Graded arc beam in light needle microscopy for axially resolved, rapid volumetric imaging without nonlinear processes.

High-speed three-dimensional (3D) imaging is essential for revealing the structure and functions of biological specimens. Confocal laser scanning microscopy has been widely employed for this purpose. However, it requires a time-consuming image-stacking procedure. As a solution, we previously developed light needle microscopy using a Bessel beam with a wavefront-engineered approach [Biomed. Opt. Express13, 1702 (2022)10.1364/BOE.449329]. However, this method applies only to multiphoton excitation microscopy because of the requirement to reduce the sidelobes of the Bessel beam. Here, we introduce a beam that produces a needle spot while eluding the intractable artifacts due to the sidelobes. This beam can be adopted even in one-photon excitation fluorescence 3D imaging. The proposed method can achieve real-time, rapid 3D observation of 200-nm particles in water at a rate of over 50 volumes per second. In addition, fine structures, such as the spines of neurons in fixed mouse brain tissue, can be visualized in 3D from a single raster scan of the needle spot. The proposed method can be applied to various modalities in biological imaging, enabling rapid 3D image acquisition.

POLArIS, a versatile probe for molecular orientation, revealed actin filaments associated with microtubule asters in early embryos.

Biomolecular assemblies govern the physiology of cells. Their function often depends on the changes in molecular arrangements of constituents, both in the positions and orientations. While recent advancements of fluorescence microscopy including super-resolution microscopy have enabled us to determine the positions of fluorophores with unprecedented accuracy, monitoring the orientation of fluorescently labeled molecules within living cells in real time is challenging. Fluorescence polarization microscopy (FPM) reports the orientation of emission dipoles and is therefore a promising solution. For imaging with FPM, target proteins need labeling with fluorescent probes in a sterically constrained manner, but because of difficulties in the rational three-dimensional design of protein connection, a universal method for constrained tagging with fluorophore was not available. Here, we report POLArIS, a genetically encoded and versatile probe for molecular orientation imaging. Instead of using a direct tagging approach, we used a recombinant binder connected to a fluorescent protein in a sterically constrained manner that can target specific biomolecules of interest by combining with phage display screening. As an initial test case, we developed POLArISact, which specifically binds to F-actin in living cells. We confirmed that the orientation of F-actin can be monitored by observing cells expressing POLArISact with FPM. In living starfish early embryos expressing POLArISact, we found actin filaments radially extending from centrosomes in association with microtubule asters during mitosis. By taking advantage of the genetically encoded nature, POLArIS can be used in a variety of living specimens, including whole bodies of developing embryos and animals, and also be expressed in a cell type/tissue specific manner.

Massive cytoplasmic transport and microtubule organization in fertilized chordate eggs.

Eggs have developed their own strategies for early development. Amphibian, teleost fish, and ascidian eggs show cortical rotation and an accompanying structure, a cortical parallel microtubule (MT) array, during the one-cell embryonic stage. Cortical rotation is thought to relocate maternal deposits to a certain compartment of the egg and to polarize the embryo. The common features and differences among chordate eggs as well as localized maternal proteins and mRNAs that are related to the organization of MT structures are described in this review. Furthermore, recent studies report progress in elucidating the molecular nature and functions of the noncentrosomal MT organizing center (ncMTOC). The parallel array of MT bundles is presumably organized by ncMTOCs; therefore, the mechanism of ncMTOC control is likely inevitable for these species. Thus, the molecules related to the ncMTOC provide clues for understanding the mechanisms of early developmental systems, which ultimately determine the embryonic axis.

Microtubule array observed in the posterior-vegetal cortex during cytoplasmic and cortical reorganization of the ascidian egg.

Body axis formation during embryogenesis results from asymmetric localization of maternal factors in the egg. Shortly before the first cleavage in ascidian eggs, cell polarity along the anteroposterior (A-P) axis is established and the cytoplasmic domain (myoplasm) relocates from the vegetal to the posterior region in a microtubule-dependent manner. Through immunostaining, tubulin accumulation during this reorganization is observable on the myoplasm cortex. However, more detailed morphological features of microtubules remain relatively unknown. In this study, we invented a new reagent that improves the immunostaining of cortical microtubules and successfully visualized a parallel array of thick microtubules. During reorganization, they covered nearly the entire myoplasm cortical region, beneath the posterior-vegetal cortex. We designated this microtubule array as CAMP (cortical array of microtubules in posterior vegetal region). During the late phase of reorganization, CAMP shrank and the myoplasm formed a crescent-like cytoplasmic domain. When the CAMP formation was inhibited by sodium azide, myoplasmic reorganization and A-P axis formation were both abolished, suggesting that CAMP is important for these two processes.

