우리는 결실 협력하고 도움 토론 앤드류 캐롤, 밝은 Chaibang, Purbasha 사카르 (에너지 Biosciences 연구소, 버클리), 바람 파빈 (로렌스 버클리 국립 연구소)와 빈센트 L. 치앙 (노스캐롤라이나 주립 대학)을 주셔서 감사합니다. 이 작품은 에너지 Biosciences 연구소에 의해 지원되었다. 분자 파운드리에서 근무가 계약 번호 DE - AC02 - 05CH1123에 따라 에너지의 미국학과의 과학 오피스, 기본 에너지 과학 사무소에 의해 지원되었다.

1. 샘플 준비 <ol><li> 마이크로톰에 수산화 식물 표본, 예를 들어 포플러 나무의 줄기 또는 Arabidopsis thaliana의 줄기를 탑재합니다. </li><li> 기본 조직의 얇은 부분을 (일반적으로 20 μm의 두께) 그만둬. </li><li> 유리 현미경 슬라이드에 공장 섹션을 전송합니다. </li><li> D 2 O의 공장 섹션을 만끽하고 D 2 O.의 증발을 방지하기 위해 현미경 슬라이드에 밀폐되어 유리 커버 슬립과 커버 공장 부분은 이제 이미지 준비 또는 그것은 나중에 사용하기 위해 저장할 수 있습니다. </li></ol> 2. 샘플 측정 <ol><li> 현미경 목표 및 / 또는 커버 슬립에 침지 기름을 적용합니다. </li><li> 장소 및 현미경 목표가 직면하고있는 커버 슬립과 현미경의 압전 스캔 단계에서 현미경 슬라이드를 확보. </li><li> 높은 수치 조리개 침지 현미경 목표를 사용하여 커버 슬립 (100x, NA = 1.40)를 통해 샘플을보고 관심 샘플 영역을 찾습니다. </li><li> 다른 모든 실험실과 현미경 광원을 전환 후, 위치 - 해결 microspectroscopic 측정이 10-30 MW의 전형적인 전원 (과 샘플에 CW - 레이저에서 밴드 - 필터링 단색 녹색 빛을 (λ = 532 nm의) 초점을 맞춤으로써 수행됩니다 ) 설치의 구조에 대한 그림 1을 참조하십시오. Autofluorescence 오래 파장 레이저 빛이 케이스 여기는 것이 좋습니다 수있는 유용한 측정을 금지할 수있는 몇 가지 샘플에 발생할 수 있습니다. </li><li> 백 흩어져 스톡 - 이동 래맨 빛이 현미경의 목적에 의해 수집, 공촛점 설정에서 공간적 필터 역할을하는 이색성 거울, 핀홀, 그리고 longpass 필터를 통해 전달하고, 격자의 슬릿에 중점을두고 있습니다 빛이 spectrally 분산 및 래맨 스펙트럼을 제공, 냉각 CCD 카메라에 의해 감지 분광계. 포플러 나무 래맨 스펙트럼은 1600과 1천7백센티미터 -1 사이의 스펙트럼 영역의 특성 리그닌 밴드, 그림 2에 표시됩니다. </li><li> 공간 리그닌 분포의 화학 이미징 및 시각화 들어, 2 차원 스펙트럼지도 래스터는 압전 스캔 무대와 레이저 초점을 통해 샘플을 검사하고 각 샘플의 위치에 대한 래맨 스펙트럼을 기록하여 인수합니다. 입체 스펙트럼지도 레이저 초점이 Z - 방향을 따라 연속적으로 강화되었다있는 2 차원지도를 스태킹에 의해 생성된 수 있습니다. </li></ol> 3. 데이터 분석 <ol><li> 화학 이미징 및 리그닌 시각화 경우, 수집된 데이터가 MATLAB를 (MathWorks, 버전 7.7)를 사용하여 분석합니다. 