Advanced easySTED microscopy based on two-photon excitation by electrical modulations of light pulse wavefronts.

We developed a compact stimulated emission depletion (STED) two-photon excitation microscopy that utilized electrically controllable components. Transmissive liquid crystal devices inserted directly in front of the objective lens converted the STED light into an optical vortex while leaving the excitation light unaffected. Light pulses of two different colors, 1.06 and 0.64 µm, were generated by laser diode-based light sources, and the delay between the two pulses was flexibly controlled so as to maximize the fluorescence suppression ratio. In our experiments, the spatial resolution of this system was up to three times higher than that obtained without STED light irradiation, and we successfully visualize the fine microtubule network structures in fixed mammalian cells without causing significant photo-damage.

Heterogeneous distribution of doublecortin-expressing cells surrounding the rostral migratory stream in the juvenile mouse.

In the postnatal mammalian brain, neural stem cells of the ventricular-subventricular zone continue to generate doublecortin (Dcx)-expressing immature neurons. Throughout life, these immature neurons migrate to the olfactory bulb through the rostral migratory stream (RMS). In this study, we investigated the distribution of these putative immature neurons using enhanced green fluorescent protein (EGFP) expression in the area surrounding the RMS of the juvenile Dcx-EGFP mice. Through the combined use of an optical clearing reagent (a 2,2'-thiodiethanol solution) and two-photon microscopy, we visualized three-dimensionally the EGFP-positive cells in the entire RMS and its surroundings. The resulting wide-field and high-definition images along with computational image processing methods developed in this study were used to comprehensively determine the position of the EGFP-positive cells. Our findings revealed that the EGFP-positive cells were heterogeneously distributed in the area surrounding the RMS. In addition, the orientation patterns of the leading process of these cells, which displayed the morphology of migrating immature neurons, differed depending on their location. These novel results provide highly precise morphological information for immature neurons and suggest that a portion of immature neurons may be detached from the RMS and migrate in various directions.

Development of 3D imaging technique of reconstructed human epidermis with immortalized human epidermal cell line.

The epidermis, the outermost layer of the skin, retains moisture and functions as a physical barrier against the external environment. Epidermal cells are continuously replaced by turnover, and thus to understand in detail the dynamic cellular events in the epidermis, techniques to observe live tissues in 3D are required. Here, we established a live 3D imaging technique for epidermis models. We first obtained immortalized human epidermal cell lines which have a normal differentiation capacity and fluorescence-labelled cytoplasm or nuclei. The reconstituted 3D epidermis was prepared with these lines. Using this culture system, we were able to observe the structure of the reconstituted epidermis live in 3D, which was similar to an in vivo epidermis, and evaluate the effect of a skin irritant. This technique may be useful for dermatological science and drug development.

Three-Dimensional Analysis of Cell Division Orientation in Epidermal Basal Layer Using Intravital Two-Photon Microscopy.

Epidermal structures are different among body sites, and proliferative keratinocytes in the epidermis play an important role in the maintenance of the epidermal structures. In recent years, intravital skin imaging has been used in mammalian skin research for the investigation of cell behaviors, but most of these experiments were performed with rodent ears. Here, we established a non-invasive intravital imaging approach for dorsal, ear, hind paw, or tail skin using R26H2BEGFP hairless mice. Using four-dimensional (x, y, z, and time) imaging, we successfully visualized mitotic cell division in epidermal basal cells. A comparison of cell division orientation relative to the basement membrane in each body site revealed that most divisions in dorsal and ear epidermis occurred in parallel, whereas the cell divisions in hind paw and tail epidermis occurred both in parallel and oblique orientations. Based on the quantitative analysis of the four-dimensional images, we showed that the epidermal thickness correlated with the basal cell density and the rate of the oblique divisions.

Transmissive liquid-crystal device for correcting primary coma aberration and astigmatism in biospecimen in two-photon excitation laser scanning microscopy.

All aberrations produced inside a biospecimen can degrade the quality of a three-dimensional image in two-photon excitation laser scanning microscopy. Previously, we developed a transmissive liquid-crystal device to correct spherical aberrations that improved the image quality of a fixed-mouse-brain slice treated with an optical clearing reagent. In this study, we developed a transmissive device that corrects primary coma aberration and astigmatism. The motivation for this study is that asymmetric aberration can be induced by the shape of a biospecimen and/or by a complicated refractive-index distribution in a sample; this can considerably degrade optical performance even near the sample surface. The device's performance was evaluated by observing fluorescence beads. The device was inserted between the objective lens and microscope revolver and succeeded in improving the spatial resolution and fluorescence signal of a bead image that was originally degraded by asymmetric aberration. Finally, we implemented the device for observing a fixed whole mouse brain with a sloping surface shape and complicated internal refractive-index distribution. The correction with the device improved the spatial resolution and increased the fluorescence signal by ?2.4×. The device can provide a simple approach to acquiring higher-quality images of biospecimens.

