Browse
Submit Data
Databases
API
Help

Dataset Information

0 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Biosensor Cell-Fit-HD^4D for correlation of single-cell fate and microscale energy deposition in complex ion beams.

ABSTRACT: We present a protocol for the biosensor Cell-Fit-HD^4D. It enables long-term monitoring and correlation of single-cell fate with subcellular-deposited energy of ionizing radiation. Cell fate tracking using widefield time-lapse microscopy is uncoupled in time from confocal ion track imaging. Registration of both image acquisition steps allows precise ion track assignment to cells and correlation with cellular readouts. For complete details on the use and execution of this protocol, please refer to Niklas et al. (2022).

SUBMITTER: Schlegel J

PROVIDER: S-EPMC9627659 | biostudies-literature | 2022 Dec

REPOSITORIES: biostudies-literature

ACCESS DATA

Json Xml

Publications

Biosensor Cell-Fit-HD4D for correlation of single-cell fate and microscale energy deposition in complex ion beams.

Schlegel Julian J Liew Hans H Rein Katrin K Dzyubachyk Oleh O Debus Jürgen J Abdollahi Amir A Niklas Martin M

STAR protocols 20221029 4

We present a protocol for the biosensor Cell-Fit-HD4D. It enables long-term monitoring and correlation of single-cell fate with subcellular-deposited energy of ionizing radiation. Cell fate tracking using widefield time-lapse microscopy is uncoupled in time from confocal ion track imaging. Registration of both image acquisition steps allows precise ion track assignment to cells and correlation with cellular readouts. For complete details on the use and execution of this protocol, plea ...[more]

PMID: 36340882

Similar Datasets

Microscale frictional strains determine chondrocyte fate in loaded cartilage.

Project description:Mounting evidence suggests that altered lubricant levels within synovial fluid have acute biological consequences on chondrocyte homeostasis. While these responses have been connected to increased friction, the mechanisms behind this response remain unknown. Here, we combine a frictional bioreactor with confocal elastography and image-based cellular assays to establish the link between cartilage friction, microscale shear strain, and acute, adverse cellular responses. Our incorporation of cell-scale strain measurements reveals that elevated friction generates high shear strains localized near the tissue surface, and that these elevated strains are closely associated with mitochondrial dysfunction, apoptosis, and cell death. Collectively, our data establish two pathways by which chondrocytes negatively respond to friction: an immediate necrotic response and a longer term pathway involving mitochondrial dysfunction and apoptosis. Specifically, in the surface region, where shear strains can exceed 0.07, cells are predisposed to acute death; however, below this surface region, cells exhibit a pathway consistent with apoptosis in a manner predicted by local shear strains. These data reveal a mechanism through which cellular damage in cartilage arises from compromised lubrication and show that in addition to boundary lubricants, there are opportunities upstream of apoptosis to preserve chondrocyte health in arthritis therapy.

| S-EPMC6367731 | biostudies-literature

Microscale biosensor for measurement of volatile fatty acids in anoxic environments.

Project description:A microscale biosensor for acetate, propionate, isobutyrate, and lactate is described. The sensor is based on the bacterial respiration of low-molecular-weight, negatively charged species with a concomitant reduction of NO(-)(3) to N(2)O. A culture of denitrifying bacteria deficient in N(2)O reductase was immobilized in front of the tip of an electrochemical N(2)O microsensor. The bacteria were separated from the outside environment by an ion-permeable membrane and supplied with nutrients (except for electron donors) from a medium reservoir behind the N(2)O sensor. The signal of the sensor, which corresponded to the rate of N(2)O production, was proportional to the supply of the electron donor to the bacterial mass. The selectivity for volatile fatty acids compared to other organic compounds was increased by selectively enhancing the transport of negatively charged compounds into the sensor by electrophoretic migration (electrophoretic sensitivity control). The sensor was susceptible to interference from O(2), N(2)O, NO(2)(-), H(2)S, and NO(-)(3). Interference from NO(-)(3) was low and could be quantified and accounted for. The detection limit was equivalent to about 1 microM acetate, and the 90% response time was 30 to 90 s. The response of the sensor was not affected by changes in pH between 5.5 and 9 and was also unaffected by changes in salinity in the range of 2 to 32 per thousand. The functioning of the sensor over a temperature span of 7 to 30 degrees C was investigated. The concentration range for a linear response was increased five times by increasing the temperature from 7 to 19.5 degrees C. The life span of the biosensor varied between 1 and 3 weeks after manufacturing.

| S-EPMC123769 | biostudies-literature

Biosensor for deconvolution of individual cell fate in response to ion beam irradiation.

