Label‐free analysis of mononuclear human blood cells in microfluidic flow by coherent imaging tools

The investigation of the physical properties of peripheral blood mononuclear cells (PBMC) is of great relevance, as they play a key role in regulating human body health. Here we report the possibility to characterize human PBMC in their physiological conditions in a microfluidic‐based measurement system. A viscoelastic polymer solution is adopted for 3D alignment of individual cells inflow. An optical signature (OS) acquisition of each flowing cell is performed using a wide angle light scattering apparatus. Besides, a quantitative phase imaging (QPI) holographic system is employed with the aim (i) to check the position in flow of individual cells using a holographic 3D cell tracking method; and (ii) to estimate their 3D morphometric features, such as their refractive index (RI). Results obtained by combining OS and QPI have been compared with literature values, showing good agreement. The results confirm the possibility to obtain sub‐micrometric details of physical cell properties in microfluidic flow, avoiding chemical staining or fluorescent labelling.

     

Emerging single-cell technologies for functional proteomics in oncology

Introduction: Cellular heterogeneity has challenged current cancer therapeutics and hindered the discovery and development of cancer drugs. The heterogeneity in functional proteome is of particular interest because many cancer drugs are developed to target signaling proteins. The complex nature of tumor systems calls for more advanced multiplexed single-cell tools to address the heterogeneity issue.

Area covered: Over the past five years, there are a few single-cell functional proteomics tools introduced with unprecedented multiplexity and performance that are transforming the oncology field. Those tools are generally categorized as cytometry-based tools and microfluidics-based tools, and we discuss the representatives in both categories.

Expert commentary: The single-cell tools have provided an avenue to understand the multifaceted differences of cancer cells, the complex signaling networks, and the relationship of intercellular interaction and tumor architecture. We also provide an outlook of single-cell tools in five years and the challenges to address before a greater impact on oncology can be made.     

TipChip: a modular,MEMS‐based platform for experimentation and phenotyping of tip‐growing cells

Large‐scale phenotyping of tip‐growing cells such as pollen tubes has hitherto been limited to very crude parameters such as germination percentage and velocity of growth. To enable efficient and high‐throughput execution of more sophisticated assays, an experimental platform, the TipChip, was developed based on microfluidic and microelectromechanical systems (MEMS) technology. The device allows positioning of pollen grains or fungal spores at the entrances of serially arranged microchannels equipped with microscopic experimental set‐ups. The tip‐growing cells (pollen tubes, filamentous yeast or fungal hyphae) may be exposed to chemical gradients, microstructural features, integrated biosensors or directional triggers within the modular microchannels. The device is compatible with Nomarski optics and fluorescence microscopy. Using this platform, we were able to answer several outstanding questions on pollen tube growth. We established that, unlike root hairs and fungal hyphae, pollen tubes do not have a directional memory. Furthermore, pollen tubes were found to be able to elongate in air, raising the question of how and where water is taken up by the cell. The platform opens new avenues for more efficient experimentation and large‐scale phenotyping of tip‐growing cells under precisely controlled, reproducible conditions.     

基于图像识别技术的金针菇表型高通量采集与分析

金针菇Flammulina filiformis作为国内产销量大、工厂化生产程度高的食用菌种类之一,其育种工作因表型信息采集效率低下而受到限制.本研究以金针菇子实体可见光图像为信息来源,基于育种工作对表型信息的需求,利用图像识别技术和深度学习模型,提出了金针菇高通量表型信息采集分析方法,并开发了相应分析系统软件.利用该...     

Microchip technology applications for blood group analysis

Blood group analysis techniques are some of the most in demand immunological applications in clinical transfusion praxis and organ transplantation. In order to aid the advance towards higher throughput and increased sensitivity, analytical solutions dealing with a minimal amount of blood samples and the miniaturization of diagnostic equipment using microchip technologies have been evolving into an optimal solution. Here we review fabrication technologies for various types of microstructure on microchips, related operating procedures, and characterization approaches. Our focus is on examples of microchip technology and instrumentation used for blood group analysis ranging from classical serological methods of glycoprotein detection and solid phase assays, to nucleic acid amplification techniques. Molecular typing using microchip-based techniques is emerging as a supplement to standard serological methods. Microchip technology will play its key role to support blood group analysis at the molecular scale by using microliters of blood samples for extremely sensitive, quantitative, and high throughput analyses.     

