[Development of Two-Photon Super-Resolution Microscopy].

Two-photon excitation microscopy enables in vivo deep-tissue imaging within organisms. This technique is based on two-photon excitation, a nonlinear optical process that uses near-infrared light for excitation, resulting in high tissue permeability. Notably, two-photon excitation occurs only near the focal plane; therefore, minimally invasive tomographic images can be obtained. Owing to these features, two-photon excitation microscopy is currently widely used in medical and life-science research, particularly in the domain of neuroscience for in vivo visualization of deep tissues. However, the use of long-wavelength excitation light in two-photon excitation microscopy has resulted in a larger focused spot size and relatively low spatial resolution, which is a limitation of this technique for further applications. Recent studies have described super-resolution microscopy techniques applied to two-photon excitation microscopy in an attempt to observe living organisms "as they are in their natural state" with high spatial resolution. We have also addressed this topic using an optical approach (two-photon stimulated emission depletion microscopy) and an image analysis approach (two-photon super-resolution radial fluctuation). Here, we describe these approaches together with a discussion of our recent accomplishments.

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.

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.

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.

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.

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.

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 [...].

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.

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.

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.

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.

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.

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.

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.

Distance between the center of the FAZ measured automatically and the highest foveal bulge using OCT-angiography in elderly healthy eyes.

The center of the fovea, termed the foveola, is the area of highest visual acuity, has the highest density of cone photoreceptors. We investigated the distance between the automatically-determined center of the foveal avascular zone (FAZ) and the manually-determined highest foveal bulge (FB) point using single swept-source optical coherence tomography angiography (OCTA) instrument. This cross-sectional study included 49 eyes of 49 individuals (34 women and 15 men; median age: 68 years) with no history of ocular disorders. The FAZ in the superficial capillary plexus was automatically determined using the Kanno-Saitama macro method, and the center of the FAZ was automatically determined using ellipse approximation. Another candidate foveal center, the highest FB point, was determined manually on the serial cross-sectional B-scan images. As a result, the foveal center was manually identified as the highest FB point on B-scan OCTA images. The center of the FAZ was more likely to be located inferior to the highest FB point (p = 0.031). In participants with a total (linear) distance of more than 50 µm between the center of the FAZ and the highest FB point, the displacement was significantly more in the horizontal direction than in the vertical direction (p = 0.017). These results can be applicable to further studies regarding the spatial relationships between the center of the FAZ and the highest FB point in various macular diseases or previously-treated eyes.

Examination of Age-Related Retinal Vascular Changes in the Macula Using Optical Coherence Tomography Angiography of the Eyes After Cataract Surgery.

PURPOSE: Optical coherence tomography angiography (OCTA) allows noninvasive observation of the retinal vasculature, and image analysis can be used to calculate the retinal vessel density and foveal avascular zone (FAZ) area. A previous study showed that macular vessel density and the signal strength index (SSI) of OCTA images increased significantly after cataract surgery. However, the effect of aging on OCTA analysis remains unclear. This study aimed to investigate age-related changes in macular vascularization while excluding the effects of cataracts. PATIENTS AND METHODS: OCTA imaging of the macula was performed in adult patients who had undergone cataract surgery between February 2018 and May 2019 and in young healthy participants between April 2017 and April 2018. RESULTS: The median (quartiles) age of the overall study population was 59.0 (27.0, 69.0) years, and no severe refractive error was observed (axial length: 24.2 ± 1.3 [mean ± SD] mm; spherical equivalent: 0.75 [-2.25, 0.00] diopter). When we investigated the relationship between macular vessel density and age after excluding the effects of cataracts, we found that the macular vessel density showed age-related changes even in the absence of the effects of the SSI. However, the FAZ area was not affected by age. CONCLUSION: Macular vessel density decreased with age, even when the effects of cataracts were excluded. The effects of age and cataracts should be considered when designing studies and interpreting OCTA findings of the retinal vasculature.

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.

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.

Comparison of central visual sensitivity between monocular and binocular testing in advanced glaucoma patients using imo perimetry.

BACKGROUND/AIM: This study aimed to compare central visual sensitivity under monocular and binocular conditions in patients with glaucoma using the new imo static perimetry. METHODS: Fifty-one consecutive eyes of 51 patients with open-angle glaucoma who were affected with at least one significant point in the central 10° were examined in this cross-sectional study. Monocular and binocular random single-eye tests were performed using the imo perimeter and the Humphrey field analyser (HFA) 24-2 and 10-2 tests. The eyes were assigned to 'better' and 'worse' categories based on the visual acuity and central visual thresholding. Central visual sensitivity results obtained by monocular, binocular random single-eye tests and binocular simultaneous both eye test were compared. RESULTS: The average mean deviation with the HFA 24-2 was -5.5 (-1.5, -14.6) dB (median, (IQR)) in the better eyes and -18.0 (-12.9, -23.8) dB in the worse eyes. The mean sensitivity in the central 4 points of the visual field (VF) of the worse eyes was lower when measured under the binocular eye condition than under the monocular condition. Conversely, this value of the better eyes was greater when measured under the binocular eye condition than under the monocular condition. CONCLUSIONS: The central sensitivity of the better eyes was better and that of the worse eyes poorer with binocular testing than with monocular testing in patients with glaucoma. Although monocular VF testing is still the most straightforward means to monocularly monitor glaucoma at clinical settings, binocular testing, such as provided with imo perimetry, may be a useful clinical tool to predict the effect of VF impairments on a patient's quality of visual life.