Mitochondrial inhibitor sodium azide inhibits the reorganization of mitochondria-rich cytoplasm and the establishment of the anteroposterior axis in ascidian embryo.

In ascidian eggs, cytoplasmic and cortical reorganization, previously called ooplasmic segregation, occurs in two phases during the first cell cycle. In the second phase of reorganization, the mitochondria-rich cytoplasm (myoplasm) moves to the future posterior side, concurrent with sperm aster migration along the egg cortex. Although this reorganization is the critical step for establishing the anteroposterior axis, its molecular mechanism is not fully understood. In this study, we showed that low concentrations of the mitochondrial inhibitor sodium azide (NaN3 ), which showed the low toxicity in sperm, inhibited the second phase of reorganization without the microtubule depolymerization. In the NaN3 -treated embryo, the sperm aster was not attracted to the cortex and altered its migration pathway; therefore, the myoplasm remained at the vegetal pole. Consequently, the anteroposterior axis was not established. Another mitochondrial inhibitor, oligomycin, did not affect these processes. These results suggest that NaN3 inhibits unknown molecules that are important for the second phase of reorganization. Identifying the target molecule of NaN3 will lead to a molecular understanding of cytoplasmic and cortical reorganization.

Multibeam continuous axial scanning two-photon microscopy for in vivo volumetric imaging in mouse brain.

This study presents an alternative approach for two-photon volumetric imaging that combines multibeam lateral scanning with continuous axial scanning using a confocal spinning-disk scanner and an electrically focus tunable lens. Using this proposed system, the brain of a living mouse could be imaged at a penetration depth of over 450 µm from the surface. In vivo volumetric Ca2+ imaging at a volume rate of 1.5 Hz within a depth range of 130-200 µm, was segmented with an axial pitch of approximately 5-µm and revealed spontaneous activity of neurons with their 3D positions. This study offers a practical microscope design equipped with compact scanners, a simple control system, and readily adjustable imaging parameters, which is crucial for the widespread adoption of two-photon volumetric imaging.

Improving two-photon excitation microscopy for sharper and faster biological imaging.

Two-photon excitation laser scanning microscopy (TPLSM) has provided many insights into the life sciences, especially for thick biological specimens, because of its superior penetration depth and less invasiveness owing to the near-infrared wavelength of its excitation laser light. This paper introduces our four kinds of studies to improve TPLSM by utilizing several optical technologies as follows: (1) A high numerical aperture objective lens significantly deteriorates the focal spot size in deeper regions of specimens. Thus, approaches to adaptive optics were proposed to compensate for optical aberrations for deeper and sharper intravital brain imaging. (2) TPLSM spatial resolution has been improved by applying super-resolution microscopic techniques. We also developed a compact stimulated emission depletion (STED) TPLSM that utilizes electrically controllable components, transmissive liquid crystal devices, and laser diode-based light sources. The spatial resolution of the developed system was five times higher than conventional TPLSM. (3) Most TPLSM systems adopt moving mirrors for single-point laser beam scanning, resulting in the temporal resolution caused by the limited physical speed of these mirrors. For high-speed TPLSM imaging, a confocal spinning-disk scanner and newly-developed high-peak-power laser light sources enabled approximately 200 foci scanning. (4) Several researchers have proposed various volumetric imaging technologies. However, most technologies require large-scale and complicated optical setups based on deep expertise for microscopic technologies, resulting in a high threshold for biologists. Recently, an easy-to-use light-needle-creating device was proposed for conventional TPLSM systems to achieve one-touch volumetric imaging.

Advanced observation of brain and nerve cells using two-photon microscopy with novel techniques.

Two-photon excitation fluorescence microscopy [two-photon microscopy (2PM)] is a robust technique for understanding physiological phenomena from the cellular to tissue level, attributable to the nonlinear excitation process induced by near-infrared ultrashort laser light pulses. Recently, we have been promoting the use of semiconductor lasers, adaptive optics, vector beams and nanomaterials to improve the observation depth or spatial resolution. The developed semiconductor-based laser light source successfully visualized the structure of the enhanced yellow fluorescent protein (EYFP)-expressing neurons at the hippocampal dentate gyrus without resecting the neocortex and neuronal activity in the hippocampal cornu ammonis (CA1) region in anesthetized mice at video rates. We also proposed using fluoropolymer nanosheets of 100-nm thickness for in vivo imaging and realized a wide field of view during anesthetized mouse brain imaging of 1-mm depth. Furthermore, the developed adaptive optical 2PM visualized single dendritic spines of EYFP-expressing neurons in cortical layer V of the secondary motor cortex, which had been difficult to observe due to the curvature of the brain surface. In addition, we combined 2PM and stimulated emission depletion microscopy to improve spatial resolution. This combined microscopy is noninvasive and has a superior spatial resolution, exceeding the diffraction limit of the conventional light. In this review, we describe our recent results and discuss the future of 2PM.

