (0° couch) and were re-calculated on all rCTs for each respective patient. WEPL and dose parameters were extracted and a statistical clustering analysis performed to identify patient sub-populations in terms of patterns in which angles were robust. RESULTS: The WEPL analysis showed a general pattern of least variation for 0° couch beam angles where three minima were found across gantry angles for the left LNs and two for the right LNs. The clustering analysis identified three patient sub-groups for the left LNs and two groups for the right LNs. The dose calculations showed similar results as the WEPL variation, e.g. for the left LNs angles around 25°-35°, 100°-110°, and 160°-170° were consistently preferable for both target and organs at risk. CONCLUSIONS: Sub-populations of patients with similar patterns of WEPL variations across beam angles were identified. The most robust angles found for WEPL variations were also confirmed by the dose/volume analysis.

Impact of beam angle choice on pencil beam scanning breath-hold proton therapy for lung lesions.

INTRODUCTION: The breath-hold technique inter alia has been suggested to mitigate the detrimental effect of motion on pencil beam scanned (PBS) proton therapy dose distributions. The aim of this study was to evaluate the robustness of incident proton beam angles to day-to-day anatomical variations in breath-hold. MATERIALS AND METHODS: Single field PBS plans at five degrees increments in the transversal plane were made and water-equivalent path lengths (WEPLs) were derived on the planning breath-hold CT (BHCT) for 30 patients diagnosed with locally-advanced non-small cell lung cancer (NSCLC), early stage NSCLC or lung metastasis. Our treatment planning system was subsequently used to recalculate the plans and derive WEPL on a BHCT scan acquired at the end of the treatment. Changes to the V95%, D95 and mean target dose were evaluated. RESULTS: The difference in WEPL as a function of the beam angle was highly patient specific, with a median of 3.3 mm (range: 0.0-41.1 mm). Slightly larger WEPL differences were located around the lateral or lateral anterior/posterior beam angles. Linear models revealed that changes in dose were associated to the changes in WEPL and the tumor baseline shift (p < 0.05). CONCLUSIONS: WEPL changes and tumor baseline shift can serve as reasonable surrogates for dosimetric uncertainty of the target coverage and are well-suited for routine evaluation of plan robustness. The two lateral beam angles are not recommended to use for PBS proton therapy of lung cancer patients treated in breath-hold, due to the poor robustness for several of the patients evaluated.

Intrafractional baseline drift during free breathing breast cancer radiation therapy.

BACKGROUND: Intrafraction motion in breast cancer radiation therapy (BCRT) has not yet been thoroughly described in the literature. It has been observed that baseline drift occurs as part of the intrafraction motion. This study aims to measure baseline drift and its incidence in free-breathing BCRT patients using an in-house developed laser system for tracking the position of the sternum. MATERIALS AND METHODS: Baseline drift was monitored in 20 right-sided breast cancer patients receiving free breathing 3D-conformal RT by using an in-house developed laser system which measures one-dimensional distance in the AP direction. A total of 357 patient respiratory traces from treatment sessions were logged and analysed. Baseline drift was compared to patient positioning error measured from in-field portal imaging. RESULTS: The mean overall baseline drift at end of treatment sessions was -1.3 mm for the patient population. Relatively small baseline drift was observed during the first fraction; however it was clearly detected already at the second fraction. Over 90% of the baseline drift occurs during the first 3 min of each treatment session. The baseline drift rate for the population was -0.5 ± 0.2 mm/min in the posterior direction the first minute after localization. Only 4% of the treatment sessions had a 5 mm or larger baseline drift at 5 min, all towards the posterior direction. Mean baseline drift in the posterior direction in free breathing BCRT was observed in 18 of 20 patients over all treatment sessions. CONCLUSIONS: This study shows that there is a substantial baseline drift in free breathing BCRT patients. No clear baseline drift was observed during the first treatment session; however, baseline drift was markedly present at the rest of the sessions. Intrafraction motion due to baseline drift should be accounted for in margin calculations.

Dosimetric Analysis of Microscopic Disease in SBRT for Lung Cancers.

OBJECTIVE: The objective of this study is to theoretically and experimentally evaluate the dosimetry in the microscopic disease regions surrounding the tumor under stereotactic body radiation therapy of lung cancer. METHODS: For simplicity, the tumor was considered moving along 1 dimension with a periodic function. The probability distribution function of the tumor position was generated according to the motion pattern and was used to estimate the delivered dose in the microscopic disease region. An experimental measurement was conducted to validate both the estimated dose with a probability function and the calculated dose from 4-dimensional computed tomography data using a dynamic thorax phantom. Four tumor motion patterns were simulated with cos4(x) and sin(x), each with 2 different amplitudes: 10 mm and 5 mm. A 7-field conformal plan was created for treatment delivery. Both films (EBT2) and optically stimulated luminescence detectors were inserted in and around the target of the phantom to measure the delivered doses. Dose differences were evaluated using gamma analysis with 3%/3 mm. RESULTS: The average gamma index between measured doses using film and calculated doses using average intensity projection simulation computed tomography was 80.8% ± 0.9%. In contrast, between measured doses using film and calculated doses accumulated from 10 sets of 4-dimensional computed tomography data, it was 98.7% ± 0.6%. The measured doses using optically stimulated luminescence detectors matched very well (within 5% of the measurement uncertainty) with the theoretically calculated doses using probability distribution function at the corresponding position. Respiratory movement caused inadvertent irradiation exposure, with 70% to 80% of the dose line wrapped around the 10 mm region outside the target. CONCLUSION: The use of static dose calculation in the treatment planning system could substantially underestimate the actual delivered dose in the microscopic disease region for a moving target. The margin for microscopic disease may be substantially reduced or even eliminated for lung stereotactic body radiation therapy.