Molecular phenotyping of aging in single yeast cells using a novel microfluidic device

Budding yeast has served as an important model organism for aging research, and previous genetic studies have led to the discovery of conserved genes/pathways that regulate lifespan across species. However, the molecular causes of aging and death remain elusive, because it is very difficult to directly observe the cellular and molecular events accompanying aging in single yeast cells by the traditional approach based on micromanipulation. We have developed a microfluidic system to track individual mother cells throughout their lifespan, allowing automated lifespan measurement and direct observation of cell cycle dynamics, cell/organelle morphologies, and various molecular markers. We found that aging of the wild-type cells is characterized by an increased general stress and a progressive lengthening of the cell cycle for the last few cell divisions; these features are much less apparent in the long-lived FOB1 deletion mutant. Following the fate of individual cells revealed that there are different forms of cell death that are characterized by different terminal cell morphologies, and associated with different levels of stress and lifespan. We have identified a molecular marker - the level of the expression of Hsp104, as a good predictor for the lifespan of individual cells. Our approach allows detailed molecular phenotyping of single cells in the process of aging and thus provides new insight into its mechanism.     

Dynamic functional and structural analysis of living cells: New tools for vital staining of nuclear DNA and for characterisation of cell motion

Increasing interest has been paid to applications of fluorescence measurements to analyze physiological mechanisms in living cells. However, few studies have taken advantage of DNA quantification by fluorometry for dynamic assessment of chromatin organization as well as cell motion during the cell cycle. This approach involves both optimal conditions for DNA staining and cell tracking methods. In this context, this report describes a stoichiometric method for nuclear DNA specific staining, using the bisbenzimidazole dye Hoechst 33342 associated with verapamil, a calcium membrane channel blocker. This method makes it possible to correlate variations of nuclear DNA content with cell motion in cells that are maintained alive. Motion measurement is the second goal of this paper and it explains the snake-spline method, and the associated cell following method.     

Comparative physiology and molecular analysis of carbonic anhydrase from the red blood cells of teleost fish

This study investigates the early evolution of vertebrate red blood cell (rbc) carbonic anhydrase (CA) by examining the physiological and molecular properties of rbc CA in teleost fish. When representatives of four different families of teleosts were compared, it was found that differences in overall rbc CA activity were due to different concentrations of CA, rather than differences in the enzymes kinetic properties. Additional molecular analysis of CA from the rbcs of rainbow trout provided further evidence that critical elements of the enzyme, such as the active site, have been highly conserved during vertebrate evolution. The active site of the trout CA differed from that of gar rbc CA at only two amino acid positions. The rainbow trout rbc CA sequence also showed high sequence homology with CA sequences from other fish tissues, and fits into an emerging group of fish CAs that are basal to mammalian CA I, II and III. Northern blot analysis of the tissue expression of the sequenced CA indicated that it is primarily found in the rbcs, but high amounts of cytosolic CA activity were also found in the gill, suggesting the presence of other cytosolic CA isozymes in this species.Abbreviations Az acetazolamide - CA carbonic anhydrase - MP maximum parsimony - NJ neighbour joining - RACE rapid amplification of cDNA ends - rbc red blood cellCommunicated by L.C.-H. Wang     

A simple method for the quantitative evaluation of functional effects of xenobiotics with cultured cells

We present a simple, noninvasive, nondestructive all-purpose method for the quantitative evaluation of functional effects of xenobiotics with cultured cells and the work station for its routine, easy implementation. At present 1 to 150 cells growing in one to six dishes can be studied in parallel or otherwise at time intervals ranging from 10 s to 6 h or more, over periods of time ranging from a few tens of minutes to 3–4 days. Any aspect of cell physiological behavior can be studied (differentiation-dedifferentiation, migration, division, degeneration, death) without preliminary staining and/or fixation provided it results in optically visible changes.     

Robust functional principal component analysis via a functional pairwise spatial sign operator

