Deal Joshua, Rich Thomas C, Leavesley Silas J
Department of Chemical & Biomolecular Engineering, University of South Alabama.
Center for Lung Biology, University of South Alabama.
Proc SPIE Int Soc Opt Eng. 2019 Feb;10881. doi: 10.1117/12.2510784. Epub 2019 Mar 4.
A major benefit of fluorescence microscopy is the now plentiful selection of fluorescent markers. These labels can be chosen to serve complementary functions, such as tracking labeled subcellular molecules near demarcated organelles. However, with the standard 3 or 4 emission channels, multiple label detection is restricted to segregated regions of the electromagnetic spectrum, as in RGB coloring. Hyperspectral imaging allows the user to discern many fluorescence labels by their unique spectral properties, provided there is significant differentiation of their emission spectra. The cost of this technique is often an increase in gain or exposure time to accommodate the signal reduction from separating the signal into many discrete excitation or emission channels. Recent advances in hyperspectral imaging have allowed the acquisition of more signal in a shorter time period by scanning the excitation spectra of fluorophores. Here, we explore the selection of optimal channels for both significant signal separation and sufficient signal detection using excitation-scanning hyperspectral imaging. Excitation spectra were obtained using a custom inverted microscope (TE-2000, Nikon Instruments) with a Xe arc lamp and thin film tunable filter array (VersaChrome, Semrock, Inc.) Tunable filters had bandwidths between 13 and 17 nm. Scans utilized excitation wavelengths between 340 nm and 550 nm. Hyperspectral image stacks were generated and analyzed using ENVI and custom MATLAB scripts. Among channel consideration criteria were: number of channels, spectral range of scan, spacing of center wavelengths, and acquisition time.
荧光显微镜的一个主要优点是现在有大量的荧光标记可供选择。这些标记可以被选择来发挥互补功能，例如追踪在划定细胞器附近的标记亚细胞分子。然而，对于标准的3个或4个发射通道，多标记检测仅限于电磁光谱的分离区域，就像RGB着色一样。高光谱成像允许用户通过其独特的光谱特性辨别许多荧光标记，前提是它们的发射光谱有显著差异。这种技术的代价通常是增加增益或曝光时间，以适应将信号分离到许多离散的激发或发射通道所导致的信号减少。高光谱成像的最新进展使得通过扫描荧光团的激发光谱在更短的时间内获取更多信号成为可能。在这里，我们使用激发扫描高光谱成像探索用于显著信号分离和充分信号检测的最佳通道选择。使用配备氙弧灯和薄膜可调滤光片阵列（VersaChrome，Semrock公司）的定制倒置显微镜（TE-2000，尼康仪器公司）获得激发光谱。可调滤光片的带宽在13至17纳米之间。扫描使用340纳米至550纳米之间的激发波长。使用ENVI和定制的MATLAB脚本生成并分析高光谱图像堆栈。通道考虑标准包括：通道数量、扫描光谱范围、中心波长间距和采集时间。
