Excitation-scanning hyperspectral imaging microscope
Excitation-scanning hyperspectral imaging microscope
复制标题
DOI：
10.1117/1.jbo.19.4.046010
复制
发表时间：
2014-04-01
影响因子：
3.5
通讯作者：
Leavesley, Silas J.
中科院分区：
文献类型：
--
作者：
Favreau, Peter F.;Hernandez, Clarissa;Leavesley, Silas J.
高光谱成像是一种多功能工具，最近已应用于各种生物医学应用，特别是活细胞和全组织信号。传统的高光谱成像方法在较宽的波长范围内过滤荧光发射，而在单一波段激发。然而，由于光谱滤波引起的光衰减，这些发射扫描方法的灵敏度降低。因此，发射扫描对时间敏感研究和光敏应用的适用性有限。在这项工作中，我们开发了一种激发扫描高光谱成像显微镜，通过提供高透射率和短采集时间来克服这些限制。这是通过过滤荧光激发而不是发射来实现的。我们测试了兴奋扫描显微镜与发射扫描在检测高度自身荧光肺组织中表达绿色荧光蛋白（GFP）的内皮细胞方面的功效。激发扫描具有更高的信噪比特性，且采集时间更短（激发扫描为300 ms/波长波段，而发射扫描为3 s/波长波段）。激发扫描还提供了核和细胞边界的更高描绘，并增加了高自身荧光组织中GFP区域的识别。这些结果表明激发扫描在广泛的时间依赖性和光敏应用中具有实用价值。(C) 2014年中国光学仪器工程师学会（SPIE）
Hyperspectral imaging is a versatile tool that has recently been applied to a variety of biomedical applications, notably live-cell and whole-tissue signaling. Traditional hyperspectral imaging approaches filter the fluorescence emission over a broad wavelength range while exciting at a single band. However, these emission-scanning approaches have shown reduced sensitivity due to light attenuation from spectral filtering. Consequently, emission scanning has limited applicability for time-sensitive studies and photosensitive applications. In this work, we have developed an excitation-scanning hyperspectral imaging microscope that overcomes these limitations by providing high transmission with short acquisition times. This is achieved by filtering the fluorescence excitation rather than the emission. We tested the efficacy of the excitation-scanning microscope in a side-by-side comparison with emission scanning for detection of green fluorescent protein (GFP)-expressing endothelial cells in highly autofluorescent lung tissue. Excitation scanning provided higher signal-to-noise characteristics, as well as shorter acquisition times (300 ms/wavelength band with excitation scanning versus 3 s/wavelength band with emission scanning). Excitation scanning also provided higher delineation of nuclear and cell borders, and increased identification of GFP regions in highly autofluorescent tissue. These results demonstrate excitation scanning has utility in a wide range of time-dependent and photosensitive applications. (C) 2014 Society of Photo-Optical Instrumentation Engineers (SPIE)