데이터는 두 공간 크기와 스펙트럼 신호를위한 3 차원으로 구성되어 있습니다 3 차원 hyperspectral 큐브에 배치됩니다. </li><li> 리그닌의 분석을 위해, 1550 및 1,700cm -1 사이의 스펙트럼 영역은 (그림 2 참조)으로 간주됩니다. 리그닌의 공간적 분포 1550에서 기준 - 수정 스펙트럼 (그림 3 참조) -1 1,700cm에 강도를 통합하여 시각입니다. 기본 수정의 대안으로, 두 번째 유도체 스펙트럼 계산하고 두 번째 파생 봉우리는 분석을 위해 사용될 수 있습니다. </li><li> . 특히 coniferaldehyde 및 coniferyl 알코올 moieties에 관한 리그닌 현지화 및 화학은, 더 (그림 2 및 심판 15 삽입된 페이지보고 1600 및 1천7백cm -1 사이의 발견 세 밴드의 장착할 가우스 봉우리 아래에있는 영역을 평가하여 분석 수 있습니다 - 17). </li><li> 다른 분광지도 사이의 강도 정규화는 참조 클러스터링 분류 K는 -을 통해 얻을 수있는 평균 루멘 스펙트럼에서 2500 cm -1 주위에 불순물 OD 스트레칭 밴드의 최고 높이로 사용하여 수행됩니다. 이것은 중요 한가 다른 측정, 티슈, 샘플과 종 사이에 리그닌 신호 강도를 비교할 수 있습니다. </li></ol> 4. 대표 결과 포플러 (포풀러스 angustifolia) 줄기 나무 대표 래맨 스펙트럼은 그림 2에 표시됩니다. 특색 리그닌 밴드는 1600과 1,700cm -1 사이의 스펙트럼 영역에서 찾을 수 있습니다. 예를 들어, 포플러 나무 단면에서 리그닌의 공간적 분포는 그림 3에 표시됩니다. 보이는 이미지에 비해, morphologically 별개의 세포 벽 지역 인해 다른 리그닌 신호 강도에 명확히 구별할됩니다. 높은 리그닌 신호 강도는 복합 중간 lamellae (CML)에, 다소 적은, 세포 코너 (CC)에 준수하고 있습니다. 리그닌의 낮은, 아직 실질이없는없는 금액은 섬유의 S2 벽 계층 내에서 관찰됩니다. 리그닌 신호 강도의 변화는 특히 섬유로 섬유, CC, CML과 S2 사이 어느 정도 발견된다. 우리가 측정의 측면 공간 해상도가 ~ 300 NM입니다. 데이터 품질 사이 lignification을 비교 잘라는 것으로추가 리그닌 화학 15 해부하다하는 샘플합니다. <img src="/files/ftp_upload/2064/2064fig1.jpg" alt="그림 1" /> 그림 1 : 기계 설치의 구조 BP :. 대역 통과 필터, DM : 이색성 거울, PH : 핀홀, LP : longpass 필터. <img src="/files/ftp_upload/2064/2064fig2.jpg" alt="그림 2" /> 그림 2 : D 2 O에 기록된 포플러 (포풀러스 angustifolia) 줄기 나무 대표 래맨 스펙트럼. 강조 분광 영역 (또한 삽입된 페이지 참조) 리그닌 세 봉우리가 구체적으로 귀책 가진 스펙트럼 영역을 표시합니다. <img src="/files/ftp_upload/2064/2064fig3.jpg" alt="그림 3" /> 그림 3 : 1550에 1천7백cm -1에서 래맨 ​​신호 강도를 통합하여 얻은, : 포플러 나무 단면의 래맨 리그닌 이미지 (아래) (보이는 이미지 상단).