All-synchronized picosecond pulses and time-gated detection improve the spatial resolution of two-photon STED microscopy in brain tissue imaging.

Super-resolution in two-photon excitation (2PE) microscopy offers new approaches for visualizing the deep inside the brain functions at the nanoscale. In this study, we developed a novel 2PE stimulated-emission-depletion (STED) microscope with all-synchronized picosecond pulse light sources and time-gated fluorescence detection, namely, all-pulsed 2PE-gSTED microscopy. The implementation of time-gating is critical to excluding undesirable signals derived from brain tissues. Even in a case using subnanosecond pulses for STED, the impact of time-gating was not negligible; the spatial resolution in the image of the brain tissue was improved by approximately 1.4 times compared with non time-gated image. This finding demonstrates that time-gating is more useful than previously thought for improving spatial resolution in brain tissue imaging. This microscopy will facilitate deeper super-resolution observation of the fine structure of neuronal dendritic spines and the intracellular dynamics in brain tissue.

Correction: Sasaki et al. Automatic Determination of the Center of Macular Hole Using Optical Coherence Tomography En Face Images. J. Clin. Med. 2022, 11, 3167.

There was an error in the original publication [...].

Cold-induced suspension and resetting of Ca2+ and transcriptional rhythms in the suprachiasmatic nucleus neurons.

Does the circadian clock keep running under such hypothermic states as daily torpor and hibernation? This fundamental question has been a research subject for decades but has remained unsettled. We addressed this subject by monitoring the circadian rhythm of clock gene transcription and intracellular Ca2+ in the neurons of the suprachiasmatic nucleus (SCN), master circadian clock, in vitro under a cold environment. We discovered that the transcriptional and Ca2+ rhythms are maintained at 22°C-28°C, but suspended at 15°C, accompanied by a large Ca2+ increase. Rewarming instantly resets the Ca2+ rhythms, while transcriptional rhythms reach a stable phase after the transient state and recover their phase relationship with the Ca2+ rhythm. We conclude that SCN neurons remain functional under moderate hypothermia but stop ticking in deep hypothermia and that the rhythms reset after rewarming. These data also indicate that stable Ca2+ oscillation precedes clock gene transcriptional rhythms in SCN neurons.

Cytosolic subunits of ATP synthase are localized to the cortical endoplasmic reticulum-rich domain of the ascidian egg myoplasm.

Previously, we revealed that p58, one of the ascidian maternal factors, is identical to the alpha-subunit of F1-ATP synthase (ATPα), a protein complex of the inner mitochondrial membrane. In the current study, we used immunological probes for ascidian mitochondria components to show that the ascidian ATPα is ectopically localized to the cytosol. Virtually all mitochondrial components were localized to the mitochondria-rich myoplasm. However, in detail, ATP synthase subunits and the matrix proteins showed different localization patterns. At least at the crescent stage, transmission electron microscopy (TEM) distinguished the mitochondria-less, endoplasmic reticulum (ER)-rich cortical region and the mitochondria-rich internal region. ATPα was enriched in the cortical region and MnSOD was limited to the internal region. Using subcellular fractionation, although all of the mitochondria components were highly enriched in the mitochondria-enriched fraction, a considerable amount of ATPα and F1-ATP synthase beta-subunit (ATPß) were recovered in the insoluble cytoplasmic fraction. Even under these conditions, F1-ATP synthase gamma-subunit (ATPγ) and F0-ATP synthase subunit b (ATPb) were not recovered in the insoluble cytoplasmic fraction. This result strongly supports the exomitochondrial localization of both ATPα and ATPß. In addition, the detergent extraction of eggs supports the idea that these cytosolic ATP synthase subunits are associated with the egg cytoskeleton. These results suggest that the subunits of ATP synthase might play dual roles at different subcellular compartments during early development.

Focusing new light on brain functions: multiphoton microscopy for deep and super-resolution imaging.

