Singularity biology is a scientific field that targets drastic state changes in multicellular systems, aiming to discover the key cells that induce the state change and investigate the mechanisms behind them. To achieve this goal, we developed a trans-scale optical imaging system (trans-scale scope), that is capable of capturing both macroscale changes across the entire system and the micro-scale behavior of individual cells, surpassing the cell observation capabilities of traditional microscopes. We developed two units of the trans-scale scope, named AMATERAS-1 and -2, which demonstrated the ability to observe multicellular systems consisting of over one million cells in a single field of view with sub-cellular resolution. This flagship instrument has been used to observe the dynamics of various cell species, with the advantage of being able to observe a large number of cells, allowing the detection and analysis of rare events and cells such as leader cells in multicellular pattern formation and cells that spontaneously initiate calcium waves. In this paper, we present the design concept of AMATERAS, the optical configuration, and several examples of observations, and demonstrate how the strength-in-numbers works in life sciences.

Identification of the neural circuits in the brain regulating animal behavior and physiology is critical for understanding brain functions and is one of the most challenging goals in neuroscience research. The fruitfly Drosophila melanogaster has often been used to identify the neural circuits involved in the regulation of specific behaviors because of the many neurogenetic tools available to express target genes in particular neurons. Neurons controlling sexual behavior, feeding behavior, and circadian rhythms have been identified, and the number of neurons responsible for controlling these phenomena is small. The search for a few neurons controlling a specific behavior is an important first step to clarify the overall picture of the neural circuits regulating that behavior. We previously found that the clock gene period (per), which is essential for circadian rhythms in Drosophila, is also essential for long-term memory (LTM). We have also found that a very limited number of per-expressing clock neurons in the adult brain are required for the consolidation and maintenance of LTM. In this review, we focus on LTM in Drosophila, introduce the concept of LTM regulation by a few clock neurons that we have recently discovered, and discuss how a few clock neurons regulate Drosophila LTM.

In a post-growth multicellular organism, the phenomenon in which a small number of rare cells can be the starting point for inducing a dramatic change in the entire system is considered a “biological singularity.” The immune response and cancer can be regarded as singularity phenomena in mammals, but their nature is fundamentally different. The immune response is considered a “programmed” singularity, whereas cancer is an “unprogrammed” singularity. These two systems perpetually engage in a cycle of attack and defense within the organism. The outcome is depending on the wining system, which determines whether the individual experiences a state resembling light or darkness. However, the overall mechanism of the competition remains unclear and is expected to be elucidated with future innovations in bioimaging technologies. Immune checkpoint blockade therapy is a means by which the two singularity balances can be artificially manipulated; therefore, mechanistic insight is necessary for cancer treatment strategies. Altogether, these findings provide a different perspective crucial for understanding the behavior of dynamic cell populations in multicellular organisms.

Understanding the temporal dynamics of T-cell transcription is crucial for insights into immune cell function and development. In this study, we show the features of the Timer-of-Cell-Kinetics-and-Activity (Tocky) system, which enables analysis of temporal dynamics of cell activities and differentiation, leveraging Fluorescent Timer protein, which spontaneously changes its emission spectrum from blue to red fluorescence in known kinetics, as reporters. The current study examines the properties of the Tocky system, highlighting the Timer-Angle approach, which is a core algorithm of Tocky analysis and converts Timer Blue and Red fluorescence into Timer Angle and Intensity by trigonometric transformation. Importantly, Tocky analyzes time-related events within individual cells by the two phases of measurements, distinguishing between (1) the temporal sequence of cellular activities and differentiation within the time domain, and (2) the transcription frequency within the frequency domain. The transition from time measurement to frequency analysis, particularly at the Persistent locus that bridges these domains, highlights that system’s unique property in what is measured and analyzed by Tocky. Intriguingly, the sustained transcriptional activities observed in cells at the Persistent locus may have unique biological features as demonstrated in activated regulatory T-cells (Treg) and pathogenic T-cells, respectively, using Foxp3-Tocky and Nr4a3-Tocky models. In conclusion, the Tocky system can provide crucial data for advancing our understanding of T-cell dynamics and function.

In collective systems, influence of individuals can permeate an entire group through indirect interactionscom-plicating any scheme to understand individual roles from observations. A typical approach to understand an individuals influence on another involves consideration of confounding factors, for example, by conditioning on other individuals outside of the pair. This becomes unfeasible in many cases as the number of individuals increases. In this article, we review some of the unforeseen problems that arise in understanding individual influence in a collective such as single cells, as well as some of the recent works which address these issues using tools from information theory.

