Excitation-Scanning Hyperspectral Imaging Microscopy to Efficiently Discriminate Fluorescence Signals
Excitation-Scanning Hyperspectral Imaging Microscopy to Efficiently Discriminate Fluorescence Signals
复制标题
DOI：
10.3791/59448
复制
发表时间：
2019-08-01
影响因子：
1.2
通讯作者：
Leavesley, Silas
中科院分区：
文献类型：
--
作者：
Deal, Joshua;Britain, Andrea;Leavesley, Silas
几种技术依赖于荧光信号的检测来识别或研究现象或阐明功能。这些荧光信号的分离被证明是繁琐的，直到高光谱成像的出现，其中荧光源可以彼此分离，以及从背景信号和自发荧光（给定其光谱特征的知识）。然而，传统的发射扫描高光谱成像由于必须对激发光和发射光两者进行滤波而遭受慢的采集时间和低的信噪比。先前已经表明，激发扫描高光谱成像减少了必要的采集时间，同时增加了采集数据的信噪比。使用市售设备，该协议描述了如何组装，校准和使用激发扫描高光谱成像显微镜系统分离信号从几个荧光源在一个单一的样品。虽然高度适用于细胞和组织的显微成像，但该技术也可用于利用荧光的任何类型的实验，其中可以改变激发波长，包括但不限于：化学成像，环境应用，眼睛护理，食品科学，法医学，医学科学和矿物学。
Several techniques rely on detection of fluorescence signals to identify or study phenomena or to elucidate functions. Separation of these fluorescence signals were proven cumbersome until the advent of hyperspectral imaging, in which fluorescence sources can be separated from each other as well as from background signals and autofluorescence (given knowledge of their spectral signatures). However, traditional, emission-scanning hyperspectral imaging suffers from slow acquisition times and low signal-to-noise ratios due to the necessary filtering of both excitation and emission light. It has been previously shown that excitation-scanning hyperspectral imaging reduces the necessary acquisition time while simultaneously increasing the signal-to-noise ratio of acquired data. Using commercially available equipment, this protocol describes how to assemble, calibrate, and use an excitation-scanning hyperspectral imaging microscopy system for separation of signals from several fluorescence sources in a single sample. While highly applicable to microscopic imaging of cells and tissues, this technique may also be useful for any type of experiment utilizing fluorescence in which it is possible to vary excitation wavelengths, including but not limited to: chemical imaging, environmental applications, eye care, food science, forensic science, medical science, and mineralogy.