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.

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.

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.

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.

Intelligent image-activated cell sorting 2.0.

The advent of intelligent image-activated cell sorting (iIACS) has enabled high-throughput intelligent image-based sorting of single live cells from heterogeneous populations. iIACS is an on-chip microfluidic technology that builds on a seamless integration of a high-throughput fluorescence microscope, cell focuser, cell sorter, and deep neural network on a hybrid software-hardware data management architecture, thereby providing the combined merits of optical microscopy, fluorescence-activated cell sorting (FACS), and deep learning. Here we report an iIACS machine that far surpasses the state-of-the-art iIACS machine in system performance in order to expand the range of applications and discoveries enabled by the technology. Specifically, it provides a high throughput of ∼2000 events per second and a high sensitivity of ∼50 molecules of equivalent soluble fluorophores (MESFs), both of which are 20 times superior to those achieved in previous reports. This is made possible by employing (i) an image-sensor-based optomechanical flow imaging method known as virtual-freezing fluorescence imaging and (ii) a real-time intelligent image processor on an 8-PC server equipped with 8 multi-core CPUs and GPUs for intelligent decision-making, in order to significantly boost the imaging performance and computational power of the iIACS machine. We characterize the iIACS machine with fluorescent particles and various cell types and show that the performance of the iIACS machine is close to its achievable design specification. Equipped with the improved capabilities, this new generation of the iIACS technology holds promise for diverse applications in immunology, microbiology, stem cell biology, cancer biology, pathology, and synthetic biology.

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.

Author Correction: A practical guide to intelligent image-activated cell sorting.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

A practical guide to intelligent image-activated cell sorting.

Intelligent image-activated cell sorting (iIACS) is a machine-intelligence technology that performs real-time intelligent image-based sorting of single cells with high throughput. iIACS extends beyond the capabilities of fluorescence-activated cell sorting (FACS) from fluorescence intensity profiles of cells to multidimensional images, thereby enabling high-content sorting of cells or cell clusters with unique spatial chemical and morphological traits. Therefore, iIACS serves as an integral part of holistic single-cell analysis by enabling direct links between population-level analysis (flow cytometry), cell-level analysis (microscopy), and gene-level analysis (sequencing). Specifically, iIACS is based on a seamless integration of high-throughput cell microscopy (e.g., multicolor fluorescence imaging, bright-field imaging), cell focusing, cell sorting, and deep learning on a hybrid software-hardware data management infrastructure, enabling real-time automated operation for data acquisition, data processing, intelligent decision making, and actuation. Here, we provide a practical guide to iIACS that describes how to design, build, characterize, and use an iIACS machine. The guide includes the consideration of several important design parameters, such as throughput, sensitivity, dynamic range, image quality, sort purity, and sort yield; the development and integration of optical, microfluidic, electrical, computational, and mechanical components; and the characterization and practical usage of the integrated system. Assuming that all components are readily available, a team of several researchers experienced in optics, electronics, digital signal processing, microfluidics, mechatronics, and flow cytometry can complete this protocol in ~3 months.

High-speed microparticle isolation unlimited by Poisson statistics.

High-speed isolation of microparticles (e.g., microplastics, heavy metal particles, microbes, cells) from heterogeneous populations is the key element of high-throughput sorting instruments for chemical, biological, industrial and medical applications. Unfortunately, the performance of continuous microparticle isolation or so-called sorting is fundamentally limited by the trade-off between throughput, purity, and yield. For example, at a given throughput, high-purity sorting needs to sacrifice yield, or vice versa. This is due to Poisson statistics of events (i.e., microparticles, microparticle clusters, microparticle debris) in which the interval between successive events is stochastic and can be very short. Here we demonstrate an on-chip microparticle sorter with an ultrashort switching window in both time (10 µs) and space (10 µm) at a high flow speed of 1 m s-1, thereby overcoming the Poisson trade-off. This is made possible by using femtosecond laser pulses that can produce highly localized transient cavitation bubbles in a microchannel to kick target microparticles from an acoustically focused, densely aligned, bumper-to-bumper stream of microparticles. Our method is important for rare-microparticle sorting applications where both high purity and high yield are required to avoid missing rare microparticles.

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.

Targeted delivery of fluorogenic peptide aptamers into live microalgae by femtosecond laser photoporation at single-cell resolution.

Microalgae-based metabolic engineering has been proven effective for producing valuable substances such as food supplements, pharmaceutical drugs, biodegradable plastics, and biofuels in the past decade. The ability to accurately visualize and quantify intracellular metabolites in live microalgae is essential for efficient metabolic engineering, but remains a major challenge due to the lack of characterization methods. Here we demonstrate it by synthesizing fluorogenic peptide aptamers with specific binding affinity to a target metabolite and delivering them into live microalgae by femtosecond laser photoporation at single-cell resolution. As a proof-of-principle demonstration of our method, we use it to characterize Euglena gracilis, a photosynthetic unicellular motile microalgal species, which is capable of producing paramylon (a carbohydrate granule similar to starch). Specifically, we synthesize a peptide aptamer containing a paramylon-binding fluorescent probe, 7-nitrobenzofurazan, and introduce it into E. gracilis cells one-by-one by suppressing their mobility with mannitol and transiently perforating them with femtosecond laser pulses at 800 nm for photoporation. To demonstrate the method's practical utility in metabolic engineering, we perform spatially and temporally resolved fluorescence microscopy of single live photoporated E. gracilis cells under different culture conditions. Our method holds great promise for highly efficient microalgae-based metabolic engineering.

Quantitative analysis of mechanical force required for cell extrusion in zebrafish embryonic epithelia.