<ol>
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관심 없음 충돌 선언하지 않습니다.

리그노셀룰로오스성 자료는 계층적 구조 및 구성 모두에 관한 이기종입니다. 화학 감도, 공간 해상도, 그것을 깊이 특성 분석 도구에 대한 기본 컨텍스트에서 해당 콘텐츠를 통찰력 바람직한된다주세요. 설명한 방법은 리그닌, 자연 상태로 가까이에 얼룩이나 샘플의 라벨없이 하위 μm의 공간입니다 해상도 리그노셀룰로오스성 식물 바이오 매스의 lignification 비교의 시각을 내실수가 있었죠. 그것...

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<td>microscope slides</td>
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<td>D2O</td>
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<td>tweezers</td>
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<td>confocal Raman microscope</td>
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label-free in situ imaging of lignification in plant cell walls

회의 성장 에너지를 안전하게 요구하고 효율적으로 눌러 글로벌 도전입니다. 따라서, 비용 효과적이고 지속 가능한 해결책을 찾기 위해 추구하고 바이오 연료 생산에 대한 연구는 시사하고 중요한 작업이되고있다. 리그노셀룰로오스성 바이오 매스는 액체 바이오 연료 1-6로 변환을 위해 바이오 매스의 주요 원천이 될 태세이다. 그러나, 비용 효과적이고 효율 저하 이러한 식물 세포 벽 재료의 말을 안들음는 바이오 연료와 화학 물질 4의 생산에서 사용하기위한 주요 장애를 제공합니다. 특히, 리그닌, 복잡하고 불규칙적인 쓰임새 phenylpropanoid heteropolymer는 리그노셀룰로오스성 바이오 매스의 postharvest 해체에 문제가된다. 바이오 연료를위한 바이오 매스 전환 예를 들어, 발효 7 간단한 설탕 생산하기위한 프로세스에서 당화을 억제. 산업 목적으로 식물 바이오 매스의 효과적인 사용은 식물 세포 벽이 lignified되는 범위에 크게 의존 사실이다. 리그닌의 제거 비용과 제한 요인 8 리그닌 따라서 세포 벽 변환을 개선하기 위해 주요 식물 육종 및 유전 공학의 대상이 될 가지고있다. 식물 세포 벽의 lignification의 정확한 빠른 특성을 허용 분석 도구는 사육 인구의 다수를 평가하기위한 점점 중요한된다. 같은 리그닌과 같은 ​​기본 구성 요소의 분리를위한 추출 절차는 크게 화학 및 구조 변경에 대해 9-11 데리고, 불가피하게 파괴하고 있습니다. 원위치 방법으로 분석 화학 따라서 리그노셀룰로오스성 물질의 구성 및 구조 특성에 대한 소중한 도구입니다. 래맨 현미경은 레이저 광자의 에너지에 변화가 분자 진동과 관련되는 레이저에서 그런식으로, 단색 광의 탄력이나 래맨 산란에 의존하고 시료의 고유 라벨 무료 분자 &quot;지문&quot;을 제시하는 기술입니다 . 래맨 현미경 원래 상태로 가까이에있는 화학 성분과 분자 구조에 대한 통찰력을주는 최소한의 샘플 준비 비 파괴적이고 비교적 저렴​​한 측정을 여유가 있습니다. 공촛점 래맨 현미경으로 화학 이미징는 이전에 다음 ID로 셀룰로오스와 나무 세포 벽 12-14에서 리그닌의 공간적 분포의 시각화에 사용되고 있습니다. 이러한 이전 결과를 바탕으로, 우리는 최근 야생 유형 및 리그닌 - 결함 유전자 변형 포풀러스 trichocarpa (블랙 커튼 우드) 줄기 나무 15 lignification을 비교하는이 방법을 채택했습니다. 1600 및 1,700cm -1, 리그닌 신호 강도와 현지화 사이의 스펙트럼 영역에서 리그닌 래맨 밴드에게 16,17 분석은 현장에 매핑되었습니다. 우리의 접근 방식은 리그닌 함량의 차이, 지역화, 그리고 화학 성분을 시각. 최근, 우리는 18 하위 μm의 해상도는 가로 Arabidopsis thaliana의 세포 벽 고분자의 래맨 이미지를 보여주었다. 여기,이 방법은 얼룩이나 조직의 상표없이 식물 세포 벽에 리그닌의 시각화 및 다른 조직, 표본 또는 수종의 lignification의 비교를 affording 제공됩니다.

Watch this Scientific Journal Video about Label-free in situ Imaging of Lignification in Plant Cell Walls at JoVE.com

Label-free in situ Imaging of Lignification in Plant Cell Walls

공촛점 래맨 현미경에 따라 방법은 레이블이없는 식물 세포 벽에 리그닌의 시각화 및 다른 조직, 표본 또는 수종의 lignification의 비교를 내실수 것을 제공됩니다.

