Multi-dimensional Dynamics of Pancreatic Islet Cells Measured by Image Mapping diSPIM
Multi-dimensional Dynamics of Pancreatic Islet Cells Measured by Image Mapping diSPIM
批准号:
10197901
负责人:
David W Piston
金额:
$31.97万
依托单位:
依托单位国家:
美国
项目类别:
财政年份:
2018
资助国家:
美国
项目状态:
已结题
起止时间:
2018-07-01 至 2024-06-30
关键词:
3-DimensionalAddressAdoptionArchitectureBeta CellBiologyBiosensorCalciumCaliberCell membraneCell physiologyCell surfaceCellsCellular biologyColorComputer softwareConfocal MicroscopyCoupledCrowdingCyclic AMPDataData AnalysesDetectionDevelopmentDimensionsDiseaseDockingEnvironmentEventExocytosisFluorescence MicroscopyFluorescent ProbesFutureGeometryGlucoseGoalsGolgi ApparatusHuman bodyImageImage AnalysisImaging DeviceIn SituInsulinInvestigationIslet CellIslets of LangerhansKnowledgeLasersLeadLengthLife Cycle StagesLightLightingMeasurementMeasuresMethodsMicroscopeMicroscopyMicrotubulesModelingMolecularMotionMovementNoisePathway interactionsPerformancePhasePhotobleachingProcessProteinsResolutionSamplingScanningSecretory VesiclesSignal TransductionSiteSpectrum AnalysisSpeedStructureStructure of beta Cell of isletSynaptic VesiclesSystemTechnologyTestingTimeVesicleWorkbasebehavior changedata acquisitionexperimental studyflexibilityfluorescence imagingimaging systemimprovedinnovationinsightinstrumentinstrumentationinsulin regulationinsulin secretionisletlight scatteringlive cell microscopynovelpanaceapancreatic islet functionspectroscopic imagingtraffickingtwo-photon
中文摘要
摘要
活细胞荧光显微镜极大地提高了我们对细胞动力学的理解。
实验。这些实验发现了小泡的运输和胞吐作用。
在功能上重要的时间和长度尺度,与胰岛功能的具体含义。活细胞
高光谱成像允许同时测量具有信噪比的多个动态过程
与基于过滤器的方法相同或更高的比率。目前,最便捷的高光谱成像
系统使用共聚焦显微镜,这受到光漂白和成像速度慢的限制。我们建议
为了开发一种新型的五维(x,y,z,t,λ)荧光成像系统,其提供高空间,
具有最小可能的光漂白的时间和光谱分辨率。我们将对其性能进行优化
用于研究长期存在的有关胰岛素分泌调节的问题。这台仪器将会结合
两种技术:产生各向同性衍射的双视图选择平面照明显微镜(DISPIM)-
在三维扩展视图上的有限成像,以及图像映射光谱(IMS)
一次快照中的全场高光谱检测。我们将构建、测试和优化这本小说
通过两个具体的目标来实施。具体目标1将专注于构建和优化新的
用于diSPIM的高光谱IMS系统,以及适用于五维数据的软件模块
获取和分析。为了证实IMS/diSPIM方法的优势,我们将获取图像
同时获得至少五种具有高时间和空间分辨率的生物传感器颜色。测试和指导
目标1、特定目标2的发展将把这一新仪器应用于β细胞生物学中无法解决的问题
用目前可用的方法解决,重点是两个问题:胰岛素囊泡运输和
分泌:a)β细胞中胰岛素囊泡的正常生命周期是什么?因为有10%的胰岛素囊泡
,假设新形成的囊泡优先分泌,我们提出
寿命较长的囊泡充当信号平台。我们将使用IMS/DiSPIM来定量测量UP
到6个荧光探针,这将使我们能够跟踪β细胞中的每个小泡,当它从高尔基体发芽时,成熟,
或者是秘密的,或者是不可逆转地移动到一个长生不老的池子里。B)做到“可随时发布”和
“储备”囊泡池导致葡萄糖刺激的胰岛素分泌的两个阶段?两个人的概念
Pools来自对突触小泡的研究,这可能不同于β细胞拥挤的环境,在那里
第一阶段的分泌事件似乎来自新到达质膜的小泡。我们
假设β细胞中的囊泡沿着微管移动到胞吐部位,这些运动是
受细胞内游离钙活性([Ca~(2+)]i)和cAMP水平调节。为了检验这一假设,我们将使用
IMS/diSPIM可同时测量多达6个荧光探针,并允许进行定量关联
胰岛素囊泡运动和分泌与[Ca2+]i、cAMP和细胞骨架结构之间的关系。
英文摘要
ABSTRACT
Our understanding of cellular dynamics has been advanced significantly by live-cell fluorescence microscopy
experiments. These experiments have yielded discoveries in vesicle trafficking and exocytosis on the
functionally important time and length scales, with specific implications for pancreatic islet function. Live-cell
hyperspectral imaging permits simultaneous measurements of multiple dynamic processes with signal-to-noise
ratios equivalent or superior to filter-based approaches. Currently, the most expedient hyperspectral imaging
systems use confocal microscopy, which is limited by photobleaching and slow imaging speeds. We propose
to develop a novel five-dimensional (x,y,z,t,λ) fluorescence imaging system that provides high spatial,