Functional principal component analysis (FPCA) has been widely used to capture major modes of variation and reduce dimensions in functional data analysis. However, standard FPCA based on the sample covariance estimator does not work well if the data exhibits heavy-tailedness or outliers. To address this challenge, a new robust FPCA approach based on a functional pairwise spatial sign (PASS) operator, termed PASS FPCA, is introduced. We propose robust estimation procedures for eigenfunctions and eigenvalues. Theoretical properties of the PASS operator are established, showing that it adopts the same eigenfunctions as the standard covariance operator and also allows recovering ratios between eigenvalues. We also extend the proposed procedure to handle functional data measured with noise. Compared to existing robust FPCA approaches, the proposed PASS FPCA requires weaker distributional assumptions to conserve the eigenspace of the covariance function. Specifically, existing work are often built upon a class of functional elliptical distributions, which requires inherently symmetry. In contrast, we introduce a class of distributions called the weakly functional coordinate symmetry (weakly FCS), which allows for severe asymmetry and is much more flexible than the functional elliptical distribution family. The robustness of the PASS FPCA is demonstrated via extensive simulation studies, especially its advantages in scenarios with nonelliptical distributions. The proposed method was motivated by and applied to analysis of accelerometry data from the Objective Physical Activity and Cardiovascular Health Study, a large-scale epidemiological study to investigate the relationship between objectively measured physical activity and cardiovascular health among older women.     

Emerging fluorescence tools for the study of proteostasis in cells

Understanding how cells maintain the functional proteome and respond to stress conditions is critical for deciphering molecular pathogenesis and developing treatments for conditions such as neurodegenerative diseases. Efforts towards finer quantification of cellular proteostasis machinery efficiency, phase transitions and local environment changes remain a priority. Herein, we describe recent developments in fluorescence-based strategy and methodology, building on the experimental toolkit, for the study of proteostasis (protein homeostasis) in cells. We hope this review can assist in bridging gaps between a multitude of research disciplines and promote interdisciplinary collaboration to address the crucial topic of proteostasis.     

Emerging tools for real-time label-free detection of interactions on functional protein microarrays

The availability of extensive genomic information and content has spawned an era of high-throughput screening that is generating large sets of functional genomic data. In particular, the need to understand the biochemical wiring within a cell has introduced novel approaches to map the intricate networks of biological interactions arising from the interactions of proteins. The current technologies for assaying protein interactions--yeast two-hybrid and immunoprecipitation with mass spectrometric detection--have met with considerable success. However, the parallel use of these approaches has identified only a small fraction of physiologically relevant interactions among proteins, neglecting all nonprotein interactions, such as with metabolites, lipids, DNA and small molecules. This highlights the need for further development of proteome scale technologies that enable the study of protein function. Here we discuss recent advances in high-throughput technologies for displaying proteins on functional protein microarrays and the real-time label-free detection of interactions using probes of the local index of refraction, carbon nanotubes and nanowires, or microelectromechanical systems cantilevers. The combination of these technologies will facilitate the large-scale study of protein interactions with proteins as well as with other biomolecules.     

High‐throughput physiological phenotyping and screening system for the characterization of plant–environment interactions

We present a simple and effective high‐throughput experimental platform for simultaneous and continuous monitoring of water relations in the soil–plant–atmosphere continuum of numerous plants under dynamic environmental conditions. This system provides a simultaneously measured, detailed physiological response profile for each plant in the array, over time periods ranging from a few minutes to the entire growing season, under normal, stress and recovery conditions and at any phenological stage. Three probes for each pot in the array and a specially designed algorithm enable detailed water‐relations characterization of whole‐plant transpiration, biomass gain, stomatal conductance and root flux. They also enable quantitative calculation of the whole plant water‐use efficiency and relative water content at high resolution under dynamic soil and atmospheric conditions. The system has no moving parts and can fit into many growing environments. A screening of 65 introgression lines of a wild tomato species (Solanum pennellii) crossed with cultivated tomato (S. lycopersicum), using our system and conventional gas‐exchange tools, confirmed the accuracy of the system as well as its diagnostic capabilities. The use of this high‐throughput diagnostic screening method is discussed in light of the gaps in our understanding of the genetic regulation of whole‐plant performance, particularly under abiotic stress.     

Simultaneous cellular and molecular phenotyping of embryonic mutants using single-cell regulatory trajectories

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Multiple functional linear model for association analysis of RNA-seq with imaging

Emerging integrative analysis of genomic and anatomical imaging data which has not been well developed, provides invaluable information for the holistic discovery of the genomic structure of disease and has the potential to open a new avenue for discovering novel disease susceptibility genes which cannot be identified if they are analyzed separately. A key issue to the success of imaging and genomic data analysis is how to reduce their dimensions. Most previous methods for imaging information extraction and RNA-seq data reduction do not explore imaging spatial information and often ignore gene expression variation at the genomic positional level. To overcome these limitations, we extend functional principle component analysis from one dimension to two dimensions (2DFPCA) for representing imaging data and develop a multiple functional linear model (MFLM) in which functional principal scores of images are taken as multiple quantitative traits and RNA-seq profile across a gene is taken as a function predictor for assessing the association of gene expression with images. The developed method has been applied to image and RNA-seq data of ovarian cancer and kidney renal clear cell carcinoma (KIRC) studies. We identified 24 and 84 genes whose expressions were associated with imaging variations in ovarian cancer and KIRC studies, respectively. Our results showed that many significantly associated genes with images were not differentially expressed, but revealed their morphological and metabolic functions. The results also demonstrated that the peaks of the estimated regression coefficient function in the MFLM often allowed the discovery of splicing sites and multiple isoforms of gene expressions.     

