Hypothesis-driven quantitative fluorescence microscopy - the importance of reverse-thinking in experimental design
Hypothesis-driven quantitative fluorescence microscopy - the importance of reverse-thinking in experimental design
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DOI：
10.1242/jcs.250027
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发表时间：
2020-11-01
影响因子：
4
通讯作者：
Chew, Teng-Leong
中科院分区：
文献类型：
--
作者：
Wait, Eric C.;Reiche, Michael A.;Chew, Teng-Leong
现代荧光显微镜面临的挑战之一是如何调和显微镜作为探测仪器的传统用途与它们作为定量工具的新兴和迅速扩大的作用。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将继续巨大。然而，利用荧光显微镜来促进定量测量以挑战假设是一个相对较新的概念，这是由于先进的光学，功能成像探针和快速增加的计算能力而成为可能。我们认为，为了充分利用显微镜在假设驱动生物学中快速发展的应用，我们不仅需要确保定量获得图像，还必须重新评估如何设计基于显微镜的实验。在本意见中，我们提出了一个反向逻辑，指导定量荧光显微镜实验的设计。这种独特的方法从确定结果开始，定量地告知假设，并将过程映射到显微镜选择。这确保了检验假设的定量方面仍然是整个实验设计的中心焦点。
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.