temporal, and spectral resolution with the minimal possible photobleaching. We will optimize its performance
for investigations of long-standing questions about regulation of insulin secretion. This instrument will combine
two technologies: dual-view Selective-Plane Illumination Microscopy (diSPIM) that yields isotropic diffraction-
limited imaging over extended views in three dimensions, and image mapping spectroscopy (IMS) that permits
whole field hyperspectral detection in a single snapshot. We will build, test, and optimize this novel
instrumentation through two specific aims. Specific aim 1 will focus on building and optimizing a new
hyperspectral IMS system for use with diSPIM, and also adapting software modules for five-dimensional data
acquisition and analysis. To substantiate the advantages of the IMS/diSPIM approach, we will acquire images
simultaneously for at least five biosensor colors with high temporal and spatial resolution. To test and guide
the developments in Aim 1, Specific aim 2 will apply this new instrument to issues in β-cell biology that cannot
be addressed with currently available methods, focusing on two questions of insulin vesicle trafficking and
secretion: a) What is the normal life cycle of an insulin vesicle in the β-cell? Since <10% of the insulin vesicles
are secreted, it has been hypothesized that newly formed vesicles are preferentially secreted, and we propose
that longer-lived vesicles act as a signaling platform. We will use the IMS/diSPIM to measure quantitatively up
to 6 fluorescent probes, which will allow us to track every vesicle in a β-cell as it buds from the Golgi, matures,
and is either secreted or moves, putatively irreversibly, into a long-lived pool. b) Do “readily releasable” and
“reserve” vesicle pools lead to the two phases of glucose-stimulated insulin secretion? The concept of two
pools comes from synaptic vesicle studies, which may differ from the crowded environment of the β-cell, where
first phase secretory events appear to come from vesicles newly arriving at the plasma membrane. We
hypothesize that vesicles in β-cells move along microtubules to sites of exocytosis, and these movements are
regulated by intracellular free calcium activity ([Ca2+]i and cAMP levels. To test this hypothesis, we will use the
IMS/diSPIM to measure up to 6 fluorescent probes simultaneously and permit quantitative correlations
between insulin vesicle motions and secretion with [Ca2+]i, cAMP, and cytoskeletal architecture.
期刊论文(3)
专著(0)
科研奖励(0)
会议论文
Fabrication of a multifaceted mapping mirror using two-photon polymerization for a snapshot image mapping spectrometer.
使用用于快照图像映射光谱仪的双光子聚合制造多面映射镜。
DOI:
10.1364/ao.495466
发表时间:
2023
期刊:
Applied optics
影响因子:
1.9
作者:
[Lu,Jiawei, Ng,XueWen, Piston,David, Tkaczyk,TomaszS]
通讯作者:
Tkaczyk,TomaszS
DOI:
10.1002/cphy.c200026
发表时间:
2021-06-30
期刊:
Comprehensive Physiology
影响因子:
5.8
作者:
[Ng XW, Chung YH, Piston DW]
通讯作者:
Piston DW
DOI:
10.1073/pnas.2302624120
发表时间:
2023-05-30
期刊:
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
影响因子:
11.1
作者:
[Polino, Alexander J., Sviben, Sanja, Melena, Isabella, Piston, David W., Hughes, Jing W.]
