Parker Marina, Annamdevula Naga S, Pleshinger Donald, Ijaz Zara, Jalkh Josephine, Penn Raymond, Deshpande Deepak, Rich Thomas C, Leavesley Silas J
Department of Chemical and Biomolecular Engineering, University of South Alabama, 150 Student Services Dr., Mobile, AL 36688, USA.
Department of Systems Engineering, University of South Alabama, 150 Student Services Dr., Mobile, AL 36688, USA.
Bioengineering (Basel). 2023 May 25;10(6):642. doi: 10.3390/bioengineering10060642.
Hyperspectral imaging (HSI) technology has been applied in a range of fields for target detection and mixture analysis. While HSI was originally developed for remote sensing applications, modern uses include agriculture, historical document authentication, and medicine. HSI has also shown great utility in fluorescence microscopy. However, traditional fluorescence microscopy HSI systems have suffered from limited signal strength due to the need to filter or disperse the emitted light across many spectral bands. We have previously demonstrated that sampling the fluorescence excitation spectrum may provide an alternative approach with improved signal strength. Here, we report on the use of excitation-scanning HSI for dynamic cell signaling studies-in this case, the study of the second messenger Ca. Time-lapse excitation-scanning HSI data of Ca signals in human airway smooth muscle cells (HASMCs) were acquired and analyzed using four spectral analysis algorithms: linear unmixing (LU), spectral angle mapper (SAM), constrained energy minimization (CEM), and matched filter (MF), and the performances were compared. Results indicate that LU and MF provided similar linear responses to increasing Ca and could both be effectively used for excitation-scanning HSI. A theoretical sensitivity framework was used to enable the filtering of analyzed images to reject pixels with signals below a minimum detectable limit. The results indicated that subtle kinetic features might be revealed through pixel filtering. Overall, the results suggest that excitation-scanning HSI can be employed for kinetic measurements of cell signals or other dynamic cellular events and that the selection of an appropriate analysis algorithm and pixel filtering may aid in the extraction of quantitative signal traces. These approaches may be especially helpful for cases where the signal of interest is masked by strong cellular autofluorescence or other competing signals.
高光谱成像（HSI）技术已应用于一系列领域进行目标检测和混合物分析。虽然HSI最初是为遥感应用而开发的，但现代用途包括农业、历史文献鉴定和医学。HSI在荧光显微镜中也显示出了巨大的实用性。然而，传统的荧光显微镜HSI系统由于需要在许多光谱带中过滤或分散发射光，信号强度有限。我们之前已经证明，对荧光激发光谱进行采样可能提供一种具有更高信号强度的替代方法。在此，我们报告了使用激发扫描HSI进行动态细胞信号研究——在这种情况下，是对第二信使Ca的研究。我们采集了人气道平滑肌细胞（HASMCs）中Ca信号的延时激发扫描HSI数据，并使用四种光谱分析算法进行分析：线性分解（LU）、光谱角映射器（SAM）、约束能量最小化（CEM）和匹配滤波器（MF），并比较了它们的性能。结果表明，LU和MF对Ca增加提供了相似的线性响应，并且都可以有效地用于激发扫描HSI。使用理论灵敏度框架对分析图像进行滤波，以拒绝信号低于最小可检测限的像素。结果表明，通过像素滤波可能揭示细微的动力学特征。总体而言，结果表明激发扫描HSI可用于细胞信号或其他动态细胞事件的动力学测量，并且选择合适的分析算法和像素滤波可能有助于提取定量信号轨迹。这些方法对于感兴趣的信号被强烈的细胞自发荧光或其他竞争信号掩盖的情况可能特别有帮助。