라벨 - 무료 현장에서</em식물 세포 벽 Lignification의> 이미징

Meeting growing energy demands safely and efficiently is a pressing global challenge. Therefore, research into biofuels production that seeks to find ...

label-free-in-situ-imaging-of-lignification-in-plant-cell-walls

Research

JoVE Journal

Biology

Label-free in situ Imaging of Lignification in Plant Cell Walls

12.3K 조회수.  University of California, Berkeley. 공촛점 래맨 현미경에 따라 방법은 레이블이없는 식물 세포 벽에 리그닌의 시각화 및 다른 조직, 표본 또는 수종의 lignification의 비교를 내실수 것을 제공됩니다.

비디오: Label-free in situ Imaging of Lignification in Plant Cell Walls

In situ Imaging of the Mouse Thymus Using 2-Photon Microscopy

Two-photon Microscopy (TPM) enables us to image deep into the thymus and document the events that are important for thymocyte development. To follow the migration of individuals in a crowd of thymocytes , we generate neonatal chimeras where less than one percent of the thymocytes are derived from a donor that is transgenic for a ubiquitously express fluorescent protein. To generate these partial hematopoetic chimeras, neonatal recipients are injected with bone marrow between 3-7 days of age. After 4-6 weeks, the mouse is sacrificed and the thymus is carefully dissected and bissected preserving the architecture of the tissue that will be imaged. The thymus is glued onto a coverslip in preparation for ex vivo imaging by TPM. During imaging the thymus is kept in DMEM without phenol red that is perfused with 95% oxygen and 5% carbon dioxide and warmed to 37&deg;C. Using this approach, we can study the events required for the generation of a diverse T cell repertoire.

We present step-by-step instructions for the generation of neonatal chimeras as well as the dissection and preparation of the thymus for ex vivo imaging by 2-Photon Microscopy.

2 광자 현미경을 사용하여 마우스 Thymus의 현장 영상에서

Two-photon Microscopy (TPM) enables us to image deep into the thymus and document the events that are important for thymocyte development.  To follow the ...

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생물학

Comprehensive Compositional Analysis of Plant Cell Walls (Lignocellulosic biomass) Part I: Lignin

The need for renewable, carbon neutral, and sustainable raw materials for industry and society has become one of the most pressing issues for the 21st century. This has rekindled interest in the use of plant products as industrial raw materials for the production of liquid fuels for transportation1 and other products such as biocomposite materials7. Plant biomass remains one of the greatest untapped reserves on the planet4. It is mostly comprised of cell walls that are composed of energy rich polymers including cellulose, various hemicelluloses (matrix polysaccharides, and the polyphenol lignin6 and thus sometimes termed lignocellulosics. However, plant cell walls have evolved to be recalcitrant to degradation as walls provide tensile strength to cells and the entire plants, ward off pathogens, and allow water to be transported throughout the plant; in the case of trees up to more the 100 m above ground level. Due to the various functions of walls, there is an immense structural diversity within the walls of different plant species and cell types within a single plant4. Hence, depending of what crop species, crop variety, or plant tissue is used for a biorefinery, the processing steps for depolymerization by chemical/enzymatic processes and subsequent fermentation of the various sugars to liquid biofuels need to be adjusted and optimized. This fact underpins the need for a thorough characterization of plant biomass feedstocks. Here we describe a comprehensive analytical methodology that enables the determination of the composition of lignocellulosics and is amenable to a medium to high-throughput analysis. In this first part we focus on the analysis of the polyphenol lignin (Figure 1). The method starts of with preparing destarched cell wall material. The resulting lignocellulosics are then split up to determine its lignin content by acetylbromide solubilization3, and its lignin composition in terms of its syringyl, guaiacyl- and p-hydroxyphenyl units5. The protocol for analyzing the carbohydrates in lignocellulosic biomass including cellulose content and matrix polysaccharide composition is discussed in Part II2.

Comprehensive Compositional Analysis of Plant Cell Walls Lignocellulosic biomass Part I: Lignin

Plant biomass is a major carbon-neutral renewable resource that could be used for the production of biofuels. Plant biomass consists mainly of cell walls, a structurally complex composite material termed lignocellulosics. Here we describe a protocol for a comprehensive analysis of the content and composition of the polyphenolic lignin.

식물 세포 벽의 종합 작곡 분석 (리그노셀룰로오스성 바이오 매스) 부 I : 리그닌

The need for renewable, carbon neutral, and sustainable raw materials for industry and society has become one of the most pressing issues for the 21st ...

