Hyperspectral imaging microscopy for identification and quantitative analysis of fluorescently-labeled cells in highly autofluorescent tissue.
Hyperspectral imaging microscopy for identification and quantitative analysis of fluorescently-labeled cells in highly autofluorescent tissue.
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DOI：
10.1002/jbio.201100066
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发表时间：
2012-01
影响因子：
2.8
通讯作者：
Alvarez, Diego F.
中科院分区：
文献类型：
--
作者：
Leavesley, Silas J.;Annamdevula, Naga;Boni, John;Stocker, Samantha;Grant, Kristin;Troyanovsky, Boris;Rich, Thomas C.;Alvarez, Diego F.
标准的荧光显微镜方法依赖于在单个激发和发射带的测量来识别特定的荧光团，并设置阈值来量化荧光团强度。由于高自发荧光，这通常不足以可靠地分辨和量化组织中的荧光标记。在这里，我们描述了使用高光谱分析技术来解决和量化荧光标记的细胞在高度自发荧光肺组织。这种方法允许精确检测绿色荧光蛋白（GFP）的发射光谱，即使当GFP的强度低至15%的自发荧光强度。GFP表达细胞易于定量，检测到零假阳性。相比之下，当使用标准（单波段）阈值方法分析相同的图像时，检测到很少的GFP细胞（高阈值）或大量的假阳性（中阈值和低阈值）。这些结果表明，高光谱分析方法独特地提供了准确和精确的检测和定量的荧光信号在高度自发荧光组织。
Standard fluorescence microscopy approaches rely on measurements at single excitation and emission bands to identify specific fluorophores and the setting of thresholds to quantify fluorophore intensity. This is often insufficient to reliably resolve and quantify fluorescent labels in tissues due to high autofluorescence. Here we describe the use of hyperspectral analysis techniques to resolve and quantify fluorescently labeled cells in highly autofluorescent lung tissue. This approach allowed accurate detection of green fluorescent protein (GFP) emission spectra, even when GFP intensity was as little as 15% of the autofluorescence intensity. GFP-expressing cells were readily quantified with zero false positives detected. In contrast, when the same images were analyzed using standard (single-band) thresholding approaches, either few GFP cells (high thresholds) or substantial false positives (intermediate and low thresholds) were detected. These results demonstrate that hyperspectral analysis approaches uniquely offer accurate and precise detection and quantification of fluorescence signals in highly autofluorescent tissues.
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影响因子：
12.3
作者：
Carpenter AE;Jones TR;Lamprecht MR;Clarke C;Kang IH;Friman O;Guertin DA;Chang JH;Lindquist RA;Moffat J;Golland P;Sabatini DM
通讯作者：
Sabatini DM
影响因子：
3.5
作者：
Chaudhari, AJ;Darvas, F;Leahy, RM
通讯作者：
Leahy, RM
影响因子：
3.5
作者：
Bernas, T;Robinson, JP;Rajwa, B
通讯作者：
Rajwa, B
影响因子：
3.7
作者：
McNamara, George;Gupta, Amit;Boswell, Carl
通讯作者：
Boswell, Carl
影响因子：
1.6
作者：
Romier, J;Selves, J;Gastellu-Etchegorry, J
通讯作者：
Gastellu-Etchegorry, J