Intelligent Image-Activated Cell Sorting.

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.

Alkyne-Tagged Dopamines as Versatile Analogue Probes for Dopaminergic System Analysis.

The dopaminergic system is essential for the function of the brain in health and disease. Therefore, detailed studies focused on unraveling the mechanisms involved in dopaminergic signaling are required. However, the lack of probes that mimic dopamine in living tissues, owing to the neurotransmitter's small size, has hampered analysis of the dopaminergic system. The current study aimed to overcome this limitation by developing alkyne-tagged dopamine compounds (ATDAs) that have a minimally invasive and uniquely identifiable alkyne group as a tag. ATDAs were established as chemically and functionally similar to dopamine and readily detectable by methods such as specific click chemistry and Raman scattering. The ATDAs developed here were verified as analogue probes that mimic dopamine in neurons and brain tissues, allowing the detailed characterization of dopamine dynamics. Therefore, ATDAs can act as safe and versatile tools with wide applicability in detailed studies of the dopaminergic system. Furthermore, our results suggest that the alkyne-tagging approach can also be applied to other small-sized neurotransmitters to facilitate characterization of their dynamics in the brain.

Multicolour chemical imaging of plant tissues with hyperspectral stimulated Raman scattering microscopy.

Recent development of stimulated Raman scattering (SRS) microscopy allows for label-free biological imaging with chemical specificity based on molecular-vibrational signatures. In particular, hyperspectral SRS imaging can acquire a molecular-vibrational spectrum at each pixel, allowing us not only to investigate the spectral difference of various biological molecules but also to discriminate different constituents based on their spectral difference. However, the number of constituents discriminated in previous label-free SRS imaging was limited to four because of the subtleness of spectral difference. Here, we report hyperspectral SRS imaging of plant tissues including leaves of Camellia japonica, roots of Arabidopsis thaliana, and thalli of a liverwort Marchantia polymorpha L. We show that SRS can discriminate as many as six components in Marchantia polymorpha L. without labeling. Our results demonstrate the effectiveness of hyperspectral SRS imaging as a tool for label-free multicolour imaging analysis of various biomolecules in plant tissues.

Time-Course Statistical Evaluation of Intercellular Adhesion Maturation by Femtosecond Laser Impulse.

The maturation of intercellular adhesion is an essential process for establishing the signal transduction network in living cells. Although the maturation is naturally considered to enhance the signal transduction, the relationship between the signal transduction and the maturation process has not been revealed in detail using time-course data. Here, using a coculture of mast cells and neurites, differences in maturation between individual cells were estimated as a function of the adhesion strength by our original single-cell measurement method utilizing a laser-induced impulsive force. When an intense femtosecond laser is focused into a culture medium under a microscope, shock and stress waves are generated at the laser focal point that exert an impulsive force on individual cells. In our method, this impulse is used to break the adhesion between a mast cell and a neurite. The magnitude of the impulse is then quantified by a local force-measurement system utilizing an atomic force microscope, and the adhesion strength is estimated from the threshold of the impulse required to break the adhesion. The measurement is conducted within 1 min/cell, and thus, data on the individual differences of the adhesion strength can be obtained within only a few hours. Coculturing of neurites and mast cells for 4 h resulted in a specific adhesion that was stronger than the nonspecific adhesions between the substrate and mast cells. In the time-course investigation, we identified two distinct temporal patterns of adhesion: 1) the strength at 24 h was the same as the initial strength; and 2) the strength increased threefold from baseline and became saturated within 24 h. Based on these results, the distribution of CADM1 adhesion molecules in the neurites was suggested to be inhomogeneous, and the relationship between adhesion maturation and the signal-transduction process was considered.

Noncontact estimation of intercellular breaking force using a femtosecond laser impulse quantified by atomic force microscopy.