Comprehensive Compositional Analysis of Plant Cell Walls (Lignocellulosic biomass) Part II: Carbohydrates

The need for renewable, carbon neutral, and sustainable raw materials for industry and society has become one of the most pressing issues for the 21st century. This has rekindled interest in the use of plant products as industrial raw materials for the production of liquid fuels for transportation2 and other products such as biocomposite materials6. Plant biomass remains one of the greatest untapped reserves on the planet4. It is mostly comprised of cell walls that are composed of energy rich polymers including cellulose, various hemicelluloses, and the polyphenol lignin5 and thus sometimes termed lignocellulosics. However, plant cell walls have evolved to be recalcitrant to degradation as walls contribute extensively to the strength and structural integrity of the entire plant. Despite its necessary rigidity, the cell wall is a highly dynamic entity that is metabolically active and plays crucial roles in numerous cell activities such as plant growth and differentiation5. Due to the various functions of walls, there is an immense structural diversity within the walls of different plant species and cell types within a single plant4. Hence, depending of what crop species, crop variety, or plant tissue is used for a biorefinery, the processing steps for depolymerisation by chemical/enzymatic processes and subsequent fermentation of the various sugars to liquid biofuels need to be adjusted and optimized. This fact underpins the need for a thorough characterization of plant biomass feedstocks. Here we describe a comprehensive analytical methodology that enables the determination of the composition of lignocellulosics and is amenable to a medium to high-throughput analysis (Figure 1). The method starts of with preparing destarched cell wall material. The resulting lignocellulosics are then split up to determine its monosaccharide composition of the hemicelluloses and other matrix polysaccharides1, and its content of crystalline cellulose7. The protocol for analyzing the lignin components in lignocellulosic biomass is discussed in Part I3.

Comprehensive Compositional Analysis of Plant Cell Walls Lignocellulosic biomass Part II: Carbohydrates

Plant biomass is a major carbon-neutral renewable resource that could be used for the production of biofuels. Plant biomass consists mainly of cell walls, a structurally complex composite material termed lignocellulosics. Here we describe a protocol for a comprehensive analysis of the content and composition of wall derived carbohydrates.

식물 세포 벽의 종합 작곡 분석 (리그노셀룰로오스성 바이오 매스) 2 부 : 탄수화물

In situ Quantification of Pancreatic Beta-cell Mass in Mice

Tracing changes of specific cell populations in health and disease is an important goal of biomedical research. The process of monitoring pancreatic beta-cell proliferation and islet growth is particularly challenging. We have developed a method to capture the distribution of beta-cells in the intact pancreas of transgenic mice with fluorescence-tagged beta-cells with a macro written for ImageJ (rsb.info.nih.gov/ij/). Following pancreatic dissection and tissue clearing, the entire pancreas is captured as a virtual slice, after which the GFP-tagged beta-cells are examined. The analysis includes the quantification of total beta-cell area, islet number and size distribution with reference to specific parameters and locations for each islet and for small clusters of beta-cells. The entire distribution of islets can be plotted in three dimensions, and the information from the distribution on the size and shape of each islet allows a quantitative and qualitative comparison of changes in overall beta-cell area at a glance.

In situ Quantification of Pancreatic Beta-cell Mass in Mice

The following protocol outlines the process of pancreatic dissection for virtual slice imaging, and the subsequent quantification of all GFP-tagged beta-cells in the entire pancreas.

마우스의 췌장 베타 세포 질량의 원위치 부량에서

Tracing changes of specific cell populations in health and disease is an important goal of biomedical research. The process of monitoring pancreatic ...