通讯作者:
Hughes, Jing W.
Nikon Confocal Microscope for Shared Biomedical Research
-
批准号:10413403
-
项目类别:
-
资助金额:$53.76万
-
财政年份:2022
-
负责人:David W Piston
-
依托单位:
High Sensitivity sCMOS Camera System for Transmission Electron Microscope
-
批准号:10414332
-
项目类别:
-
资助金额:$14.7万
-
财政年份:2022
-
负责人:David W Piston
-
依托单位:
Zeiss LSM 980 Airyscan 2 Microscope for Shared Mental Health Research
-
批准号:10282117
-
项目类别:
-
资助金额:$60.0万
-
财政年份:2021
-
负责人:David W Piston
-
依托单位:
Regulation of Glucagon Secretion from Pancreatic Islets
-
批准号:10675668
-
项目类别:
-
资助金额:$39.38万
-
财政年份:2020
-
负责人:David W Piston
-
依托单位:
Regulation of Glucagon Secretion from Pancreatic Islets
-
批准号:10468865
-
项目类别:
-
资助金额:$39.38万
-
财政年份:2020
-
负责人:David W Piston
-
依托单位:
Regulation of Glucagon Secretion from Pancreatic Islets
-
批准号:10264101
-
项目类别:
-
资助金额:$39.38万
-
财政年份:2020
-
负责人:David W Piston
-
依托单位:
Cellular Imaging Core
-
批准号:10704277
-
项目类别:
-
资助金额:$11.66万
-
财政年份:2018
-
负责人:David W Piston
-
依托单位:
Pancreatic Islets Dynamics Regulating Glucagon Secretion
-
批准号:9068608
-
项目类别:
-
资助金额:$33.17万
-
财政年份:2015
-
负责人:David W Piston
-
依托单位:
Dopamine Action in Pancreatic Islet Function
-
批准号:9068606
-
项目类别:
-
资助金额:$12.75万
-
财政年份:2015
-
负责人:David W Piston
-
依托单位:
Pancreatic Islets Dynamics Regulating Glucagon Secretion
-
批准号:9116182
-
项目类别:
-
资助金额:$33.17万
-
财政年份:2015
-
负责人:David W Piston
-
依托单位:
Pancreatic Islets Dynamics Regulating Glucagon Secretion
-
批准号:8630762
-
项目类别:
-
资助金额:$33.87万
-
财政年份:2013
-
负责人:David W Piston
-
依托单位:
PANCREATIC ISLETS DYNAMICS REGULATING GLUCAGON SECRETION
-
批准号:9539011
-
项目类别:
-
资助金额:$22.88万
-
财政年份:2013
-
负责人:David W Piston
-
依托单位:
Parallel Computer with High Memory Nodes
-
批准号:8447324
-
项目类别:
-
资助金额:$59.75万
-
财政年份:2013
-
负责人:David W Piston
-
依托单位:
Pancreatic Islets Dynamics Regulating Glucagon Secretion
-
批准号:8735943
-
项目类别:
-
资助金额:$34.09万
-
财政年份:2013
-
负责人:David W Piston
-
依托单位:
Cell and Tissue Imaging Core
-
批准号:10630361
-
项目类别:
-
资助金额:$14.87万
-
财政年份:2012
-
负责人:David W Piston
-
依托单位:
Multiphoton microscope upgrade
-
批准号:8247216
-
项目类别:
-
资助金额:$43.59万
-
财政年份:2012
-
负责人:David W Piston
-
依托单位:
Dopamine action in pancreatic islet function
-
批准号:8053760
-
项目类别:
-
资助金额:$31.97万
-
财政年份:2010
-
负责人:David W Piston
-
依托单位:
Dopamine action in pancreatic islet function
-
批准号:7767594
-
项目类别:
-
资助金额:$38.72万
-
财政年份:2010
-
负责人:David W Piston
-
依托单位:
Dopamine action in pancreatic islet function
-
批准号:8248311
-
项目类别:
-
资助金额:$32.02万
-
财政年份:2010
-
负责人:David W Piston
-
依托单位:
Cell Imaging Shared Resource
-
批准号:8180560
-
项目类别:
-
资助金额:$7.39万
-
财政年份:2010
-
负责人:David W Piston
-
依托单位:
海外基金