A fundamental challenge of biology is to understand the vast heterogeneity of cells, particularly how cellular composition, structure, and morphology are linked to cellular physiology. Unfortunately, conventional technologies are limited in uncovering these relations. We present a machine-intelligence technology based on a radically different architecture that realizes real-time image-based intelligent cell sorting at an unprecedented rate. This technology, which we refer to as intelligent image-activated cell sorting, integrates high-throughput cell microscopy, focusing, and sorting on a hybrid software-hardware data-management infrastructure, enabling real-time automated operation for data acquisition, data processing, decision-making, and actuation. We use it to demonstrate real-time sorting of microalgal and blood cells based on intracellular protein localization and cell-cell interaction from large heterogeneous populations for studying photosynthesis and atherothrombosis, respectively. The technology is highly versatile and expected to enable machine-based scientific discovery in biological, pharmaceutical, and medical sciences.

2Plants can acclimate by using tropisms to link the direction of growth to 41 environmental conditions. Hydrotropism allows roots to forage for water, a process 42 known to depend on abscisic acid (ABA) but whose molecular and cellular basis 43 remains unclear. Here, we show that hydrotropism still occurs in roots after laser 44 ablation removed the meristem and root cap. Additionally, targeted expression 45 studies reveal that hydrotropism depends on the ABA signalling kinase, SnRK2.2, and 46 the hydrotropism-specific MIZ1, both acting specifically in elongation zone cortical 47 cells. Conversely, hydrotropism, but not gravitropism, is inhibited by preventing 48 differential cell-length increases in the cortex, but not in other cell types. We conclude 49 that root tropic responses to gravity and water are driven by distinct tissue-based 50 mechanisms. In addition, unlike its role in root gravitropism, the elongation zone 51 performs a dual function during a hydrotropic response, both sensing a water 52 potential gradient and subsequently undergoing differential growth. 53 3 Tropic responses are differential growth mechanisms that roots use to explore the 54 surrounding soil efficiently. In general, a tropic response can be divided into several steps, 55 comprising perception, signal transduction, and differential growth. All of these steps have 56 been well characterized for gravitropism, where gravity sensing cells in the columella of the 57 root cap generate a lateral auxin gradient, whilst adjacent lateral root cap cells transport 58 auxin to epidermal cells in the elongation zone, thereby triggering the differential growth that 59 drives bending [1][2][3][4] . In gravi-stimulated roots, the lateral auxin gradient is transported 60 principally by AUX1 and PIN carriers [3][4][5] . 61Compared with gravitropism, the tropic response to asymmetric water availability, i.e., 62 hydrotropism, has been far less studied. Previously, it was reported that surgical removal or 63 ablation of the root cap reduces hydrotropic bending in pea [6][7][8] and Arabidopsis thaliana 9 , 64suggesting that the machinery for sensing moisture gradients resides in the root cap. It has 65 also been reported that hydrotropic bending occurs due to differential growth in the 66 elongation zone 7,10 . However unlike gravitropism, hydrotropism in A. thaliana is independent 67 of AUX1 and PIN-mediated auxin transport 11,12 . Indeed, roots bend hydrotropically in the 68 absence of any redistribution of auxin detectable by auxin-responsive reporters 13,14 . 18,19 . 83However it is unclear whether this broad expression pattern is necessary for MIZ1's function 84 in hydrotropism or whether ABA signal transduction components in general have to be 85 expressed in specific root tip tissues for a hydrotropic response. The present study describes 86 a series of experiments in A. thaliana designed to identify the root tissues essential for a 87 hydrotropic response. We report that MIZ1 and a key ABA signal-transduction component 88SnRK2....