When a femtosecond laser pulse (fsLP) is focused through an objective lens into a culture medium, an impulsive force (fsLP-IF) is generated that propagates from the laser focal point (O(f)) in a micron-sized space. This force can detach individual adherent cells without causing considerable cell damage. In this study, an fsLP-IF was reflected in the vibratory movement of an atomic force microscopy (AFM) cantilever. Based on the magnitude of the vibration and the geometrical relationship between O(f) and the cantilever, the fsLP-IF generated at O(f) was calculated as a unit of impulse [N-s]. This impulsive force broke adhesion molecule-mediated intercellular interactions in a manner that depended on the adhesion strength that was estimated by the cell aggregation assay. The force also broke the interactions between streptavidin-coated microspheres and a biotin-coated substrate with a measurement error of approximately 7%. These results suggest that fsLP-IF can be used to break intermolecular and intercellular interactions and estimate the adhesion strength. The fsLP-IF was used to break intercellular contacts in two biologically relevant cultures: a coculture of leukocytes seeded over on an endothelial cell monolayer, and a polarized monolayer culture of epithelial cells. The impulses needed to break leukocyte-endothelial and interepithelial interactions, which were calculated based on the geometrical relationship between O(f) and the adhesive interface, were on the order of 10(-13) and 10(-12) N-s, respectively. When the total impulse at O(f) is well-defined, fsLP-IF can be used to estimate the force required to break intercellular adhesions in a noncontact manner under biologically relevant conditions.

Enhanced nerve-mast cell interaction by a neuronal short isoform of cell adhesion molecule-1.

Close apposition of nerve and mast cells is viewed as a functional unit of neuro-immune mechanisms, and it is sustained by trans-homophilic binding of cell adhesion molecule-1 (CADM1), an Ig superfamily member. Cerebral nerve-mast cell interaction might be developmentally modulated, because the alternative splicing pattern of four (a-d) types of CADM1 transcripts drastically changed during development of the mouse cerebrum: developing cerebrums expressed CADM1b and CADM1c exclusively, while mature cerebrums expressed CADM1d additionally and predominantly. To probe how individual isoforms are involved in nerve-mast cell interaction, Neuro2a neuroblastoma cells that express CADM1c endogenously were modified to express additionally either CADM1b (Neuro2a-CADM1b) or CADM1d (Neuro2a-CADM1d), and they were cocultured with mouse bone marrow-derived mast cells (BMMCs) and BMMC-derived cell line IC-2 cells, both of which expressed CADM1c. BMMCs were found to adhere to Neuro2a-CADM1d neurites more firmly than to Neuro2a-CADM1b neurites when the adhesive strengths were estimated from the femtosecond laser-induced impulsive forces minimally required for detaching BMMCs. GFP-tagging and crosslinking experiments revealed that the firmer adhesion site consisted of an assembly of CADM1d cis-homodimers. When Neuro2a cells were specifically activated by histamine, intracellular Ca(2+) concentration was increased in 63 and 38% of CADM1c-expressing IC-2 cells that attached to the CADM1d assembly site and elsewhere, respectively. These results indicate that CADM1d is a specific neuronal isoform that enhances nerve-mast cell interaction, and they suggest that nerve-mast cell interaction may be reinforced as the brain grows mature because CADM1d becomes predominant.

Pulsed laser activated impulse response encoder (PLAIRE): sensitive evaluation of surface cellular stiffness on zebrafish embryos.

Mechanical properties of cells and tissues closely link to their architectures and physiological functions. To obtain the mechanical information of submillimeter scale small biological objects, we recently focused on the object vibration responses when excited by a femtosecond laser-induced impulsive force. These responses are monitored by the motion of an AFM cantilever placed on top of a sample. In this paper, we examined the surface cellular stiffness of zebrafish embryos based on excited vibration forms in different cytoskeletal states. The vibration responses were more sensitive to their surface cellular stiffness in comparison to the Young's modulus obtained by a conventional AFM force curve measurement.

Cellular internalization mechanism of novel Raman probes designed for plant cells.

Diphenylacetylene derivatives containing different polymeric components, poly(l-lysine) (pLys) or tetra(ethylene glycol) (TEG) were designed as novel Raman imaging probes with high Raman sensitivity and low cytotoxicity in living plant cells. The pLys-conjugated probe is internalized via an endocytosis-dependent pathway, whereas TEG-conjugated probe most likely induces direct penetration into the plant cells.

Raman image-activated cell sorting.

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.