Semi-automated 3D fluorescence speckle analyzer (3D-Speckler) for microscope calibration and nanoscale measurement.
Semi-automated 3D fluorescence speckle analyzer (3D-Speckler) for microscope calibration and nanoscale measurement.
复制标题
DOI：
10.1083/jcb.202202078
复制
发表时间：
2023-04-03
期刊：
影响因子：
--
通讯作者：
中科院分区：
文献类型：
--
作者：
Loi等人介绍了3D-Speckler，这是一种功能强大的高通量图像分析软件，具有用户友好的界面，可提供荧光颗粒的半自动化，多维测量和校正，包括2D/3D尺寸，纳米精度的定位以及常规显微镜校准和生物结构定量分析的强度。荧光显微镜的广泛使用促进了旨在提高生物学问题研究的分辨率和定量准确性的工具的持续发展。当前用于荧光图像的校准和量化工具面临可用性/用户体验、缺乏自动化和全面的多维测量/校正能力的问题。在这里，我们开发了3D-Speckler，一个多功能的，高通量的图像分析软件，可以提供荧光斑点定量测量，如2D/3D粒度，空间位置/方向，并通过半自动化在一个单一的，用户友好的界面强度。集成的分析选项，如2D/3D局部背景校正、色差校正和颗粒匹配/过滤，也可用于提高精度和准确度。我们展示了3D-Speckler显微镜校准能力，通过确定色差，场照明均匀性，并响应纳米级发射器以上和以下的衍射极限，我们的成像系统使用多光谱珠。此外，我们展示了3D-Speckler定量功能，可深入了解细胞中的蛋白质结构和组成。
Loi et al. introduce 3D-Speckler, a powerful and high-throughput image analysis software with a user-friendly interface that offers semi-automated, multi-dimensional measurements and corrections of fluorescent particles including 2D/3D size, localization with nanometer accuracy, and intensity for routine microscope calibration and quantitative analyses of biological structures. The widespread use of fluorescence microscopy has prompted the ongoing development of tools aiming to improve resolution and quantification accuracy for study of biological questions. Current calibration and quantification tools for fluorescence images face issues with usability/user experience, lack of automation, and comprehensive multidimensional measurement/correction capabilities. Here, we developed 3D-Speckler, a versatile, and high-throughput image analysis software that can provide fluorescent puncta quantification measurements such as 2D/3D particle size, spatial location/orientation, and intensities through semi-automation in a single, user-friendly interface. Integrated analysis options such as 2D/3D local background correction, chromatic aberration correction, and particle matching/filtering are also encompassed for improved precision and accuracy. We demonstrate 3D-Speckler microscope calibration capabilities by determining the chromatic aberrations, field illumination uniformity, and response to nanometer-scale emitters above and below the diffraction limit of our imaging system using multispectral beads. Furthermore, we demonstrated 3D-Speckler quantitative capabilities for offering insight into protein architectures and composition in cells.
登录
查看更多内容
DOI：
10.1083/jcb.201004104
发表时间：
2010-05-31
期刊：
The Journal of cell biology
影响因子：
--
作者：
Linkert M;Rueden CT;Allan C;Burel JM;Moore W;Patterson A;Loranger B;Moore J;Neves C;Macdonald D;Tarkowska A;Sticco C;Hill E;Rossner M;Eliceiri KW;Swedlow JR
通讯作者：
Swedlow JR
DOI：
10.1083/jcb.200912022
发表时间：
2010-06-14
期刊：
The Journal of cell biology
影响因子：
--
作者：
Johnston K;Joglekar A;Hori T;Suzuki A;Fukagawa T;Salmon ED
通讯作者：
Salmon ED
影响因子：
6
作者：
Hickey SM;Ung B;Bader C;Brooks R;Lazniewska J;Johnson IRD;Sorvina A;Logan J;Martini C;Moore CR;Karageorgos L;Sweetman MJ;Brooks DA
通讯作者：
Brooks DA
DOI：
10.7171/jbt.15-2602-001
发表时间：
2015-07-01
期刊：
Journal of biomolecular techniques : JBT
影响因子：
--
作者：
Brown, Claire M;Reilly, Andrew;Cole, Richard W
通讯作者：
Cole, Richard W
实时3D单分子使用实验点扩散函数的定位。
影响因子：
48
作者：
Li Y;Mund M;Hoess P;Deschamps J;Matti U;Nijmeijer B;Sabinina VJ;Ellenberg J;Schoen I;Ries J
通讯作者：
Ries J