University of South Alabama, Department of Chemical and Biomolecular Engineering, 150 Jaguar Dr., SH 4129, Mobile, Alabama 36688bUniversity of South Alabama, Center for Lung Biology, 150 Jaguar Dr., SH 4129, Mobile, Alabama 36688.
University of South Alabama, Department of Chemical and Biomolecular Engineering, 150 Jaguar Dr., SH 4129, Mobile, Alabama 36688.
J Biomed Opt. 2014 Apr;19(4):046010. doi: 10.1117/1.JBO.19.4.046010.
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.
高光谱成像是一种多功能工具，最近已应用于多种生物医学应用，特别是活细胞和整个组织信号。传统的高光谱成像方法在单个波段激发的同时，对宽波长范围的荧光发射进行滤波。然而，由于光谱滤波引起的光衰减，这些发射扫描方法显示出灵敏度降低。因此，发射扫描对于时间敏感的研究和光敏感的应用的适用性有限。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，通过提供高传输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。我们通过在高度自发荧光的肺组织中检测表达绿色荧光蛋白 (GFP) 的内皮细胞，对激发扫描显微镜的功效进行了与发射扫描的并排比较测试。激发扫描提供了更高的信噪比特性，以及更短的采集时间（激发扫描的每个波长带宽为 300 ms，而发射扫描的每个波长带宽为 3 s）。激发扫描还提供了更好的核和细胞边界划分，并增加了在高度自发荧光组织中 GFP 区域的识别。这些结果表明，激发扫描在广泛的时间相关和光敏感应用中具有实用性。
