Two-photon excitation fluorescence microscopy has become widely used in various life science fields in this decade. In the field of neuroscience in particular, in vivo two-photon microscopy has provided vital information on neural activity and brain function. In the current era of connectomics, visualization of the morphology and activity of numerous neurons in ever larger regions of the living brain are required within short periods. Based on this viewpoint, we discuss the fundamentals, advantages and potential of two-photon excitation fluorescence microscopy for the investigation of neural circuit functions.
In this paper, we proposed the possibility of implementing a single chip device for realizing optoelectronic integrated circuits (OEICs). Micro-light-emitting-diode (LED) arrays and a complementary metal–oxide–semiconductor (CMOS) pulse width modulation (PWM) silicon driver were proposed, designed, and fabricated on a single chip. The micro-LED arrays were separated by a dry etching method into 64 pixels of 8×8, each with a size of 30×30 µm2 and operated in 3 V at 100 µA. The PWM Si driver was well operated and modulated using various control signals. Furthermore, to investigate the driver for handling massive parallel information, a simple multifunctional driver was designed, fabricated, and flip-chip-bonded using a gold compliant bump and anisotropic conductive adhesive with micro-LED arrays.
For the field of neuroscience, laser-scanning florescence microscopy utilizing two-(or multi-) photon excitation process (two-(multi-)photon excitation laser scanning microscopy, two-(multi-)photon microscopy) has become widely used as an essential tool for biological and medical research including cancer, and immune researches. Especially, "in vivo" two-photon microscopy has revealed vital information on neural activity for brain function, even in light of its limitation in imaging events at depths greater than a several hundred micrometers from the brain surface. To break the limit of this penetration depth, we introduced a novel light source based on a semiconductor laser. The light source successfully visualized not only cortex layer V pyramidal neurons spreading to all cortex layers at a superior S/N ratio, but visualize hippocampal CA1 neurons in young adult mice. In addition, we developed liquid crystal devices to convert linearly polarized beams (LP) to vector beams. A liquid device generated a vector beam called higher-order radially polarized (HRP) beam, that enabled us to identify individual fluorescent beads of which diameter is 170 nm; smaller than classical PSF width. HRP beam also visualized finer structures of microtubules in fixed cells. Here, we will discuss these improvements and future application on the basis of our recent data.
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