Microscopy is better in color: development of a streamlined spectral light path for real-time multiplex fluorescence microscopy
Microscopy is better in color: development of a streamlined spectral light path for real-time multiplex fluorescence microscopy
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DOI：
10.1364/boe.453657
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发表时间：
2022-07-01
影响因子：
3.4
通讯作者：
Leavesley, Silas J.
中科院分区：
文献类型：
--
作者：
Browning, Craig M.;Mayes, Samantha;Leavesley, Silas J.
光谱图像数据为许多领域提供了分子辨别，包括：遥感、食品安全和生物医学成像。尽管获取光谱数据的技术多种多样，但在获取数据时仍然存在权衡。通常，光谱成像要么需要较长的采集时间来收集具有高光谱特异性的图像堆栈，要么以减少光谱带或减少空间采样为代价来缩短采集时间。因此，需要新的光谱成像显微镜平台来帮助减轻这些限制。荧光激发扫描光谱成像就是这样一种新技术，与同类发射扫描光谱成像系统相比，它可以检测到更多的发射信号。在这里，我们开发了一种新的光学几何结构，可为激发扫描光谱成像显微镜系统提供光谱照明。这是通过使用特定波长的 LED 阵列来获取光谱图像数据来实现的。通过模拟和台式测试以及与宽场荧光显微镜集成时的成像性能评估，评估了基于 LED 的光谱照明器的可行性。光线追踪模拟 (TracePro) 用于确定最佳光学元件选择和几何形状。使用一系列 6 标记荧光载玻片评估光谱成像的可行性。基于 LED 的系统响应与之前测试的基于薄膜可调谐滤波器 (TFTF) 的系统进行了比较。光谱分解成功地区分了从 LED 和 TFTF 系统获取的光谱图像数据中的所有荧光成分。因此，基于 LED 的光谱照明器提供了具有可比信息内容的光谱图像数据集，以便识别每种荧光成分。这些结果提供了原理验证，证明了使用双镜（卡塞格伦式）光学配置组合来自许多离散波长 LED 光源的输出的能力，该光学配置可以进一步修改以实现高速、视频速率光谱图像采集。实时光谱荧光显微镜将允许监测快速细胞信号传导过程（即 Ca-2(+) 和其他第二信使信号传导），并有可能转化为临床成像平台。 (C) 2022 Optica Publishing Group 根据 Optica 开放获取出版协议的条款
Spectroscopic image data has provided molecular discrimination for numerous fields including: remote sensing, food safety and biomedical imaging. Despite the various technologies for acquiring spectral data, there remains a trade-off when acquiring data. Typically, spectral imaging either requires long acquisition times to collect an image stack with high spectral specificity or acquisition times are shortened at the expense of fewer spectral bands or reduced spatial sampling. Hence, new spectral imaging microscope platforms are needed to help mitigate these limitations. Fluorescence excitation-scanning spectral imaging is one such new technology, which allows more of the emitted signal to be detected than comparable emission-scanning spectral imaging systems. Here, we have developed a new optical geometry that provides spectral illumination for use in excitation-scanning spectral imaging microscope systems. This was accomplished using a wavelength-specific LED array to acquire spectral image data. Feasibility of the LED-based spectral illuminator was evaluated through simulation and benchtop testing and assessment of imaging performance when integrated with a widefield fluorescence microscope. Ray tracing simulations (TracePro) were used to determine optimal optical component selection and geometry. Spectral imaging feasibility was evaluated using a series of 6-label fluorescent slides. The LED-based system response was compared to a previously tested thin-film tunable filter (TFTF)-based system. Spectral unmixing successfully discriminated all fluorescent components in spectral image data acquired from both the LED and TFTF systems. Therefore, the LED-based spectral illuminator provided spectral image data sets with comparable information content so as to allow identification of each fluorescent component. These results provide proof-of-principle demonstration of the ability to combine output from many discrete wavelength LED sources using a double-mirror (Cassegrain style) optical configuration that can be further modified to allow for high speed, video-rate spectral image acquisition. Real-time spectral fluorescence microscopy would allow monitoring of rapid cell signaling processes (i.e., Ca-2(+) and other second messenger signaling) and has potential to be translated to clinical imaging platforms. (C) 2022 Optica Publishing Group under the terms of the Optica Open Access Publishing Agreement