In this talk, I introduce high-throughput intelligence-powered image-activated cell sorting or an “imaging” version of fluorescence-activated cell sorting [Cell 175, 1 (2018)] that realizes real-time image-based intelligent cell sorting at an unprecedented rate of ~100 events per second. This technology integrates high-speed cell microscopy, focusing, and sorting methods on rapid hybrid software-hardware data-management infrastructure, enabling real-time automated operation for data acquisition, data processing, decision-making, and actuation. I present two unique applications in microbiology and hematology enabled by the technology and discuss how technology enables machine-based scientific discovery in diverse biological and medical sciences.
I present a newly developed machine intelligence technology called “Intelligent Image-Activated Cell Sorting” [Cell 175, 1 (2018)] that achieves real-time fluorescence-image-based intelligent cell sorting at a high throughput of ~100 events per second. This technology builds on a unique integration of high-throughput cell microscopy, focusing, and sorting techniques on a unique software-hardware platform and hence performs fully automated operation for data acquisition, data processing, decision making, and actuation. Also, I introduce the technology’s broad utility to addressing a fundamental biological question that cannot be answered by conventional technologies – how molecular architectures of cells are connected with their physiological functions.
When an intense femtosecond laser pulse is focused into a culture medium through an objective lens, an impulsive force is loaded on the cells with generations of the shock and stress waves at the laser focal point. The shock and stress waves were acted to single cells in the vicinity of the laser focal point as an impulsive force. We have applied the impulsive force to manipulate single cells. As the transient intensity of the impulsive force is over 1000 times stronger than the force due to optical tweezers, drastic single manipulation which is difficult by the optical tweezers can be realized. The generation process of the impulsive force and behavior of animal cell after loading the impulsive force were reviewed, and then our original quantification method of the impulsive force utilizing atomic force microscope (AFM) was introduced with its applications for evaluating adhesions between animal cells and between sub-organelles in plant cell.
Single nerve cell’s mechanical response is an important issue for understanding function of nerve system, though, the response has been rarely clear. One of the factors is difficulty to stimulate the single cells by quantitative and controllable mechanical stress with subcellular spatial selectivity. As such mechanical stimulator, our group has focused on shock and stress waves generated by focusing the femtosecond laser under a microscope. When those waves impact on the biological cell, they act as an impulsive force. Although the impulsive force is available as a mechanical manipulator of the single cells, it was not confirmed that it could stimulate the nerve cells. Here we investigated the issue using neuro2a cells extending their neurite as an experimental model of nerve cell. Our results indicated that the impulsive force could be available as the stimulator to cause the mechanical response of the neuro2a cell.
A new methodology to estimate the dynamics of femtosecond laser-induced impulsive force generated into water under microscope was developed. In this method, the position shift of the bead in water before and after the femtosecond laser irradiation was investigated experimentally and compared with motion equation assuming stress wave propagation with expansion and collapse the cavitation bubble. In the process of the comparison, parameters of force and time of the stress wave were determined. From these results, dynamics of propagations of shock and stress waves, cavitation bubble generation, and these actions to micro-objects were speculated.
We developed a local force measurement system of a femtosecond laser-induced impulsive force, which is due to shock
and stress waves generated by focusing an intense femtosecond laser into water with a highly numerical aperture
objective lens. In this system, the force localized in micron-sized region was detected by bending movement of a
cantilever of atomic force microscope (AFM). Here we calculated the bending movement of the AFM cantilever when
the femtosecond laser is focused in water at the vicinity of the cantilever and the impulsive force is loaded on the
cantilever. From the result, a method to estimate the total of the impulsive force at the laser focal point was suggested
and applied to estimate intercellular adhesion strength.
Injection and delivery of small amount reagent in aqueous solution for cell chip was performed utilizing regeneratively
amplified femtosecond laser system. In our new trial, the reagent integrated on a solid strip are released and delivered to
targeted cells with the femutosecond laser-induced impulsive-force. The reagent was fixed in poly(vinyl alcohol) or
polystyrene film on a glass-substrate strip. When a single pulsed femtosecond laser was focused in the solution, the film
near the focal point was fragmented and the reagent was dispersed in 45-μm φ area at 50 μm from the surface of the
reagent strip. As examples cardiomyocyte beating cells of P19CL6 were bombed with epinephrine and acetylcholine,
and as a result the beating ratio of the cells were quickly stimulated and suppressed, respectively. The results
demonstrate that the present method is a promising key nano/micro technology for diagnosis and drug discovery.
We demonstrate preparations of zinc porphyrin nanoparticles by reprecipitation method and their spectroscopic
analysis by dark-field light scattering microspectroscopy. The size distribution of the prepared nanoparticles was 80-150
nm. By using dark-field illumination the nanoparticles could be observed as bright points in dark background and could
be examined by their Rayleigh scattering spectra at single particle level. The spectra differed from particle to particle,
which would be ascribed to their size and crystalline phase difference. Thus we have performed this single particle
spectroscopic technique to remove the ambiguity about the spectroscopic information owing to distributions of particles
and to improve the space selectivility. In addition, we have successfully demonstrated the detection of amine molecules
in water at single particle level. These results indicate that the detection technique using the single porphyrin
nanoparticles can be applied to chemical and biological sensors with nanometer scale.