Multiphoton microscopy has become a powerful tool for visualizing neurobiological phenomena such as the dynamics of individual synapses and the functional activities of neurons. Owing to its near-infrared excitation laser wavelength, multiphoton microscopy achieves greater penetration depth and is less invasive than single-photon excitation. Here, we review the principles of two-photon microscopy and its technical limitations (penetration depth and spatial resolution) on brain tissue imaging. We then describe the technological improvements of two-photon microscopy that enable deeper imaging with higher spatial resolution for investigating unrevealed brain functions.

Automatic Determination of the Center of Macular Hole Using Optical Coherence Tomography En Face Images.

To evaluate the automated determination of the center of an idiopathic macular hole (MH) by using swept-source optical coherence tomography (OCT) images with new macro-based algorithms in ImageJ and to compare the difference between the MH center measurements obtained automatically and manually. This cross-sectional study included 39 eyes of 39 elderly individuals (22 women, 17 men) with stage 3 and 4 MH. The MH center was automatically determined using the ImageJ macro. The foveal center was also manually identified by two masked examiners using horizontal and vertical serial B-scan OCT angiography images. The mean age was 68.8 ± 8.3 years. After adjusting for the effect of magnification, the mean distance between the MH center determined manually by Examiner 1 and that determined automatically was 15.5 ± 9.9 µm. The mean distance between the two manually determined measurements of the MH center was 20.3 ± 19.7 µm. These two mean distance values did not differ significantly (Welch t-test, p = 0.27) and was non-inferior (p < 0.0001). The automated ImageJ-based method for determining the MH center was comparable to manual methods. This study showed that automated measurements were non-inferior to manual measurements, and demonstrated a substitutable usefulness, at least for use in clinical practice.

Relationship Between Deep Retinal Macular Vessel Density and Bipolar Cell Function in Glaucomatous Eyes.

Purpose: To evaluate the correlation between macular retinal function and the changes in the macular retinal vascular structure in glaucomatous eyes. Methods: The study included patients with glaucoma who visited Saitama Medical University and underwent optical coherence tomography angiography, and multifocal electroretinographic examinations at the same time between February 2020 and April 2021. Correlations among the ocular parameters, macular vessel density, and multifocal electroretinographic parameters were evaluated using a mixed model. Results: Forty-one eyes (mean deviation, -12.4 ± 7.8 dB) of 24 subjects (mean age, 75.2 ± 8.3 years) were included in the analysis. There were no significant correlations for macular vessel density in the superficial retinal layer. However, macular vessel density in the deep retinal layer showed a significant positive correlation with P1-N1 amplitude (coefficient = 0.724; P = 0.001). There were no significant correlations between the optical coherence tomography parameters and any of the multifocal electroretinographic parameters. Conclusions: A decrease in N1-P1 amplitude was observed in glaucomatous eyes in relation to a reduction in macular vessel density in the deep retinal layer, which suggests that ischemia-induced bipolar cell dysfunction may be involved in the intermediate retinal dysfunction associated with glaucoma. Translational Relevance: Intermediate retinal dysfunction in glaucoma is related to the changes in deep retinal microvasculature.

Deep Learning with a Dataset Created Using Kanno Saitama Macro, a Self-Made Automatic Foveal Avascular Zone Extraction Program.

The extraction of the foveal avascular zone (FAZ) from optical coherence tomography angiography (OCTA) images has been used in many studies in recent years due to its association with various ophthalmic diseases. In this study, we investigated the utility of a dataset for deep learning created using Kanno Saitama Macro (KSM), a program that automatically extracts the FAZ using swept-source OCTA. The test data included 40 eyes of 20 healthy volunteers. For training and validation, we used 257 eyes from 257 patients. The FAZ of the retinal surface image was extracted using KSM, and a dataset for FAZ extraction was created. Based on that dataset, we conducted a training test using a typical U-Net. Two examiners manually extracted the FAZ of the test data, and the results were used as gold standards to compare the Jaccard coefficients between examiners, and between each examiner and the U-Net. The Jaccard coefficient was 0.931 between examiner 1 and examiner 2, 0.951 between examiner 1 and the U-Net, and 0.933 between examiner 2 and the U-Net. The Jaccard coefficients were significantly better between examiner 1 and the U-Net than between examiner 1 and examiner 2 (p < 0.001). These data indicated that the dataset generated by KSM was as good as, if not better than, the agreement between examiners using the manual method. KSM may contribute to reducing the burden of annotation in deep learning.

Efficient visible/NIR light-driven uncaging of hydroxylated thiazole orange-based caged compounds in aqueous media.