Imaging Cell Shape Change in Living Drosophila Embryos

The developing Drosophila melanogaster embryo undergoes a number of cell shape changes that are highly amenable to live confocal imaging. Cell shape changes in the fly are analogous to those in higher organisms, and they drive tissue morphogenesis. So, in many cases, their study has direct implications for understanding human disease (Table 1)1-5. On the sub-cellular scale, these cell shape changes are the product of activities ranging from gene expression to signal transduction, cell polarity, cytoskeletal remodeling and membrane trafficking. Thus, the Drosophila embryo provides not only the context to evaluate cell shape changes as they relate to tissue morphogenesis, but also offers a completely physiological environment to study the sub-cellular activities that shape cells.
The protocol described here is designed to image a specific cell shape change called cellularization. Cellularization is a process of dramatic plasma membrane growth, and it ultimately converts the syncytial embryo into the cellular blastoderm. That is, at interphase of mitotic cycle 14, the plasma membrane simultaneously invaginates around each of ~6000 cortically anchored nuclei to generate a sheet of primary epithelial cells. Counter to previous suggestions, cellularization is not driven by Myosin-2 contractility6, but is instead fueled largely by exocytosis of membrane from internal stores7. Thus, cellularization is an excellent system for studying membrane trafficking during cell shape changes that require plasma membrane invagination or expansion, such as cytokinesis or transverse-tubule (T-tubule) morphogenesis in muscle.
Note that this protocol is easily applied to the imaging of other cell shape changes in the fly embryo, and only requires slight adaptations such as changing the stage of embryo collection, or using "embryo glue" to mount the embryo in a specific orientation (Table 1)8-19. In all cases, the workflow is basically the same (Figure 1). Standard methods for cloning and Drosophila transgenesis are used to prepare stable fly stocks that express a protein of interest, fused to Green Fluorescent Protein (GFP) or its variants, and these flies provide a renewable source of embryos. Alternatively, fluorescent proteins/probes are directly introduced into fly embryos via straightforward micro-injection techniques9-10. Then, depending on the developmental event and cell shape change to be imaged, embryos are collected and staged by morphology on a dissecting microscope, and finally positioned and mounted for time-lapse imaging on a confocal microscope.

Imaging Cell Shape Change in Living Drosophila Embryos

Early development of the fruit fly, Drosophila melanogaster, is characterized by a number of cell shape changes that are well suited for imaging approaches. This article will describe basic tools and methods required for live confocal imaging of Drosophila embryos, and will focus on a cell shape change called cellularization.

생활의 이미징 셀 모양 변경 Drosophila 태아

The developing Drosophila melanogaster embryo undergoes a number of cell shape changes that are highly amenable to live confocal imaging.  Cell shape ...

Glycan Profiling of Plant Cell Wall Polymers using Microarrays

Plant cell walls are complex matrixes of heterogeneous glycans which play an important role in the physiology and development of plants and provide the raw materials for human societies (e.g. wood, paper, textile and biofuel industries)1,2. However, understanding the biosynthesis and function of these components remains challenging.
Cell wall glycans are chemically and conformationally diverse due to the complexity of their building blocks, the glycosyl residues. These form linkages at multiple positions and differ in ring structure, isomeric or anomeric configuration, and in addition, are substituted with an array of non-sugar residues. Glycan composition varies in different cell and/or tissue types or even sub-domains of a single cell wall3. Furthermore, their composition is also modified during development1, or in response to environmental cues4.
In excess of 2,000 genes have Plant cell walls are complex matrixes of heterogeneous glycans been predicted to be involved in cell wall glycan biosynthesis and modification in Arabidopsis5. However, relatively few of the biosynthetic genes have been functionally characterized 4,5. Reverse genetics approaches are difficult because the genes are often differentially expressed, often at low levels, between cell types6. Also, mutant studies are often hindered by gene redundancy or compensatory mechanisms to ensure appropriate cell wall function is maintained7. Thus novel approaches are needed to rapidly characterise the diverse range of glycan structures and to facilitate functional genomics approaches to understanding cell wall biosynthesis and modification.
Monoclonal antibodies (mAbs)8,9 have emerged as an important tool for determining glycan structure and distribution in plants. These recognise distinct epitopes present within major classes of plant cell wall glycans, including pectins, xyloglucans, xylans, mannans, glucans and arabinogalactans. Recently their use has been extended to large-scale screening experiments to determine the relative abundance of glycans in a broad range of plant and tissue types simultaneously9,10,11.
Here we present a microarray-based glycan screening method called Comprehensive Microarray Polymer Profiling (CoMPP) (Figures 1 &amp; 2)10,11 that enables multiple samples (100 sec) to be screened using a miniaturised microarray platform with reduced reagent and sample volumes. The spot signals on the microarray can be formally quantified to give semi-quantitative data about glycan epitope occurrence. This approach is well suited to tracking glycan changes in complex biological systems12 and providing a global overview of cell wall composition particularly when prior knowledge of this is unavailable.

