Super-resolution imaging of lateral distribution for the blue-light emission of an InGaN single-quantum-well structure utilizing the stimulated emission depletion effect.

We have observed a remarkable decrease in photoluminescence (PL) from a blue-light emitting InGaN single-quantum-well (SQW) structure under the radiation of a green laser due to the stimulated emission depletion (STED) phenomenon. By extending the observed STED effect, super-resolution imaging of the blue-light emission lateral distribution was demonstrated for the InGaN-SQW structure through co-irradiation using a doughnut-shaped green light beam and a Gaussian-shaped violet excitation light beam. We measured point-spread functions (PSFs) to evaluate the spatial resolution of the system by imaging a small emission area. A lateral PSF size of ~150 nm was confirmed, which was approximately 40% smaller than that without the STED beam. This demonstrates that the STED technique is applicable for PL imaging of semiconductor quantum structures. The present approach may make possible a new strategy for characterizing and investigating the spatial inhomogeneity of emission properties and carrier dynamics in InGaN-based quantum wells, as well as in other semiconductor materials exhibiting quantum confinement effects.

7-ps optical pulse generation from a 1064-nm gain-switched laser diode and its application for two-photon microscopy.

In this study, we investigated the picosecond optical pulse generation from a 1064-nm distributed feedback laser diode under strong gain switching. The spectrum of the generated optical pulses was manipulated in two different ways: (i) by extracting the short-wavelength components of the optical pulse spectrum and (ii) by compensating for spectral chirping in the extracted mid-spectral region. Both of these methods shortened the optical pulse duration to approximately 7 ps. These optical pulses were amplified to over 20-kW peak power for two-photon microscopy. We obtained clear two-photon images of neurons in a fixed brain slice of H-line mouse expressing enhanced yellow fluorescent protein. Furthermore, a successful experiment was also confirmed for in vivo deep region H-line mouse brain neuron imaging.

Gravity-driven microfluidic device placed on a slow-tilting table enables constant unidirectional perfusion culture of human induced pluripotent stem cells.

Gravity-driven microfluidics, which utilizes gravity force to drive liquid flow, offers portability and multi-condition setting flexibility because they do not require pumps or connection tubes to drive the flow. However, because the flow rate decreases with time in gravity-driven microfluidics, it is not suitable for stem cell experiments, which require long-term (at least a day) stability. In this study, gravity-driven microfluidics and a slow-tilting table were developed to culture cells under constant unidirectional perfusion. The microfluidic device was placed on a slow-tilting table, which tilts unidirectionally at a rate of approximately 7° per day to compensate for the reduction in the flow rate. Computational simulations showed that the pulsation of the flow arising from the stepwise movement of the table was less than 0.2%, and the flow was laminar. Hydrophilization of the tanks increased the flow rate, which is consistent with the theoretical values. We showed that vitronectin is better than laminin 511 fragments as a coating material for adhering human induced pluripotent stem cells on a microchamber made of polydimethylsiloxane, and succeeded in culturing the cells for 3 days. It is believed that the system offers easy-to-use cell culture tools, such as conventional multiwell culture vessels, and enables the control of the cell microenvironment.

Stepwise construction of dynamic microscale concentration gradients around hydrogel-encapsulated cells in a microfluidic perfusion culture device.

Inside living organisms, concentration gradients dynamically change over time as biological processes progress. Therefore, methods to construct dynamic microscale concentration gradients in a spatially controlled manner are needed to provide more realistic research environments. Here, we report a novel method for the construction of dynamic microscale concentration gradients in a stepwise manner around cells in micropatterned hydrogel. In our method, cells are encapsulated in a photodegradable hydrogel formed inside a microfluidic perfusion culture device, and perfusion microchannels are then fabricated in the hydrogel by micropatterned photodegradation. The cells in the micropatterned hydrogel can then be cultured by perfusing culture medium through the fabricated microchannels. By using this method, we demonstrate the simultaneous construction of two dynamic concentration gradients, which allowed us to expose the cells encapsulated in the hydrogel to a dynamic microenvironment.

High cell density suppresses BMP4-induced differentiation of human pluripotent stem cells to produce macroscopic spatial patterning in a unidirectional perfusion culture chamber.

Spatial pattern formation is a critical step in embryogenesis. Bone morphogenetic protein 4 (BMP4) and its inhibitors are major factors for the formation of spatial patterns during embryogenesis. However, spatial patterning of the human embryo is unclear because of ethical issues and isotropic culture environments resulting from conventional culture dishes. Here, we utilized human pluripotent stem cells (hiPSCs) and a simple anisotropic (unidirectional perfusion) culture chamber, which creates unidirectional conditions, to measure the cell community effect. The influence of cell density on BMP4-induced differentiation was explored during static culture using a conventional culture dish. Immunostaining of the early differentiation marker SSEA-1 and the mesendoderm marker BRACHYURY revealed that high cell density suppressed differentiation, with small clusters of differentiated and undifferentiated cells formed. Addition of five-fold higher concentration of BMP4 showed similar results, suggesting that suppression was not caused by depletion of BMP4 but rather by high cell density. Quantitative RT-PCR array analysis showed that BMP4 induced multi-lineage differentiation, which was also suppressed under high-density conditions. We fabricated an elongated perfusion culture chamber, in which proteins were transported unidirectionally, and hiPSCs were cultured with BMP4. At low density, the expression was the same throughout the chamber. However, at high density, SSEA-1 and BRACHYURY were expressed only in upstream cells, suggesting that some autocrine/paracrine factors inhibited the action of BMP4 in downstream cells to form the spatial pattern. Human iPSCs cultured in a perfusion culture chamber might be useful for studying in vitro macroscopic pattern formation in human embryogenesis.

In vivo two-photon imaging of mouse hippocampal neurons in dentate gyrus using a light source based on a high-peak power gain-switched laser diode.

In vivo two-photon microscopy is an advantageous technique for observing the mouse brain at high resolution. In this study, we developed a two-photon microscopy method that uses a 1064-nm gain-switched laser diode-based light source with average power above 4 W, pulse width of 7.5-picosecond, repetition rate of 10-MHz, and a high-sensitivity photomultiplier tube. Using this newly developed two-photon microscope for in vivo imaging, we were able to successfully image hippocampal neurons in the dentate gyrus and obtain panoramic views of CA1 pyramidal neurons and cerebral cortex, regardless of age of the mouse. Fine dendrites in hippocampal CA1 could be imaged with a high peak-signal-to-background ratio that could not be achieved by titanium sapphire laser excitation. Finally, our system achieved multicolor imaging with neurons and blood vessels in the hippocampal region in vivo. These results indicate that our two-photon microscopy system is suitable for investigations of various neural functions, including the morphological changes undergone by neurons during physiological phenomena.