Advanced phenotyping offers opportunities for improved breeding of forage and turf species

Background and Aims

Advanced phenotyping, i.e. the application of automated, high-throughput methods to characterize plant architecture and performance, has the potential to accelerate breeding progress but is far from being routinely used in current breeding approaches. In forage and turf improvement programmes, in particular, where breeding populations and cultivars are characterized by high genetic diversity and substantial genotype × environment interactions, precise and efficient phenotyping is essential to meet future challenges imposed by climate change, growing demand and declining resources.

Scope

This review highlights recent achievements in the establishment of phenotyping tools and platforms. Some of these tools have originally been established in remote sensing, some in precision agriculture, while others are laboratory-based imaging procedures. They quantify plant colour, spectral reflection, chlorophyll-fluorescence, temperature and other properties, from which traits such as biomass, architecture, photosynthetic efficiency, stomatal aperture or stress resistance can be derived. Applications of these methods in the context of forage and turf breeding are discussed.

Conclusions

Progress in cutting-edge molecular breeding tools is beginning to be matched by progress in automated non-destructive imaging methods. Joint application of precise phenotyping machinery and molecular tools in optimized breeding schemes will improve forage and turf breeding in the near future and will thereby contribute to amended performance of managed grassland agroecosystems.     

Resistance to the Brownian movement of red blood cells on flat horizontal surfaces

The movements of red blood cells (RBC), suspended in plasma, on plastic, glass, rhodium metal plate, siliconized glass, and siliconized rhodium were recorded on cinéfilm and analyzed. Values for the drag coefficient were calculated, using Einstein's theory of Brownian movement, and compared with the theoretical Stokes' hydrodynamic drag. The difference between the computed and Stokes' values gave the frictional coefficient or resistance resulting from the interaction of the cells, with the test surface. Of the three uncoated test surfaces, plastic was found to have the least interaction with the RBC. The frictional coefficient for plastic was found to be 1.75×10⁻⁷ N s m⁻¹ compared with a value of 2.82×10⁻⁷ N s m⁻¹ for rhodium metal, which had the largest interaction. Upon siliconization of the test surfaces, the interaction decreased by 40%. Reduction in the pH of the suspending plasma increased the interaction between the cells and the uncoated test surfaces, but the pH effect of diminished when the surfaces were siliconized.     

Cytotoxic cell function and phenotypic analysis of peripheral blood mononuclear cells in cancer patients treated with low-dose interleukin-2 and mitomycin C

We previously found that the ability of peripheral blood mononuclear cells (PBM) of cancer patients to generate lymphokine-activated killer (LAK) cells became remarkably augmented after mitomycin C administration. On the basis of the clinical finding, we designed a treatment regimen comprised of 12 mg/m² mitomycin C i. v. on day 1 and 700 U/m² recombinant interleukin-2 (IL-2) i.v. every 12 h from day 4 through day 8. Of 25 patients with advanced carcinoma, 9 had a partial response and 3 had a minor response. Cytotoxic cell function, including natural killer activity, lymphokine-activated killer (LAK) activity, and the ability to generate LAK cells, and lymphocyte subsets in PBM was measured 1 day before and after either the first or second course of this therapy. The relationship between these parameters and the clinical antitumor response to this treatment was examined. Although the cytotoxic activities were significantly augmented after either the first or second treatment course, no positive correlation was observed between the changes in these cytotoxic activities and the clinical response to this therapy, when patients who either showed a partial response or whose disease remission was partial or minor were defined as responders. Further, phenotypic analysis showed a significant increase in CD2⁺, CD3⁺ CD4⁺ and CD4⁺Leu8^– cells after the firs course, and CD25⁺ cells after either the first or second course of this treatment. The precentages of CD2⁺ and CD25⁺ cells were significantly elevated only in responders but not in nonresponders, suggesting the increase in these subsets was related to clinical response.