Advanced Imaging Center, Howard Hughes Medical Institute, Janelia Research Campus, Ashburn, VA 20147, USA.
Advanced Imaging Center, Howard Hughes Medical Institute, Janelia Research Campus, Ashburn, VA 20147, USA
J Cell Sci. 2020 Nov 5;133(21):jcs250027. doi: 10.1242/jcs.250027.
One of the challenges in modern fluorescence microscopy is to reconcile the conventional utilization of microscopes as exploratory instruments with their emerging and rapidly expanding role as a quantitative tools. The contribution of microscopy to observational biology will remain enormous owing to the improvements in acquisition speed, imaging depth, resolution and biocompatibility of modern imaging instruments. However, the use of fluorescence microscopy to facilitate the quantitative measurements necessary to challenge hypotheses is a relatively recent concept, made possible by advanced optics, functional imaging probes and rapidly increasing computational power. We argue here that to fully leverage the rapidly evolving application of microscopes in hypothesis-driven biology, we not only need to ensure that images are acquired quantitatively but must also re-evaluate how microscopy-based experiments are designed. In this Opinion, we present a reverse logic that guides the design of quantitative fluorescence microscopy experiments. This unique approach starts from identifying the results that would quantitatively inform the hypothesis and map the process backward to microscope selection. This ensures that the quantitative aspects of testing the hypothesis remain the central focus of the entire experimental design.
现代荧光显微镜面临的挑战之一是如何调和传统上显微镜作为探索工具的使用方式，以及它们作为定量工具的新兴和快速扩展的作用。由于现代成像仪器在采集速度、成像深度、分辨率和生物相容性方面的改进，显微镜对观察生物学的贡献仍然是巨大的。然而，利用荧光显微镜来促进定量测量，以挑战假设，这是一个相对较新的概念，这得益于先进的光学、功能成像探针和快速增长的计算能力。在这里，我们认为，要充分利用显微镜在假设驱动生物学中的快速发展应用，我们不仅需要确保图像被定量获取，还必须重新评估基于显微镜的实验设计。在本观点中，我们提出了一种反向逻辑来指导定量荧光显微镜实验的设计。这种独特的方法从确定将定量告知假设的结果开始，并将该过程反向映射到显微镜选择。这确保了测试假设的定量方面仍然是整个实验设计的核心焦点。