In photoactivation strategies with bioactive molecules, one-photon visible or two-photon near-infrared light-sensitive caged compounds are desirable tools for biological applications because they offer reduced phototoxicity and deep tissue penetration. However, visible-light-sensitive photoremovable protecting groups (PPGs) reported so far have displayed high hydrophobicity and low uncaging cross sections (ÎµΦ < 50) in aqueous media, which can obstruct the control of bioactivity with high spatial and temporal precision. In this study, we developed hydroxylated thiazole orange (HTO) derivatives as visible-light-sensitive PPGs with high uncaging cross sections (ÎµΦ ≈ 370) in aqueous solution. In addition, 2PE photolysis reactions of HTO-caged glutamate were achieved using a NIR laser (940 nm). Moreover, HTO-caged glutamate can activate N-methyl-d-aspartic acid receptors in Xenopus oocytes and mammalian cells with green-light illumination, thus allowing optical control of biological functions.

Single-scan volumetric imaging throughout thick tissue specimens by one-touch installable light-needle creating device.

Biological tissues and their networks frequently change dynamically across large volumes. Understanding network operations requires monitoring their activities in three dimensions (3D) with single-cell resolution. Several researchers have proposed various volumetric imaging technologies. However, most technologies require large-scale and complicated optical setups, as well as deep expertise for microscopic technologies, resulting in a high threshold for biologists. In this study, we propose an easy-to-use light-needle creating device for conventional two-photon microscopy systems. By only installing the device in one position for a filter cube that conventional fluorescent microscopes have, single scanning of the excitation laser light beam excited fluorophores throughout over 200 µm thickness specimens simultaneously. Furthermore, the developed microscopy system successfully demonstrated single-scan visualization of the 3D structure of transparent YFP-expressing brain slices. Finally, in acute mouse cortical slices with a thickness of approximately 250 µm, we detected calcium activities with 7.5 Hz temporal resolution in the neuronal population.

OCT angiography measured changes in the foveal avascular zone area after glaucoma surgery.

BACKGROUND/AIMS: To evaluate quantitative changes in the foveal avascular zone (FAZ) area after glaucoma surgery using swept-source optical coherence tomography angiography (SS-OCTA). METHODS: Fifty-four consecutive patients with primary open-angle glaucoma (POAG) who met the inclusion criteria and underwent unilateral glaucoma surgery to reduce intraocular pressure (IOP) between April 2018 and July 2019.Eyes underwent IOP-lowering glaucoma surgery and their fellow (non-surgical) eyes were included. OCTA of the macula was performed in both eyes before glaucoma surgery and 3 months postoperatively. Two blinded examiners reviewed the image quality. Within- and between-group comparisons of the FAZ area and correlation of the FAZ area with age, IOP, central sensitivity and clinical variables. RESULTS: The mean (±SD) age was 66.7±11.3 years. After surgery, the IOP and FAZ area significantly decreased from 22.1±9.5 mmHg to 10.3±3.5 mmHg and from 0.485±0.193 mm2 to 0.446±0.174 mm2, respectively (both p<0.001). Conversely, in the non-surgery group, the preoperative and postoperative mean FAZ areas (0.398±0.119 mm2 and 0.396±0.110 mm2, respectively) did not significantly differ (p=0.469). Change in the FAZ area significantly correlated with the preoperative FAZ area, preoperative foveal sensitivity and change in IOP (all p<0.05). CONCLUSIONS: The FAZ area is decreased with IOP-lowering surgery in patients with POAG, and change in the FAZ area was significantly correlated with both preoperative foveal sensitivity and change in IOP.

Dynamic organization of cortical actin filaments during the ooplasmic segregation of ascidian Ciona eggs.

Spatial reorganization of cytoplasm in zygotic cells is critically important for establishing the body plans of many animal species. In ascidian zygotes, maternal determinants (mRNAs) are first transported to the vegetal pole a few minutes after fertilization and then to the future posterior side of the zygotes in a later phase of cytoplasmic reorganization, before the first cell division. Here, by using a novel fluorescence polarization microscope that reports the position and the orientation of fluorescently labeled proteins in living cells, we mapped the local alignments and the time-dependent changes of cortical actin networks in Ciona eggs. The initial cytoplasmic reorganization started with the contraction of vegetal hemisphere approximately 20 s after the fertilization-induced [Ca2+] increase. Timing of the vegetal contraction was consistent with the emergence of highly aligned actin filaments at the cell cortex of the vegetal hemisphere, which ran perpendicular to the animal-vegetal axis. We propose that the cytoplasmic reorganization is initiated by the local contraction of laterally aligned cortical actomyosin in the vegetal hemisphere, which in turn generates the directional movement of cytoplasm within the whole egg.