When cells in epithelial sheets are damaged by intrinsic or extrinsic causes, they are eliminated by extrusion from the sheet. Cell extrusion, which is required for maintenance of tissue integrity, is the consequence of contraction of actomyosin rings, as demonstrated by both molecular/cellular biological experimentation and numerical simulation. However, quantitative evaluation of actomyosin contraction has not been performed because of the lack of a suitable direct measurement system. In this study, we developed a new method using a femtosecond laser to quantify the contraction force of the actomyosin ring during cell extrusion in zebrafish embryonic epithelia. In this system, an epithelial cell in zebrafish embryo is first damaged by direct femtosecond laser irradiation. Next, a femtosecond laser-induced impulsive force is loaded onto the actomyosin ring, and the contraction force is quantified to be on the order of kPa as a unit of pressure. We found that cell extrusion was delayed when the contraction force was slightly attenuated, suggesting that a relatively small force is sufficient to drive cell extrusion. Thus, our method is suitable for the relative quantitative evaluation of mechanical dynamics in the process of cell extrusion, and in principle the method is applicable to similar phenomena in different tissues and organs of various species.

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.

An all-glass 12 µm ultra-thin and flexible micro-fluidic chip fabricated by femtosecond laser processing.

This study investigated and established a method, using femtosecond laser processing, to fabricate a 100%-glass-based 12 µm ultra-thin and flexible micro-fluidic chip. First we investigated the suitable pulse energy of the laser to fabricate ultra-thin glass sheets and then we fabricated a prototype glass micro-fluidic chip. Two 1 mm-in-diameter orifices for facilitating alignment in the bonding step and one channel with a width of 20 µm and a length of 25 mm were fabricated in a 4 µm thickness ultra-thin glass sheet using the femtosecond laser; this forms layer 2 in the completed device. Next, the glass sheet with the channel was sandwiched between another glass sheet having an inlet hole and an outlet hole (layer 1) and a base glass sheet (layer 3); the three sheets were bonded to each other, resulting in a flexible, 100%-glass micro-fluidic chip with a thickness of approximately 12 µm and a weight of 3.6 mg. The basic function of the glass micro-fluidic chip was confirmed by flowing 1 and 2 µm in-diameter bead particles through the channel. The fabrication method clearly scales down the thickness limitation of flexible glass devices and offers a possible element technology for fabricating ultra-thin glass devices that can be applied to convection-enhanced delivery, implantable medical devices, wearable devices, and high-resolution imaging of small biological objects such as bacteria and proteins in the channel.

Physical interaction between peroxisomes and chloroplasts elucidated by in situ laser analysis.

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.

On fundamental cellular processes for emergence of collective epithelial movement.

In all animals, collective cell movement is an essential process in many events, including wound healing and embryonic development. However, our understanding of what characterizes the emergence of multicellular collective behavior is still far from complete. In this article we showed the fundamental cellular processes that drive collective cell movement by means of integrated approaches, including precise quantification measurements and mathematical modeling of measured data. First, we observed the dependence of the collective behaviors of cultured human skin cells on Ca(2+) concentrations. When the culturing area confined by a PDMS sheet was suddenly expanded by removing the sheet, the group of cells moved to the expanded area with higher collectivity at higher Ca(2+) concentrations. Next, we quantitatively measured cellular responses to the Ca(2+) treatments, such as cell growth, cell division, and the strength of intercellular adhesion. Using a femtosecond-laser-based assay, an original method for estimating intercellular adhesion, we found that the strength of intercellular adhesion has an approximately 13-fold range in our treatments. Incorporating the quantitative data into a mathematical model, we then confirmed that the model well reproduced the multicellular behaviors we observed, demonstrating that the strength of intercellular adhesion sufficiently determines the generation of collective cell movement. Finally, we performed extensive numerical experiments, and the results suggested that the emergence of collective cell movement is derived by an optimal balance between the strength of intercellular adhesion and the intensity of cell migration.

Effect of the distance between adherent mesenchymal stem cell and the focus of irradiation of femtosecond laser on cell replication capacity.

The effect of femtosecond laser irradiation on adherent mesenchymal stem cell was investigated with the aim to develop a novel noninvasive cell purification system. A single mesenchymal stem cell was irradiated with a femtosecond laser on the center of the cell using several energy values and the cell lost its replication capacity with one time irradiation with an energy of 2.0 µJ. Besides at a neighbor point in the major axis, when irradiated in the minor axis at a distance shorter than 10 µm, the cell stopped its replication capacity. The accumulation effect of cell damage caused by multiple laser shots at a neighbor point in the minor axis was correlated with the critical distance at which the cell lost its replication capacity. Finally, a novel equation of laser cell damage as a function of laser pulse energy and laser shot number is proposed.

Photoporation of biomolecules into single cells in living vertebrate embryos induced by a femtosecond laser amplifier.

Introduction of biomolecules into cells in living animals is one of the most important techniques in molecular and developmental biology research, and has potentially broad biomedical implications. Here we report that biomolecules can be introduced into single cells in living vertebrate embryos by photoporation using a femtosecond laser amplifier with a high pulse energy and a low repetition rate. First, we confirmed the efficiency of this photoporation technique by introducing dextran, morpholino oligonucleotides, or DNA plasmids into targeted single cells of zebrafish, chick, shark, and mouse embryos. Second, we demonstrated that femtosecond laser irradiation efficiently delivered DNA plasmids into single neurons of chick embryos. Finally, we successfully manipulated the fate of single neurons in zebrafish embryos by delivering mRNA. Our observations suggest that photoporation using a femtosecond laser with a high pulse energy and low repetition rate offers a novel way to manipulate the function(s) of individual cells in a wide range of vertebrate embryos by introduction of selected biomolecules.

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.