Correcting spherical aberrations in a biospecimen using a transmissive liquid crystal device in two-photon excitation laser scanning microscopy.

Two-photon excitation laser scanning microscopy has enabled the visualization of deep regions in a biospecimen. However, refractive-index mismatches in the optical path cause spherical aberrations that degrade spatial resolution and the fluorescence signal, especially during observation at deeper regions. Recently, we developed transmissive liquid-crystal devices for correcting spherical aberration without changing the basic design of the optical path in a conventional laser scanning microscope. In this study, the device was inserted in front of the objective lens and supplied with the appropriate voltage according to the observation depth. First, we evaluated the device by observing fluorescent beads in single- and two-photon excitation laser scanning microscopes. Using a 25× water-immersion objective lens with a numerical aperture of 1.1 and a sample with a refractive index of 1.38, the device recovered the spatial resolution and the fluorescence signal degraded within a depth of 0.6 mm. Finally, we implemented the device for observation of a mouse brain slice in a two-photon excitation laser scanning microscope. An optical clearing reagent with a refractive index of 1.42 rendered the fixed mouse brain transparent. The device improved the spatial resolution and the yellow fluorescent protein signal within a depth of 0-0.54 mm.

STED microscopy--super-resolution bio-imaging utilizing a stimulated emission depletion.

One of the most popular super-resolution microscopies that breaks the diffraction barrier is stimulated emission depletion (STED) microscopy. As the optical set-up of STED microscopy is based on a laser scanning microscopy (LSM) system, it potentially has several merits of LSM like confocal or two-photon excitation LSM. In this article, we first describe the principles of STED microscopy and then describe the features of our newly developed two-photon excitation STED microscopy. On the basis of our recent results and those of other researchers, we conclude by discussing future research and new technologies in this field.

Multi-point scanning two-photon excitation microscopy by utilizing a high-peak-power 1042-nm laser.

The temporal resolution of a two-photon excitation laser scanning microscopy (TPLSM) system is limited by the excitation laser beam's scanning speed. To improve the temporal resolution, the TPLSM system is equipped with a spinning-disk confocal scanning unit. However, the insufficient energy of a conventional Ti:sapphire laser source restricts the field of view (FOV) for TPLSM images to a narrow region. Therefore, we introduced a high-peak-power Yb-based laser in order to enlarge the FOV. This system provided three-dimensional imaging of a sufficiently deep and wide region of fixed mouse brain slices, clear four-dimensional imaging of actin dynamics in live mammalian cells and microtubule dynamics during mitosis and cytokinesis in live plant cells.

A rapid optical clearing protocol using 2,2'-thiodiethanol for microscopic observation of fixed mouse brain.

Elucidation of neural circuit functions requires visualization of the fine structure of neurons in the inner regions of thick brain specimens. However, the tissue penetration depth of laser scanning microscopy is limited by light scattering and/or absorption by the tissue. Recently, several optical clearing reagents have been proposed for visualization in fixed specimens. However, they require complicated protocols or long treatment times. Here we report the effects of 2,2'-thiodiethanol (TDE) solutions as an optical clearing reagent for fixed mouse brains expressing a yellow fluorescent protein. Immersion of fixed brains in TDE solutions rapidly (within 30 min in the case of 400-µm-thick fixed brain slices) increased their transparency and enhanced the penetration depth in both confocal and two-photon microscopy. In addition, we succeeded in visualizing dendritic spines along single dendrites at deep positions in fixed thick brain slices. These results suggest that our proposed protocol using TDE solution is a rapid and useful method for optical clearing of fixed specimens expressing fluorescent proteins.

Two-photon excitation fluorescence microscopy and its application in functional connectomics.

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.

Ultrasensitive imaging of Ca2+ dynamics in pancreatic acinar cells of yellow cameleon-nano transgenic mice.