Project description:Clonogenic survival assay constitutes the gold standard method for quantifying radiobiological effects. However, it neglects cellular radiation response variability and heterogeneous energy deposition by ion beams on the microscopic scale. We introduce "Cell-Fit-HD4D" a biosensor that enables a deconvolution of individual cell fate in response to the microscopic energy deposition as visualized by optical microscopy. Cell-Fit-HD4D enables single-cell dosimetry in clinically relevant complex radiation fields by correlating microscopic beam parameters with biological endpoints. Decrypting the ion beam's energy deposition and molecular effects at the single-cell level has the potential to improve our understanding of radiobiological dose concepts as well as radiobiological study approaches in general.

| S-EPMC9017136 | biostudies-literature

A model for relative biological effectiveness of therapeutic proton beams based on a global fit of cell survival data.

Project description:We introduce an approach for global fitting of the recently published high-throughput and high accuracy clonogenic cell-survival data for therapeutic scanned proton beams. Our fitting procedure accounts for the correlation between the cell-survival, the absorbed (physical) dose and the proton linear energy transfer (LET). The fitting polynomials and constraints have been constructed upon generalization of the microdosimetric kinetic model (gMKM) adapted to account for the low energy and high lineal-energy spectrum of the beam where the current radiobiological models may underestimate the reported relative biological effectiveness (RBE). The parameters (α, β) of the linear-quadratic (LQ) model calculated by the presented method reveal a smooth transition from low to high LETs which is an advantage of the current method over methods previously employed to fit the same clonogenic data. Finally, the presented approach provides insight into underlying microscopic mechanisms which, with future study, may help to elucidate radiobiological responses along the Bragg curve and resolve discrepancies between experimental data and current RBE models.

| S-EPMC5567137 | biostudies-literature

Illuminate the hidden: in vivo mapping of microscale pH in the mycosphere using a novel whole-cell biosensor.

Project description:The pH of an environment is both a driver and the result of diversity and functioning of microbial habitats such as the area affected by fungal hyphae (mycosphere). Here we used a novel pH-sensitive bioreporter, Synechocystis sp. PCC6803_peripHlu, and ratiometric fluorescence microscopy, to spatially and temporally resolve the mycosphere pH at the micrometre scale. Hyphae of the basidiomycete Coprionopsis cinerea were allowed to overgrow immobilised and homogeneously embedded pH bioreporters in an agarose microcosm. Signals of >700 individual cells in an area of 0.4 × 0.8 mm were observed over time and used to create highly resolved (3 × 3 µm) pH maps using geostatistical approaches. C. cinerea changed the pH of the agarose from 6.9 to ca. 5.0 after 48 h with hyphal tips modifying pH in their vicinity up to 1.8 mm. pH mapping revealed distinct microscale spatial variability and temporally stable gradients between pH 4.4 and 5.8 over distances of ≈20 µm. This is the first in vivo mapping of a mycosphere pH landscape at the microscale. It underpins the previously hypothesised establishment of pH gradients serving to create spatially distinct mycosphere reaction zones.

| S-EPMC9723660 | biostudies-literature

Global H3.3 dynamic deposition defines its bimodal role in cell fate transition.

Project description:H3.3 is a histone variant, which is deposited on genebodies and regulatory elements, by Hira, marking active transcription. Moreover, H3.3 is deposited on heterochromatin by Atrx/Daxx complex. The exact role of H3.3 in cell fate transition remains elusive. Here, we investigate the dynamic changes in the deposition of the histone variant H3.3 during cellular reprogramming. H3.3 maintains the identities of the parental cells during reprogramming as its removal at early time-point enhances the efficiency of the process. We find that H3.3 plays a similar role in transdifferentiation to hematopoietic progenitors and neuronal differentiation from embryonic stem cells. Contrastingly, H3.3 deposition on genes associated with the newly reprogrammed lineage is essential as its depletion at the later phase abolishes the process. Mechanistically, H3.3 deposition by Hira, and its K4 and K36 modifications are central to the role of H3.3 in cell fate conversion. Finally, H3.3 safeguards fibroblast lineage by regulating Mapk cascade and collagen synthesis.

| S-EPMC5906632 | biostudies-literature

A Real-Time Biosensor for ERK Activity Reveals Signaling Dynamics during C. elegans Cell Fate Specification.

Project description:Kinase translocation reporters (KTRs) are genetically encoded fluorescent activity sensors that convert kinase activity into a nucleocytoplasmic shuttling equilibrium for visualizing single-cell signaling dynamics. Here, we adapt the first-generation KTR for extracellular signal-regulated kinase (ERK) to allow easy implementation in vivo. This sensor, "ERK-nKTR," allows quantitative and qualitative assessment of ERK activity by analysis of individual nuclei and faithfully reports ERK activity during development and neural function in diverse cell contexts in Caenorhabditis elegans. Analysis of ERK activity over time in the vulval precursor cells, a well-characterized paradigm of epidermal growth factor receptor (EGFR)-Ras-ERK signaling, has identified dynamic features not evident from analysis of developmental endpoints alone, including pulsatile frequency-modulated signaling associated with proximity to the EGF source. The toolkit described here will facilitate studies of ERK signaling in other C. elegans contexts, and the design features will enable implementation of this technology in other multicellular organisms.

| S-EPMC5595649 | biostudies-literature

Comparison of detector performance in small 6 MV and 6 MV FFF beams using a Versa HD accelerator.