Several kinds of manipulation of biological cells were performed utilizing regeneratively amplified femtosecond laser
system. When single-shot pulse of an amplified Ti: Sapphire femtosecond laser pulse is focused on a culture medium,
shockwave and cavitation bubble are generated with little heating. An impulsive force resulting in these phenomena was
applied to pttern specific cells form a culture substrate. Furthermore, laser trapping of cells was realized using high-repetition
rate pulses from the laser oscillator. Although the cell was trapped stably when the laser power was less than
100 mW, the cell was burst above the threshold laser power. The bursting would be due to heating inside cell, on which
the laser was focused and multiphoton absorption was induced. On the bases of these results, we propose a new
methodology to pattern biological cells, which is speedy and flexible when compared with previous micropatterning
methods.
PC12 cells, which are derived from a rat pheochromocytoma, were independently patterned utilizing an impulsive force
resulting in impulsive shockwave and cavitation bubble generation by focused femtosecond laser irradiation. Since the
PC12 cells respond reversibly to nerve growth factor by induction of the neuronal phenotype, we can assess an influence
that the impulsive force gives to the bioactivity in term of the cell differentiation. The patterned cells were accumulated
on an intact dish and cultured for 3 days. The behavior of appearance and cell differentiation was observed by multipoint
time-lapse system. On bases of these results, it was proved that the biological activity of the cell is unaffected by the
femtosecond laser patterning.
We have prepared nanoparticles of five organic dye molecules by laser ablation of their microcrystals in poor solvents
using the third harmonics of nanosecond Nd3+:YAG laser as an excitation light source. Their colloidal solutions were
stable for longer than 1 week without any surfactants. The mean size was almost common to all the dyes (about 50 nm)
and its distribution was narrow, which was confirmed by SEM observation. By applying electrophoretic deposition, the
homogeneous thin film of quinacridone nanoparticles was fabricated on an indium-tin-oxide electrode. It was
demonstrated that the films with different grain size and crystalline phase can be fabricated arbitrarily by using different
nanoparticles. Laser ablation is also useful for crystallization of organic molecules, which was demonstrated for a
representative organic nonlinear optical material.
Micro-patterning of proteins has been attracted much attention as a potential technique to realize bio-microdevice. In this work, as a new method to realize non-destructive micro-patterning of proteins, laser transfer printing for a um-sized protein crystal was developed by utilizing focused femtosecond laser. The micro-patterning was performed to transfer the protein crystal which was adhered on a source substrate to a target substrate which was underlaid on the source substrate. An 800-nm femtosecond laser was focused in a water between the source and target substrates on an inverted microscope with a 100x objective lens. When the laser focal point was scanned at the position with distance of a few um far from the source substrate, the protein crystals were detached by a shockwave and cavitation bubble generation at the circumstance of the focal point and transferred to the target substrate forming a line pattern. The line width of the protein crystal was a few tens um with the scanning speed of 90 μm/sec. Furthermore, multi-patterning of several kinds of protein crystals was realized by this method. The pattering resolution is comparable or better than that by another multi-material transfer printing, such as ink jet printing, micro-printing, and laser direct writing.
Laser manipulation system combining microfluidic and microimaging devices was developed, in which a cell sorting in a transparent microchip was successfully demonstrated. The microchip containing two microchambers was prepared with laser microfabrication, in which a solution containing yeast cells was injected as a sample. In the microchip, a cell transfer from one chamber to another one was performed by using single, fixed trapping laser beam. Furthermore, to realize an efficient cell sorting, the trapping laser bam was split into two by a polarizing beam splitter and each beam was modulated independently; one trapping beam was used to trap individual cells and to move them, which is freely controlled by a mouse pointer, and another was used to store the selected yeast cells with its liner scanning. In this method, the cells on a locus of the scanned beam were isolated to transfer in the microchip. From these results, it is concluded that shortening of the cell sorting time in microchip by a few time was realized by using dual-beam laser manipulation.
Time-resolved interferometry, surface light scattering imaging, and optical microscopic imaging have been developed and applied to amorphous and multicrystalline films upon intense pulsed excitation. Nanosecond interferometry of neat polystyrene film gives interesting expansion dynamics followed by complete recovery. Femtosecond surface light scattering imaging reveals the roughing processes of Copper phthalocyanine (CuPc) films. Femtosecond optical microscopic imaging of a single anthracene microcrystal shows dynamics of its laser-induced fracture and destruction. All the morphological behaviors have been directly measured in the time domain by these newly developed pump-probe methods. Ablation of CuPc crystalline and amorphous films show novel dependence of etch profile on excitation pulse width; fs etch depth increases stepwise with laser fluence, while ns etch depth becomes gradually deep as fluence does. The results are discussed in view of how electronic excitation energy evolves to morphological change.
Laser ablation and etching of microcrystalline Cu- phthalocyanine thin films were examined by changing pulse duration (170 fs, 250 ps, 100 ns) of a 780 nm Ti:sapphire laser. Above fs (40 mJ/cm2) and ps (50 mJ/cm2) ablation thresholds, the etch depth becomes constant and is almost independent of laser fluence, and further increase in the fs fluence results in complete removal of the film. We name the unique ablation phenomenon discrete etching. On the other hand, the depth etched by ns laser excitation increases gradually with the fluence above its ablation threshold (80 mJ/cm2. In order to reveal the difference between the fs and ns etching behaviors, we measured directly excitation energy relaxation and surface morphology change with time-resolved absorption spectroscopy and time- resolved surface scattering imaging, respectively. The fs discrete etching phenomenon and its mechanism were considered in view of time evolutions from highly intense fs laser excitation to the step-wise etching. On the basis of the results, we propose an fs laser ablation model that ultrafast stress increase brings about mechanical disruption leading to the discrete etching behavior.
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