Department of Electrical and Computer Engineering, Herbert Wertheim College of Engineering, University of Florida, Gainesville, Florida, United States of America.
Department of Electrical and Computer Engineering, University of California, Santa Barbara, California, United States of America.
PLoS One. 2022 Jul 28;17(7):e0266098. doi: 10.1371/journal.pone.0266098. eCollection 2022.
Automatic operations of multi-functional and time-lapse live-cell imaging are necessary for the biomedical science community to study active, multi-faceted, and long-term biological phenomena. To achieve automatic control, most existing solutions often require the purchase of extra software programs and hardware that rely on the manufacturers' own specifications. However, these software programs are usually non-user-programmable and unaffordable for many laboratories. To address this unmet need, we have developed a novel open-source software program, titled Automatic Multi-functional Integration Program (AMFIP), as a new Java-based and hardware-independent system that provides proven advantages over existing alternatives to the scientific community. Without extra hardware, AMFIP enables the functional synchronization of the μManager software platform, the Nikon NIS-Elements platform, and other 3rd party software to achieve automatic operations of most commercially available microscopy systems, including but not limited to those from Nikon. AMFIP provides a user-friendly and programmable graphical user interface (GUI), opening the door to expanding the customizability for myriad hardware and software systems according to user-specific experimental requirements and environments. To validate the intended purposes of developing AMFIP, we applied it to elucidate the question whether single cells, prior to their full spreading, can sense and respond to a soft solid substrate, and if so, how does the interaction depend on the cell spreading time and the stiffness of the substrate. Using a CRISPR/Cas9-engineered human epithelial Beas2B (B2B) cell line that expresses mNeonGreen2-tagged mechanosensitive Yes-associated protein (YAP), we show that single B2B cells develop distinct substrate-stiffness-dependent YAP expressions within 10 hours at most on the substrate, suggesting that cells are able to sense, distinguish, and respond to mechanical cues prior to the establishment of full cell spreading. In summary, AMFIP provides a reliable, open-source, and cost-free solution that has the validated long-term utility to satisfy the need of automatic imaging operations in the scientific community.
多功能和延时活细胞成像的自动化操作对于生物医学科学界研究活跃、多方面和长期的生物现象是必要的。为了实现自动控制，大多数现有的解决方案通常需要购买额外的软件程序和硬件，这些硬件依赖于制造商自己的规格。然而，这些软件程序通常是不可用户编程的，许多实验室都负担不起。为了解决这一未满足的需求，我们开发了一种新的开源软件程序，名为自动多功能集成程序（Automatic Multi-functional Integration Program，AMFIP），它是一个基于 Java 的、与硬件无关的系统，为科学界提供了比现有替代方案更具优势的功能。不需要额外的硬件，AMFIP 能够使 μManager 软件平台、Nikon NIS-Elements 平台和其他第三方软件的功能同步，从而实现大多数市售显微镜系统的自动化操作，包括但不限于尼康的显微镜系统。AMFIP 提供了一个用户友好和可编程的图形用户界面（Graphical User Interface，GUI），为根据用户特定的实验要求和环境扩展各种硬件和软件系统的可定制性打开了大门。为了验证开发 AMFIP 的目的，我们应用它来阐明这样一个问题，即在细胞完全铺展之前，单细胞是否能够感知和响应软固体底物，如果可以，细胞的这种相互作用如何取决于细胞铺展时间和底物的硬度。我们使用表达 mNeonGreen2 标记的机械敏感 Yes 相关蛋白（Yes-associated protein，YAP）的 CRISPR/Cas9 工程人类上皮 Beas2B（B2B）细胞系，证明了在底物上最多 10 小时内，单个 B2B 细胞会根据底物的硬度表现出明显的依赖于底物硬度的 YAP 表达，这表明细胞在完全铺展之前能够感知、区分和响应机械线索。总之，AMFIP 提供了一种可靠的、开源的、免费的解决方案，具有经过验证的长期效用，可以满足科学界对自动成像操作的需求。
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