Considering the fundamental mechanism causing singularity phenomena, we performed the following abduction: Assuming that a multicellular system is driven by spontaneous fluctuation of each cell and dynamic interaction of the cells, state transition of the system would be experimentally predictable from cellular heterogeneity. This study evaluates the abductive hypothesis by analyzing cellular heterogeneity to distinguish pre-state of state transition of differentiating cells with Raman spectroscopy and human induced pluripotent stem cells (hiPSCs) technique. Herein, we investigated the time development of cellular heterogeneity in Raman spectra during cardiomyogenesis of six hiPSC lines and tested two types of analyses for heterogeneity. As expected, some spectral peaks, possibly attributed to glycogen, correctively exhibited higher heterogeneity, prior to intensity changes of the spectrum in the both analyses in the all cell-lines tested. The combination of spectral data and heterogeneity-based analysis will be an approach to the arrival of biology that uses not only signal intensity but also heterogeneity as a biological index.

Bioluminescence imaging has recently attracted great attention as a highly sensitive and non-invasive analytical method. However, weak signal and low chemical stability of the luciferin are conventional drawbacks of bioluminescence imaging. In this review article, we describe the recent progress on the development and applications of bioluminescent probes for overcoming the aforementioned limitations, thereby enabling spatiotemporal trans-scale imaging. The detailed molecular design for manipulation of their luminescent properties and functions enabled a variety of applications, including in vivo deep tissue imaging, long-term imaging, and chemical sensor.

Singularity phenomena are rare events that occur only with a probability of one in tens of thousands and yet play an important role in the fate of the entire system. Recently, an ultra-wide-field microscopy imaging systems, AMATERAS, have been developed to reliably capture singularity phenomena. However, to determine whether a rare phenomenon captured by microscopy is a true singularity phenomenon—one with a significant impact on the entire system—, causal analysis is required. In this section, we introduce the CALI method, which uses light to inactivate molecules as one of the techniques enabling causal analysis. In addition, we discuss the technical innovations of the CALI method that are required to contribute to the future development of singularity biology.

Single-molecule imaging in living cells is an effective tool for elucidating the mechanisms of cellular phenomena at the molecular level. However, the analysis was not designed for throughput and requires high expertise, preventing it from reaching large scale, which is necessary when searching for rare cells that induce singularity phenomena. To overcome this limitation, we have automated the imaging procedures by combining our own focusing device, artificial intelligence, and robotics. The apparatus, called automated in-cell single-molecule imaging system (AiSIS), achieves a throughput that is a hundred-fold higher than conventional manual imaging operations, enabling the analysis of molecular events by individual cells across a large population. Here, using AiSIS, we demonstrate the single-molecule imaging of molecular behaviors and reactions related to tau protein aggregation, which is considered a singularity phenomenon in neurological disorders. Changes in the dynamics and kinetics of molecular events were observed inside and on the basal membrane of cells after the induction of aggregation. Additionally, to detect rare cells based on the molecular behavior, we developed a method to identify the state of individual cells defined by the quantitative distribution of molecular mobility and clustering. Using this method, cellular variations in receptor behavior were shown to decrease following ligand stimulation. This cell state analysis based on large-scale single-molecule imaging by AiSIS will advance the study of molecular mechanisms causing singularity phenomena.

During embryogenesis, human hematopoietic stem cells (HSCs) first emerge in the aorta-gonad-mesonephros (AGM) region via transformation of specialized hemogenic endothelial (HE) cells into premature HSC precursors. This process is termed endothelial-to-hematopoietic transition (EHT), in which the HE cells undergo drastic functional and morphological changes from flat, anchorage-dependent endothelial cells to free-floating round hematopoietic cells. Despite its essential role in human HSC development, molecular mechanisms underlying the EHT are largely unknown. This is due to lack of methods to visualize the emergence of human HSC precursors in real time in contrast to mouse and other model organisms. In this study, by inducing HE from human pluripotent stem cells in feeder-free monolayer cultures, we achieved real-time observation of the human EHT in vitro. By continuous observation and single-cell tracking in the culture, it was possible to visualize a process that a single endothelial cell gives rise to a hematopoietic cell and subsequently form a hematopoietic-cell cluster. The EHT was also confirmed by a drastic HE-to-HSC switching in molecular marker expressions. Notably, HSC precursor emergence was not linked to asymmetric cell division, whereas the hematopoietic cell cluster was formed through proliferation and assembling of the floating cells after the EHT. These results reveal unappreciated dynamics in the human EHT, and we anticipate that our human EHT model in vitro will provide an opportunity to improve our understanding of the human HSC development.