The analysis of in situ single cells in tissue is crucial for the study of tumors. However, simultaneous analysis of multiple biomolecules for single cells by fluorescence immunohistochemistry is ...challenging. Spontaneous Raman spectroscopy provides biochemical fingerprints of individual cells in a label-free and nondestructive manner, yet it is not well applied to single cells in tissue. Here, we develop an in situ single-cell spontaneous Raman spectroscopy (SRS) system that can localize a single cell in tissue with fluorescence imaging, while obtaining the single-cell Raman spectra for molecular analysis. Our system is verified by measuring the standard polystyrene beads of different sizes. Measurements of in situ single macrophages in clinical cancerous and para-cancerous tissues are then performed. By using discriminant analysis, it is shown that CD68+ macrophages in cancerous and para-cancerous tissues are classified with an accuracy of 98.3 %. The molecular content of single macrophages in tissue is analyzed by using multivariate curve resolution-alternating least squares method. We find that the lipid content of tumor-associated macrophages in cancerous tissues is higher while both the contents of protein and nucleic acid are lower than those in para-cancerous tissues, which is challenging to achieve by conventional immunohistochemistry. Our in situ single-cell SRS system has the capability for the obtaining of molecular information of a localized single cell in tissue, which may find potential applications for clinical cancer diagnosis.
•An in situ single-cell spontaneous Raman spectroscopy (SRS) system is developed.•In situ single-cell SRS localizes a single cell in tissue, and obtains its Raman spectra for molecular analysis.•Single CD68+ macrophages in liver cancerous and para-cancerous tissues are classified.•Various molecular substances of single CD68+ macrophages in liver cancerous and para-cancerous tissues are analyzed.
Channel models are important tools to evaluate the performance of new concepts in mobile communications. However, there is a tradeoff between complexity and accuracy. In this paper, we extend the ...popular Wireless World Initiative for New Radio (WINNER) channel model with new features to make it as realistic as possible. Our approach enables more realistic evaluation results at an early stage of algorithm development. The new model supports 3-D propagation, 3-D antenna patterns, time evolving channel traces of arbitrary length, scenario transitions and variable terminal speeds. We validated the model by measurements in a coherent LTE advanced testbed in downtown Berlin, Germany. We then reproduced the same scenario in the model and compared several channel parameters (delay spread, path gain, K-factor, geometry factor and capacity). The results match very well and we can accurately predict the performance for an urban macro-cell setup with commercial high-gain antennas. At the same time, the computational complexity does not increase significantly and we can use all existing WINNER parameter tables. These artificial channels, having equivalent characteristics as measured data, enable virtual field trials long before prototypes are available.
The understanding of biomolecular function is coupled to knowledge about the structure and dynamics of these biomolecules, preferably acquired under native conditions. In this regard, pulsed dipolar ...EPR spectroscopy (PDS) in conjunction with site‐directed spin labeling (SDSL) is an important method in the toolbox of biophysical chemistry. However, the currently available spin labels have diverse deficiencies for in‐cell applications, for example, low radical stability or long bioconjugation linkers. In this work, a synthesis strategy is introduced for the derivatization of trityl radicals with a maleimide‐functionalized methylene group. The resulting trityl spin label, called SLIM, yields narrow distance distributions, enables highly sensitive distance measurements down to concentrations of 90 nm, and shows high stability against reduction. Using this label, the guanine‐nucleotide dissociation inhibitor (GDI) domain of Yersinia outer protein O (YopO) is shown to change its conformation within eukaryotic cells.
SLIM fit: A trityl label with a short linker and high reduction stability, called SLIM, was synthesized. It enables highly sensitive pulsed dipolar EPR measurements down to low nanomolar concentrations and yields narrow distance distributions. Its exemplary use in in‐cell measurements showed that YopO preferably adopts one of the two conformations found in vitro.