A technique called Comprehensive Microarray Polymer Profiling (CoMPP) for the characterisation of plant cell wall glycans is described. This method combines the specificity of monoclonal antibodies directed to defined glycan-epitopes with a miniature microarray analytical platform allowing screening of glycan occurrence in a broad range of biological contexts.

Microarrays를 사용하여 식물 세포의 벽 폴리머의 Glycan 프로파일

Plant cell walls are complex matrixes of heterogeneous glycans which play an important role in the physiology and development of plants and provide the ...

Live Imaging of Apoptotic Cell Clearance during Drosophila Embryogenesis

The proper elimination of unwanted or aberrant cells through apoptosis and subsequent phagocytosis (apoptotic cell clearance) is crucial for normal development in all metazoan organisms. Apoptotic cell clearance is a highly dynamic process intimately associated with cell death; unengulfed apoptotic cells are barely seen in vivo under normal conditions. In order to understand the different steps of apoptotic cell clearance and to compare 'professional' phagocytes - macrophages and dendritic cells to 'non-professional' - tissue-resident neighboring cells, in vivo live imaging of the process is extremely valuable. Here we describe a protocol for studying apoptotic cell clearance in live Drosophila embryos. To follow the dynamics of different steps in phagocytosis we use specific markers for apoptotic cells and phagocytes. In addition, we can monitor two phagocyte systems in parallel: 'professional' macrophages and 'semi-professional' glia in the developing central nervous system (CNS). The method described here employs the Drosophila embryo as an excellent model for real time studies of apoptotic cell clearance.

Live Imaging of Apoptotic Cell Clearance during Drosophila Embryogenesis

Here we describe an effective method for studying dynamics of apoptotic cell clearance in vivo. This method employs live Drosophila embryos as a powerful model for monitoring phagocytosis of apoptotic cells using specific labeling of apoptotic cells and phagocytes.

사멸 세포 간극의 이미지를하는 동안 라이브<em&gt; 초파리</em&gt; 배아

The  proper elimination of unwanted or aberrant cells through apoptosis and  subsequent phagocytosis (apoptotic cell clearance) is crucial for normal  ...

High Resolution Quantification of Crystalline Cellulose Accumulation in Arabidopsis Roots to Monitor Tissue-specific Cell Wall Modifications

Plant cells are surrounded by a cell wall, the composition of which determines their final size and shape. The cell wall is composed of a complex matrix containing polysaccharides that include cellulose microfibrils that form both crystalline structures and cellulose chains of amorphous organization. The orientation of the cellulose fibers and their concentrations dictate the mechanical properties of the cell. Several methods are used to determine the levels of crystalline cellulose, each bringing both advantages and limitations. Some can distinguish the proportion of crystalline regions within the total cellulose. However, they are limited to whole-organ analyses that are deficient in spatiotemporal information. Others relying on live imaging, are limited by the use of imprecise dyes. Here, we report a sensitive polarized light-based system for specific quantification of relative light retardance, representing crystalline cellulose accumulation in cross sections of Arabidopsis thaliana roots. In this method, the cellular resolution and anatomical data are maintained, enabling direct comparisons between the different tissues composing the growing root. This approach opens a new analytical dimension, shedding light on the link between cell wall composition, cellular behavior and whole-organ growth.

High Resolution Quantification of Crystalline Cellulose Accumulation in Arabidopsis Roots to Monitor Tissue-specific Cell Wall Modifications

Crystalline cellulose is an important constituent of the plant cell wall. However, its quantification at a cellular resolution is technically challenging. Here, we report the use of polarized light technology and root cross sections to obtain information of cell wall composition at a spatiotemporal resolution.

에서 결정 셀룰로오스 축적의 높은 해상도 정량<em&gt; 애기 장대</em&gt; 뿌리 조직 특이 세포 벽 수정을 모니터링하는

Plant cells are surrounded by a cell wall, the composition of which determines their final size and shape. The cell wall is composed of a complex matrix ...

