Optical microscopic imaging, manipulation, and analysis methods for morphogenesis research

Microscopy, 2024, 73(3), 226–242 DOI: https://doi.org/10.1093/jmicro/dfad059 Advance Access Publication Date: 15 December 2023 Review Optical microscopic imaging, manipulation, and analysis methods for morphogenesis research Takanobu A. Katoh 1,†,* , Yohsuke T. Fukai 2,†,* and Tomoki Ishibashi 3,†,* 1 Department of Cell Biology, Graduate School of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan Nonequilibrium Physics of Living Matter RIKEN Hakubi Research Team, RIKEN Center for Biosystems Dynamics Research, 2-2-3 Minatojima-minamimachi, Chuo-ku, Kobe, Hyogo 650-0047, Japan 3 Laboratory for Physical Biology, RIKEN Center for Biosystems Dynamics Research, 2-2-3 Minatojima-minamimachi, Chuo-ku, Kobe, Hyogo 650-0047, Japan 2 To whom correspondence should be addressed. E-mail: , , These authors equally contributed to this review. Authors’ note: The authors wish it to be known that, in their opinion, the three authors should be regarded as joint first and corresponding authors. The order of the authors was determined by lottery. The authors can prioritize their names when adding this paper’s reference to their CVs. † Abstract Morphogenesis is a developmental process of organisms being shaped through complex and cooperative cellular movements. To understand the interplay between genetic programs and the resulting multicellular morphogenesis, it is essential to characterize the morphologies and dynamics at the single-cell level and to understand how physical forces serve as both signaling components and driving forces of tissue deformations. In recent years, advances in microscopy techniques have led to improvements in imaging speed, resolution and depth. Concurrently, the development of various software packages has supported large-scale, analyses of challenging images at the single-cell resolution. While these tools have enhanced our ability to examine dynamics of cells and mechanical processes during morphogenesis, their effective integration requires specialized expertise. With this background, this review provides a practical overview of those techniques. First, we introduce microscopic techniques for multicellular imaging and image analysis software tools with a focus on cell segmentation and tracking. Second, we provide an overview of cutting-edge techniques for mechanical manipulation of cells and tissues. Finally, we introduce recent findings on morphogenetic mechanisms and mechanosensations that have been achieved by effectively combining microscopy, image analysis tools and mechanical manipulation techniques. Key words: optical microscopy, segmentation tool, tracking tool, morphogenesis, mechanobiology Introduction The formation of ordered multicellular structures during embryonic development has long been a significant subject of research. Early studies attempted to elucidate the morphogenetic mechanisms during development through histological observations [1]. In the post-Genome Project era, major progress has been made in uncovering the genetic and signaling pathways that govern the morphogenesis of diverse yet robust organ and tissue structures. Morphogenesis is driven by entities that are interlaced with gene regulatory networks, specifically, the cell shape alterations and movements that are propelled by mechanical forces [2]. To understand how genetic programs orchestrate the formation of structures and trace the causal relationships behind the process, we must both characterize the morphologies and dynamics at the single-cell scale and comprehend how physical forces serve as signaling components and drivers of deformation. The field has seen the development of sophisticated microscopy techniques that improve observation speed, resolution and imaging depth. Various image analysis techniques have also been developed to support large-scale, single-cell-level analyses. Particularly, the development of machine-learning methods has enabled the quantification of challenging images [3,4]. Efforts have also been made to make those techniques widely accessible. Furthermore, advancements in imaging, manipulation and analysis techniques have facilitated the comprehensive examinations of mechanical processes during morphogenesis [5–9]. Specifically, the integration of diverse technologies has revealed intricate interactions between shapes, forces and signaling pathways [10,11]. Despite the importance of combining different techniques, this growing sophistication demands increased expertise. With this background, this review aims to provide an overview of these techniques and briefly introduce them for practical applications. First, we introduce microscopies for multicellular imaging. Next, we review image analysis techniques and software tools, focusing on cell instance segmentation and tracking which are essential for uncovering the relationships between microscopic cellular properties and macroscopic morphogenetic dynamics. Then we introduce cuttingedge mechanical manipulation techniques to dissect mechanical processes in living systems. Finally, we discuss the latest Received 30 June 2023; Revised 20 November 2023; Editorial Decision 23 November 2023; Accepted 11 December 2023 © The Author(s) 2024. Published by Oxford University Press on behalf of The Japanese Society of Microscopy. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. * Microscopy, 2024, Vol. 73, No. 3 227 research findings morphogenesis and illustrate how our understanding has progressed through the use of these imaging and analysis techniques. Three-dimensional imaging microscopy for morphogenesis research Spinning-disk confocal microscopy Conventional confocal microscopy has been used as a standard method for acquiring 3D images. However, the speed and Two-photon microscopy Two-photon excitation microscopy is suitable for deep-tissue observation (Table 1, Fig. 1b). Unlike the majority of fluorescent microscopes that rely on the absorption of a single photon to excite fluorophores, this technique uses two-photon absorption. Two-photon excitation refers to the excitation of a single fluorophore by simultaneous absorption of two or more photons with lower energy than that of the fluorescent light. In contrast to single-photon excitation, for which the excitation light needs to have a shorter wavelength Table 1. Typical features of microscopy techniques Microscopy Conventional confocal Objective Speed Resolution (x × z) Field Imaging depth Spinning-disk Confocal Two-photon Light-sheet 4×/0.16 <∼30 fps ∼1.5 × 40 μm 100×/1.45 4×/0.16 <∼1000 fps ∼0.2 × 1 μm 2 × 100 μm 100×/1.45 10×/0.6 Obj <∼30 fps ∼0.2 × 1 μm >0.7 × 7 μm 25×/1.0 Obj 5×/0.1&5×/0.16 <∼100 fps >0.4 × 2.5 μm ∼2 × 14 μm∼ 10×/0.2&20×/1.0 ∼4500 μm <100 μ (...truncated)