During development, morphogens provide extracellular cues allowing cells to select a specific fate by inducing complex transcriptional programs. The mating pathway in budding yeast offers simplified ...settings to understand this process. Pheromone secreted by the mating partner triggers the activity of a MAPK pathway, which results in the expression of hundreds of genes. Using a dynamic expression reporter, we quantified the kinetics of gene expression in single cells upon exogenous pheromone stimulation and in the physiological context of mating. In both conditions, we observed striking differences in the timing of induction of mating‐responsive promoters. Biochemical analyses and generation of synthetic promoter variants demonstrated how the interplay between transcription factor binding and nucleosomes contributes to determine the kinetics of transcription in a simplified cell‐fate decision system.
Synopsis
Quantitative and dynamic single cell measurements uncover a complex temporal orchestration of gene expression events in the yeast mating response. The promoter architecture controls the timing of gene expression relative to the time of fusion.
Exogenous stimulations of yeast cells with pheromone have allowed to classify mating promoters in three categories, based on their gene expression dynamics.
The number and affinity of transcription factor binding sites in nucleosome depleted regions regulate the timing of gene induction.
In presence of mating partners, early genes are expressed during the sensing phase, while late genes are induced shortly before fusion.
Quantitative and dynamic single cell measurements uncover a complex temporal orchestration of gene expression events in the yeast mating response. The promoter architecture controls the timing of gene expression relative to the time of fusion.
Sudden stress often triggers diverse, temporally structured gene expression responses in microbes, but it is largely unknown how variable in time such responses are and if genes respond in the same ...temporal order in every single cell. Here, we quantified timing variability of individual promoters responding to sublethal antibiotic stress using fluorescent reporters, microfluidics, and time‐lapse microscopy. We identified lower and upper bounds that put definite constraints on timing variability, which varies strongly among promoters and conditions. Timing variability can be interpreted using results from statistical kinetics, which enable us to estimate the number of rate‐limiting molecular steps underlying different responses. We found that just a few critical steps control some responses while others rely on dozens of steps. To probe connections between different stress responses, we then tracked the temporal order and response time correlations of promoter pairs in individual cells. Our results support that, when bacteria are exposed to the antibiotic nitrofurantoin, the ensuing oxidative stress and SOS responses are part of the same causal chain of molecular events. In contrast, under trimethoprim, the acid stress response and the SOS response are part of different chains of events running in parallel. Our approach reveals fundamental constraints on gene expression timing and provides new insights into the molecular events that underlie the timing of stress responses.
Synopsis
Single‐cell measurements of individual promoter timing variability and the temporal order of gene expression in response to antibiotic stress in Escherichia coli reveal connections between promoters and provide a detailed view into the temporal organization of stress responses.
Cell‐cell variability in the timing of gene expression is quantified for dozens of E. coli promoters upon sudden exposure to different antibiotics.
Timing variability increases with response time and has clear lower and upper bounds, which are interpreted using statistical kinetics.
A dual‐reporter approach reveals the temporal order of responses at the single‐cell level, corroborating or refuting mechanistic couplings between different cellular response systems.
Single‐cell measurements of individual promoter timing variability and the temporal order of gene expression in response to antibiotic stress in Escherichia coli reveal connections between promoters and provide a detailed view into the temporal organization of stress responses.