Identification of Novel Regulators of Plant Transpiration by Large-Scale Thermal Imaging Screening in Helianthus Annuus

Plant adaptation to biotic and abiotic stresses is governed by a variety of factors, among which the regulation of stomatal aperture in response to water deficit or pathogens plays a crucial role. Identifying small molecules that regulate stomatal movement can therefore contribute to understanding the physiological basis by which plants adapt to their environment. Large-scale screening approaches that have been used to identify regulators of stomatal movement have potential limitations: some rely heavily on the abscisic acid (ABA) hormone signaling pathway, therefore excluding ABA-independent mechanisms, while others rely on the observation of indirect, long-term physiological effects such as plant growth and development. The screening method presented here allows the large-scale treatment of plants with a library of chemicals coupled with a direct quantification of their transpiration by thermal imaging. Since evaporation of water through transpiration results in leaf surface cooling, thermal imaging provides a non-invasive approach to investigate changes in stomatal conductance over time. In this protocol, Helianthus annuus seedlings are grown hydroponically and then treated by root feeding, in which the primary root is cut and dipped into the chemical being tested. Thermal imaging followed by statistical analysis of cotyledonary temperature changes over time allows for the identification of bioactive molecules modulating stomatal aperture. Our proof-of-concept experiments demonstrate that a chemical can be carried from the cut root to the cotyledon of the sunflower seedling within 10 minutes. In addition, when plants are treated with ABA as a positive control, an increase in leaf surface temperature can be detected within minutes. Our method thus allows the efficient and rapid identification of novel molecules regulating stomatal aperture.

Identification of Novel Regulators of Plant Transpiration by Large-Scale Thermal Imaging Screening in Helianthus Annuus

We provide a method for identifying modulators of foliar transpiration by large-scale screening of a compound library.

헬리안투스 아누스의 대규모 열화상 스크리닝에 의한 새로운 식물 증발 규제 기관 식별

Plant adaptation to biotic and abiotic stresses is governed by a variety of factors, among which the regulation of stomatal aperture in response to water ...

Label-Free Imaging of Lipid Storage Dynamics in Caenorhabditis elegans using Stimulated Raman Scattering Microscopy

Lipid metabolism is a fundamental physiological process necessary for cellular and organism health. Dysregulation of lipid metabolism often elicits obesity and many associated diseases including cardiovascular disorders, type II diabetes, and cancer. To advance the current understanding of lipid metabolic regulation, quantitative methods to precisely measure in vivo lipid storage levels in time and space have become increasingly important and useful. Traditional approaches to analyze lipid storage are semi-quantitative for microscopic assessment or lacking spatio-temporal information for biochemical measurement. Stimulated Raman scattering (SRS) microscopy is a label-free chemical imaging technology that enables rapid and quantitative detection of lipids in live cells with a subcellular resolution. As the contrast is exploited from intrinsic molecular vibrations, SRS microscopy also permits four-dimensional tracking of lipids in live animals. In the last decade, SRS microscopy has been widely used for small molecule imaging in biomedical research and overcome the major limitations of conventional fluorescent staining and lipid extraction methods. In the laboratory, we have combined SRS microscopy with the genetic and biochemical tools available to the powerful model organism, Caenorhabditis elegans, to investigate the distribution and heterogeneity of lipid droplets across different cells and tissues and ultimately to discover novel conserved signaling pathways that modulate lipid metabolism. Here, we present the working principles and the detailed setup of the SRS microscope and provide methods for its use in quantifying lipid storage at distinct developmental timepoints of wild-type and insulin signaling deficient mutant C. elegans.

Label-Free Imaging of Lipid Storage Dynamics in Caenorhabditis elegans using Stimulated Raman Scattering Microscopy

Stimulated Raman scattering (SRS) microscopy allows selective, label-free imaging of specific chemical moieties and it has been effectively employed to image lipid molecules in vivo. Here, we provide a brief introduction to the principle of SRS microscopy and describe methods for its use in imaging lipid storage in Caenorhabditis elegans.

자극라만 산란 현미경 검사를 사용하여 Caenorhabditis elegans의 지질 저장 역학의 라벨 프리 이미징

Lipid metabolism is a fundamental physiological process necessary for cellular and organism health. Dysregulation of lipid metabolism often elicits ...

라벨 - 무료 현장에서 이미징