Project description:1. background & purposeInvestigate the applicability of a series of detectors in small field dosimetry and the possible differences between their responses to FF and FFF beams. This work extends upon the series of detectors used by other authors to also include metal-oxide-semiconductor field-effect transistors (MOSFETs) detectors and radiochromic film. We also included a later correction of output factors (OFs) recommended by the recently published IAEA´s code of practice TRS 483 on dosimetry of small static fields used in external beam radiotherapy.2. materials & methodsThe OFs, profiles, and PDDs of 6 MV and 6 MV FFF beams were measured with 11 different detectors using field sizes between 0.6 × 0.6 cm2 and 10 × 10 cm2.3. resultsThe OFs of the FFF beams were lower than those of the FF beams for field sizes larger than 3 × 3 cm2 but higher for field sizes smaller than 3 × 3 cm2. After applying the IAEA´s TRS 483 corrections, the final OFs were compatible with our initial results when considering uncertainties involved. Small-volume detectors are preferable for measuring the penumbra of these small fields where this attribute is higher in the crossline direction than in the inline direction. The R100 of equivalent-quality FFF beams was higher compared to the corresponding flattened beams.4. conclusionsWe observed no difference for the dose responses between 6 MV and 6 MV FFF beams for any of the detectors. OF results, profiles and PDDs were clearly consistent with the previously published literature regarding the Versa HD linac. Correcting our first OFs, taken as ratio of detector charges, with the IAEA´s TRS 483 corrections to obtain the final OFs, did not make the former significantly different.

| S-EPMC6411166 | biostudies-literature

Dosimetric properties of equivalent-quality flattening filter-free (FFF) and flattened photon beams of Versa HD linear accelerator.

Project description:This study presents the basic dosimetric properties of photon beams of a Versa HD linear accelerator (linac), which is capable of delivering flattening filter-free (FFF) beams with a beam quality equivalent to the corresponding flattened beams based on comprehensive beam data measurement. The analyzed data included the PDDs, profiles, penumbra, out-of-field doses, surface doses, output factors, head and phantom scatter factors, and MLC transmissions for both FFF and flattened beams of 6 MV and 10 MV energy from an Elekta Versa HD linac. The 6MVFFF and 10MVFFF beams had an equivalent mean energy to the flattened beams and showed less PDD variations with the field sizes. Compared with their corresponding flattened beams, Dmax was deeper for FFF beams for all field sizes; the ionization ratio variations with the field size were lower for FFF beams; the out-of-field doses were lower and the penumbras were sharper for the FFF beams; the off-axis profile variations with the depths were lesser for the FFF beams. Further, the 6MVFFF and 10MVFFF beams had 35.7% and 40.9% less variations in output factor with the field size, respectively. The collimator exchange effect was reduced in the FFF mode. The head scatter factor showed 59.1% and 73.6% less variations, on average, for the 6MVFFF and 10MVFFF beams, respectively; the variations in the phantom scatter factor were also smaller. The surface doses for all beams increased linearly with the field size. The 6MVFFF and 10MVFFF beams had higher surface doses than the corresponding flattened beams for field sizes of up to 10 ×10cm2 but had lower surface doses for larger fields. Both FFF beams had lower average MLC transmissions than the flattened beams. The finding that the FFF beams were of equivalent quality to the corresponding flattened beams indicates a significant dif-ference from the data on unmatched FFF beams.

| S-EPMC5690903 | biostudies-literature

cBAF complex components and MYC cooperate early in CD8+ T cell fate.

Project description:The identification of mechanisms to promote memory T (Tmem) cells has important implications for vaccination and anti-cancer immunotherapy1-4. Using a CRISPR-based screen for negative regulators of Tmem cell generation in vivo5, here we identify multiple components of the mammalian canonical BRG1/BRM-associated factor (cBAF)6,7. Several components of the cBAF complex are essential for the differentiation of activated CD8+ T cells into T effector (Teff) cells, and their loss promotes Tmem cell formation in vivo. During the first division of activated CD8+ T cells, cBAF and MYC8 frequently co-assort asymmetrically to the two daughter cells. Daughter cells with high MYC and high cBAF display a cell fate trajectory towards Teff cells, whereas those with low MYC and low cBAF preferentially differentiate towards Tmem cells. The cBAF complex and MYC physically interact to establish the chromatin landscape in activated CD8+ T cells. Treatment of naive CD8+ T cells with a putative cBAF inhibitor during the first 48 h of activation, before the generation of chimeric antigen receptor T (CAR-T) cells, markedly improves efficacy in a mouse solid tumour model. Our results establish cBAF as a negative determinant of Tmem cell fate and suggest that manipulation of cBAF early in T cell differentiation can improve cancer immunotherapy.

| S-EPMC9623036 | biostudies-literature