CdS thin films have been grown on glass substrate at 250o C employing pulsed laser deposition method. The effect of laser pulses number on the crystalline structure, surface morphology, optical ...properties, and films thickness have been studied. XRD analysis shows that the CdS films have polycrystalline and hexagonal nanostructure with three notable peaks along (100), (002), and (101) planes and preferentially orientated along (101). The crystallite size of the preferred orientation was in the range of (21.4 - 27.3 nm). With small pulses number, XRD pattern confirms the formation of CdO with three peaks (111), (200), and (220). Theses peaks gradually reduce with the increasing of the pulses. The absorbance of the films is in the visible part of the spectrum. The band gap of the synthesized films reduces by rising the number of laser pulses. AFM studies indicate that the grain size and surface roughness increase with the film thickness. Due to the good crystalline structure and optical properties of the film of the highest thickness, it has been grown on a wafer silicon substrate for solar cell applications measurements. Hall measurements indicate low resistivity of 0.3×10-2 (Ω.m) and high conductivity of 3.3×10+2 (Ω.m)-1. The efficiency of the n-CdS/ p-Si junction has been calculated to be 3.4 % using I-V characteristic measurement. Keywords: pulsed laser, thin films, structural, optical, morphology, solar cell measurements
The complexity of the cellular medium can affect proteins’ properties, and, therefore, in-cell characterization of proteins is essential. We explored the stability and conformation of the first ...baculoviral IAP repeat (BIR) domain of X chromosome-linked inhibitor of apoptosis (XIAP), BIR1, as a model for a homodimer protein in human HeLa cells. We employed double electron–electron resonance (DEER) spectroscopy and labeling with redox stable and rigid Gd3+ spin labels at three representative protein residues, C12 (flexible region), E22C, and N28C (part of helical residues 26 to 31) in the N-terminal region. In contrast to predictions by excluded-volume crowding theory, the dimer–monomer dissociation constant KD was markedly higher in cells than in solution and dilute cell lysate. As expected, this increase was partially recapitulated under conditions of high salt concentrations, given that conserved salt bridges at the dimer interface are critically required for association. Unexpectedly, however, also the addition of the crowding agent Ficoll destabilized the dimer while the addition of bovine serum albumin (BSA) and lysozyme, often used to represent interaction with charged macromolecules, had no effect. Our results highlight the potential of DEER for in-cell study of proteins as well as the complexities of the effects of the cellular milieu on protein structures and stability.
The understanding of biomolecular function is coupled to knowledge about the structure and dynamics of these biomolecules, preferably acquired under native conditions. In this regard, pulsed dipolar ...EPR spectroscopy (PDS) in conjunction with site‐directed spin labeling (SDSL) is an important method in the toolbox of biophysical chemistry. However, the currently available spin labels have diverse deficiencies for in‐cell applications, for example, low radical stability or long bioconjugation linkers. In this work, a synthesis strategy is introduced for the derivatization of trityl radicals with a maleimide‐functionalized methylene group. The resulting trityl spin label, called SLIM, yields narrow distance distributions, enables highly sensitive distance measurements down to concentrations of 90 nm, and shows high stability against reduction. Using this label, the guanine‐nucleotide dissociation inhibitor (GDI) domain of Yersinia outer protein O (YopO) is shown to change its conformation within eukaryotic cells.
SLIM fit: A trityl label with a short linker and high reduction stability, called SLIM, was synthesized. It enables highly sensitive pulsed dipolar EPR measurements down to low nanomolar concentrations and yields narrow distance distributions. Its exemplary use in in‐cell measurements showed that YopO preferably adopts one of the two conformations found in vitro.
Microbial growth and division are fundamental processes relevant to many areas of life science. Of particular interest are homeostasis mechanisms, which buffer growth and division from accumulating ...fluctuations over multiple cycles. These mechanisms operate within single cells, possibly extending over several division cycles. However, all experimental studies to date have relied onmeasurements pooled from many distinct cells. Here, we disentangle long-term measured traces of individual cells from one another, revealing subtle differences between temporal and pooled statistics. By analyzing correlations along up to hundreds of generations, we find that the parameter describing effective cell size homeostasis strength varies significantly among cells. At the same time, we find an invariant cell size, which acts as an attractor to all individual traces, albeit with different effective attractive forces. Despite the common attractor, each cell maintains a distinct average size over its finite lifetime with suppressed temporal fluctuations around it, and equilibration to the global average size is surprisingly slow (>150 cell cycles). To show a possible source of variable homeostasis strength, we construct a mathematical model relying on intracellular interactions, which integrates measured properties of cell size with those of highly expressed proteins. Effective homeostasis strength is then influenced by interactions and by noise levels and generally varies among cells. A predictable and measurable consequence of variable homeostasis strength appears as distinct oscillatory patterns in cell size and protein content over many generations. We discuss implications of our results to understanding mechanisms controlling division in single cells and their characteristic timescales.
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