Yellow Cameleons are genetically encoded Ca2+ indicators in which cyan and yellow fluorescent proteins and calmodulin work together as a fluorescence (Förster) resonance energy transfer Ca2+-sensor probe. To achieve ultrasensitive Ca2+ imaging for low resting Ca2+ or small Ca2+ transients in various organs, we generated a transgenic mouse line expressing the highest-sensitive genetically encoded Ca2+ indicator (Yellow Cameleon-Nano 15) in the whole body. We then focused on the mechanism of exocytotic events mediated by intracellular Ca2+ signaling in acinar cells of the mice with an agonist and observed them by two-photon excitation microscopy. In the results, two-photon excitation imaging of Yellow Cameleon-Nano 15 successfully visualized intracellular Ca2+ concentration under stimulation with the agonist at nanomolar levels. This is the first demonstration for application of genetically encoded Ca2+ indicators to pancreatic acinar cells. We also simultaneously observed exocytotic events and an intracellular Ca2+ concentration under in vivo condition. Yellow Cameleon-Nano 15 mice are healthy and no significant deteriorative effect was observed on physiological response regarding the pancreatic acinar cells. The dynamic range of 165% was calculated from Rmax and Rmin values under in vivo condition. The mice will be useful for ultrasensitive Ca2+ imaging in vivo.

Two-photon excitation STED microscopy by utilizing transmissive liquid crystal devices.

Transmissive liquid crystal devices (tLCDs) enable the modification of optical properties, such as phase, polarization, and laser light intensity, over a wide wavelength region at a high conversion efficiency. By utilizing tLCDs, we developed a new two-photon excitation stimulated emission depletion microscopy technique based on a conventional two-photon microscope. Spatial resolution was improved by compensating for phase shifts distributed in the optical path. Using this technique, we observed the fine structures of microtubule networks in fixed biological cells.

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.

Improvement of lateral resolution and extension of depth of field in two-photon microscopy by a higher-order radially polarized beam.

The spatial resolution of laser scanning microscopes depends on the focal spot size. As previously reported, we successfully improved the lateral spatial resolution in confocal microscopy using liquid crystal devices (LCDs) to convert a linearly polarized (LP) beam into a higher-order radially polarized (HRP) beam. Taking advantage of the fact that those LCDs can be utilized at various wavelengths, including near-infrared, we employed a near-infrared HRP beam to improve the resolution in two-photon microscopy. Point-spread functions estimated from fluorescent beads embedded in agarose gel showed that an HRP beam at 800-nm excitation improved lateral resolution to 230 nm from 294 nm, which was obtained using an LP beam at the same wavelength. Furthermore, at the glass-water interface, the lateral resolution was considerably improved to 188 nm using the HRP beam, whereas it degraded to 510 nm while using the LP beam. The HRP beams visualized fine intracellular structures not only in fixed cells stained with various dyes but also in living cells. Moreover, the HRP beam significantly extended the depth of field, which facilitated obtaining in-focus images, especially during time-lapse observations of living cells. These results indicate that our method is applicable to various biological applications.

Visualizing hippocampal neurons with in vivo two-photon microscopy using a 1030 nm picosecond pulse laser.

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 several hundred micrometers from the brain surface. We developed a novel semiconductor-laser-based light source with a wavelength of 1030 nm that can generate pulses of 5-picosecond duration with 2-W output power, and a 20-MHz repetition rate. We also developed a system to secure the head of the mouse under an upright microscope stage that has a horizontal adjustment mechanism. We examined the penetration depth while imaging the H-Line mouse brain and demonstrated that our newly developed laser successfully images not only cortex pyramidal neurons spreading to all cortex layers at a superior signal-to-background ratio, but also images hippocampal CA1 neurons in a young adult mouse.

Lateral resolution enhancement of laser scanning microscopy by a higher-order radially polarized mode beam.

We demonstrate that the lateral resolution of confocal laser scanning microscopy is dramatically improved by a higher-order radially polarized (HRP) beam with six concentric rings. This beam was generated simply by inserting liquid crystal devices in front of an objective lens. An HRP beam visualized aggregated 0.17 µm beads individually and is also applicable to biological imaging. This method can extend the capability of conventional laser scanning microscopes without modification of the system, with the exception of the addition of the liquid crystal devices in the optical path.

Downregulation of functional Reelin receptors in projection neurons implies that primary Reelin action occurs at early/premigratory stages.