Life on earth relies upon photosynthesis, which consumes carbon dioxide and generates oxygen and carbohydrates. Photosynthesis is sustained by a dynamic environment within the plant cell involving numerous organelles with cytoplasmic streaming. Physiological studies of chloroplasts, mitochondria and peroxisomes show that these organelles actively communicate during photorespiration, a process by which by-products produced by photosynthesis are salvaged. Nevertheless, the mechanisms enabling efficient exchange of metabolites have not been clearly defined. We found that peroxisomes along chloroplasts changed shape from spherical to elliptical and their interaction area increased during photorespiration. We applied a recent femtosecond laser technology to analyse adhesion between the organelles inside palisade mesophyll cells of Arabidopsis leaves and succeeded in estimating their physical interactions under different environmental conditions. This is the first application of this estimation method within living cells. Our findings suggest that photosynthetic-dependent interactions play a critical role in ensuring efficient metabolite flow during photorespiration.

The advent of image-activated cell sorting and imaging-based cell picking has advanced our knowledge and exploitation of biological systems in the last decade. Unfortunately, they generally rely on fluorescent labeling for cellular phenotyping, an indirect measure of the molecular landscape in the cell, which has critical limitations. Here we demonstrate Raman image-activated cell sorting by directly probing chemically specific intracellular molecular vibrations via ultrafast multicolor stimulated Raman scattering (SRS) microscopy for cellular phenotyping. Specifically, the technology enables real-time SRS-image-based sorting of single live cells with a throughput of up to~100 events per second without the need for fluorescent labeling. To show the broad utility of the technology, we show its applicability to diverse cell types and sizes. The technology is highly versatile and holds promise for numerous applications that are previously difficult or undesirable with fluorescence-based technologies.

Actin and actin-associated proteins migrate within various cell types. To uncover the mechanism of their migration, we analyzed actin waves, which translocate actin and actin-associated proteins along neuronal axons toward the growth cones. We found that arrays of actin filaments constituting waves undergo directional assembly and disassembly, with their polymerizing ends oriented toward the axonal tip, and that the lateral side of the filaments is mechanically anchored to the adhesive substrate. A combination of live-cell imaging, molecular manipulation, force measurement, and mathematical modeling revealed that wave migration is driven by directional assembly and disassembly of actin filaments and their anchorage to the substrate. Actin-associated proteins co-migrate with actin filaments by interacting with them. Furthermore, blocking this migration, by creating an adhesion-free gap along the axon, disrupts axonal protrusion. Our findings identify a molecular mechanism that translocates actin and associated proteins toward the cell's leading edge, thereby promoting directional cell motility.

The upgraded version of intelligent image-activated cell sorting (iIACS) has enabled higher-throughput and more sensitive intelligent image-based sorting of single live cells from heterogeneous populations.

A mechanism of femtosecond laser-induced crystallization was investigated using a supersaturated solution of anthracene. When a single-shot femtosecond laser pulse with a pulse energy above 3.1 µJ/pulse was shot into a sufficiently supersaturated solution, crystallization of anthracene was induced immediately after irradiation at the vicinity of the laser focal point. The threshold energy of the crystallization (3.1 µJ/pulse) was in agreement with that of laser-induced bubble formation, which was a sequential process after shockwave emission, cavitation bubble formation, and collapse. Sufficient supersaturation for crystallization decreased with an increase in the pulse energy. These results suggest that crystallization is triggered in the processes resulting in bubble formation. Furthermore, crystallization was enhanced at the surface of the bubble. The crystallization mechanism was completely different from that reported previously based on photochemical reactions or molecular alignment due to a strong optical field.

The crystallization of urea was triggered using an intense 800 nm femtosecond laser that was focused to a supersaturated solution through an objective lens. An explosive crystallization proceeded in the entire sample glass tube for a few seconds at a concentration that no spontaneous nucleation occurred even after a few days. The crystallization was precisely monitored using a high-speed complementary metal oxide semiconductor (CMOS) camera attached to a microscope with a time resolution of 100 µs. On the basis of the results, the dynamic process of crystallization triggered by femtosecond laser ablation was discussed.