Reelin signaling is essential for correct development of the mammalian brain. Reelin binds to apolipoprotein E receptor 2 and very low-density lipoprotein receptor and induces phosphorylation of Dab1. However, when and where these reactions occur is essentially unknown, and the primary function(s) of Reelin remain unclear. Here, we used alkaline phosphatase fusion of the receptor-binding region of Reelin to quantitatively investigate the localization of functional Reelin receptors (i.e., those on the plasma membrane as mature forms) in the developing brain. In the wild-type cerebral cortex, they are mainly present in the intermediate and subventricular zones, as well as in radial fibers, but much less in the cell bodies of the cortical plate. Functional Reelin receptors are much more abundant in the Reelin-deficient cortical plate, indicating that Reelin induces their downregulation and that it begins before the neurons migrate out of the intermediate zone. In the wild-type cerebellum, functional Reelin receptors are mainly present in the cerebellar ventricular zone but scarcely expressed by Purkinje cells that have migrated out of it. It is thus strongly suggested that Reelin exerts critical actions on migrating projection neurons at their early/premigratory stages en route to their final destinations, in the developing cerebral cortex and cerebellum.

The N-terminal fragment of Reelin is generated after endocytosis and released through the pathway regulated by Rab11.

Reelin is a large secreted glycoprotein essential for brain formation, but its trafficking and function at the molecular level remain incompletely understood. After binding to its receptor, Reelin is internalized by endocytosis. Here we show that internalized Reelin is subject to specific proteolysis within the cell and its N-terminal fragment is re-secreted. This re-secretion is inhibited by bafilomycin A(1) or by expression of a mutant of Rab11, a regulator of the recycling pathway. As the N-terminal fragment does not bind to Reelin receptor but has homology to F-spondin, its recycling may be involved in the regulation of extracellular matrix.

Splicing variations in the ligand-binding domain of ApoER2 results in functional differences in the binding properties to Reelin.

Reelin plays critical roles in brain formation by binding to apolipoprotein E receptor 2 (ApoER2) and very low-density lipoprotein receptor. Several isoforms and fragments of Reelin are generated by alternative splicing and proteolytic cleavage. In addition, two splice variants of ApoER2 have been recognized, namely, LA1237 and LA12378, that differ in the number of ligand-binding type A (LA) repeats. Here, we quantitatively investigated the affinity between various isoforms/fragments of Reelin and the ApoER2 splice variants. ApoER2-LA1237 bound rather strongly to the Reelin central fragment than to the fragment bearing Reelin repeat 8 (RR8). ApoER2-LA12378 bound comparably to all Reelin fragments without the C-terminal region. These findings suggest that LA8 of ApoER2 and RR8 interfere with the interaction between the Reelin central fragment and ApoER2. Using a monoclonal antibody that only recognizes ApoER2-LA12378, we found that this variant of ApoER2 was expressed in the cerebral cortical wall and in the internal granule cells of the cerebellum during development. Primary-cultured cortical neurons did not express ApoER2-LA12378, and the extent of signal activation by Reelin fragments was well correlated with their affinity for ApoER2-LA1237. Therefore, proteolytic cleavage of Reelin and alternative splicing of ApoER2 may be involved in the fine regulation of Reelin signaling.

The extremely conserved C-terminal region of Reelin is not necessary for secretion but is required for efficient activation of downstream signaling.

Reelin is a very large secreted glycoprotein essential for correct development of the mammalian brain. It is also implicated in higher functions and diseases of human brain. However, whether or not secretion of Reelin is regulated and how Reelin transmits signals remain largely unknown. Reelin protein is composed of an N-terminal F-spondin-like domain, Reelin repeats, and a short and highly basic C-terminal region (CTR). The primary sequence of CTR is almost completely conserved among vertebrates except fishes, indicating its importance. A prevailing idea regarding the function of CTR is that it is required for the secretion of Reelin, although this remains unproven. Here we aimed to clarify the function of Reelin CTR. Neither deleting most of CTR nor replacing CTR with unrelated amino acids affected secretion efficiency, indicating that CTR is not absolutely required for the secretion of Reelin. We also found that Reelin mutants without CTR were less potent in activating the downstream signaling in cortical neurons. Although these mutants were able to bind to the Reelin receptor ectodomain as efficiently as wild-type Reelin, quite interestingly, their ability to bind to the isolated cell membrane bearing Reelin receptors or receptor-expressing cells (including cortical neurons) was much weaker than that of wild-type Reelin. Therefore, it is concluded that the CTR of Reelin is not essential for its secretion but is required for efficient activation of downstream signaling events, presumably via binding to an unidentified "co-receptor" molecule(s) on the cell membrane.