Search

Two-photon high-speed fluorescence calcium imaging stands as a mainstream technique in neuroscience for capturing neural activities with high spatiotemporal resolution. However, challenges arise from the inherent tradeoff between acquisition speed and image quality, grappling with a low signal-to-noise ratio (SNR) due to limited signal photon flux. Here, a contrast-enhanced video-rate volumetric system, integrating a tunable acoustic gradient (TAG) lens-based high-speed microscopy with a TAG-SPARK denoising algorithm is demonstrated. The former facilitates high-speed dense z-sampling at sub-micrometer-scale intervals, allowing the latter to exploit the spatial redundancy of z-slices for self-supervised model training. This spatial redundancy-based approach, tailored for 4D (xyzt) dataset, not only achieves >700% SNR enhancement but also retains fast-spiking functional profiles of neuronal activities. High-speed plus high-quality images are exemplified by in vivo Purkinje cells calcium observation, revealing intriguing dendritic-to-somatic signal convolution, i.e., similar dendritic signals lead to reverse somatic responses. This tailored technique allows for capturing neuronal activities with high SNR, thus advancing the fundamental comprehension of neuronal transduction pathways within 3D neuronal architecture., (© 2024 The Author(s). Advanced Science published by Wiley‐VCH GmbH.)

Competing Interests: The authors have no conflicts of interest relevant to this article.

Tissue-clearing and labeling techniques have revolutionized brain-wide imaging and analysis, yet their application to clinical formalin-fixed paraffin-embedded (FFPE) blocks remains challenging. We introduce HIF-Clear, a novel method for efficiently clearing and labeling centimeter-thick FFPE specimens using elevated temperature and concentrated detergents. HIF-Clear with multi-round immunolabeling reveals neuron circuitry regulating multiple neurotransmitter systems in a whole FFPE mouse brain and is able to be used as the evaluation of disease treatment efficiency. HIF-Clear also supports expansion microscopy and can be performed on a non-sectioned 15-year-old FFPE specimen, as well as a 3-month formalin-fixed mouse brain. Thus, HIF-Clear represents a feasible approach for researching archived FFPE specimens for future neuroscientific and 3D neuropathological analyses., Competing Interests: YL, LW, YC, YC, SH, SW, CC, GH, SY, SC, KW, CL, PH, HC, BC, LC No competing interests declared, (© 2024, Lin et al.)

Precise measurement and control of local heating in plasmonic nanostructures are vital for diverse nanophotonic devices. Despite significant efforts, challenges in understanding temperature-induced plasmonic nonlinearity persist, particularly in light absorption and near-field enhancement due to the absence of suitable measurement techniques. This study presents an approach allowing simultaneous measurements of light absorption and near-field enhancement through angle-resolved near-field scanning optical microscopy with iterative opto-thermal analysis. We revealed gold thin films exhibit sublinear nonlinearity in near-field enhancement due to nonlinear opto-thermal effects, while light absorption shows both sublinear and superlinear behaviors at varying thicknesses. These observations align with predictions from a simple harmonic oscillation model, in which changes in damping parameters affect light absorption and field enhancement differently. The sensitivity of our method was experimentally examined by measuring the opto-thermal responses of three-dimensional nanostructure arrays. Our findings have direct implications for advancing plasmonic applications, including photocatalysis, photovoltaics, photothermal effects, and surface-enhanced Raman spectroscopy.

Raman scattering is sensitive to local temperature and thus offers a convenient tool for non-contact and non-destructive optical thermometry at the nanoscale. In turn, all-dielectric nanostructures, such as silicon particles, exhibit strongly enhanced photothermal heating due to Mie resonances, which leads to the strong modulation of elastic Rayleigh scattering intensity through subsequent thermo-optical effects. However, the influence of the complex photo-thermo-optical effect on inelastic Raman scattering has yet to be explored for resonant dielectric nanostructures. In this work, we experimentally demonstrate that the strong photo-thermo-optical interaction results in the nonlinear dependence of the Raman scattering signal intensity from a crystalline silicon nanoparticle via the thermal reconfiguration of the resonant response. Our results reveal a crucial role of the Mie resonance spectral sensitivity to temperature, which modifies not only the conversion of the incident light into heat but also Raman scattering efficiency. The developed comprehensive model provides the mechanism for thermal modulation of Raman scattering, shedding light on the photon-phonon interaction physics of resonant material, which is essential for the validation of Raman nanothermometry in resonant silicon structures under a strong laser field., Competing Interests: Conflict of interest: The authors declare no conflict of interest., (© 2024 the author(s), published by De Gruyter, Berlin/Boston.)

Saturated excitation microscopy, which collects nonlinear fluorescence signals generated by saturation, has been proposed to improve three-dimensional spatial resolution. Differential saturated excitation (dSAX) microscopy can further improve the detection efficiency of a nonlinear fluorescence signal. By comparing signals obtained at different saturation levels, high spatial resolution can be achieved in a simple and efficient manner. High-resolution multiplane microscopy is perquisite for volumetric imaging of thick samples. To the best of our knowledge, no reports of multiplane dSAX have been made. Our aim is to obtain multiplane high-resolution optically sectioned images by adapting differential saturated excitation in confocal laser scanning fluorescence microscopy. To perform multiplane dSAX microscopy, a variable focus lens is employed in a telecentric design to achieve focus tunability with constant magnification and contrast throughout the axial scanning range. Multiplane fluorescence imaging of two different types of pollen grains shows improved resolution and contrast. Our system's imaging performance is evaluated using standard targets, and the results are compared with standard confocal microscopy. Using a simple and efficient method, we demonstrate multiplane high-resolution fluorescence imaging. We anticipate that high-spatial resolution combined with high-speed focus tunability with invariant contrast and magnification will be useful in performing 3D imaging of thick biological samples., Competing Interests: The authors declare no conflicts of interest., (© 2024 Optica Publishing Group.)

We demonstrated optical bistability in an amorphous silicon Mie resonator with a size of ∼100 nm and Q -factor as low as ∼4 by utilizing photothermal and thermo-optical effects. We not only experimentally confirmed the steep intensity transition and the hysteresis in the scattering response from silicon nanocuboids but also established a physical model to numerically explain the underlying mechanism based on temperature-dependent competition between photothermal heating and heat dissipation. The transition between the bistable states offered particularly steep superlinearity of scattering intensity, reaching an effective nonlinearity order of ∼100th power over excitation intensity, leading to the potential of advanced optical switching devices and super-resolution microscopy.

Label-free super-resolution (LFSR) imaging relies on light-scattering processes in nanoscale objects without a need for fluorescent (FL) staining required in super-resolved FL microscopy. The objectives of this Roadmap are to present a comprehensive vision of the developments, the state-of-the-art in this field, and to discuss the resolution boundaries and hurdles which need to be overcome to break the classical diffraction limit of the LFSR imaging. The scope of this Roadmap spans from the advanced interference detection techniques, where the diffraction-limited lateral resolution is combined with unsurpassed axial and temporal resolution, to techniques with true lateral super-resolution capability which are based on understanding resolution as an information science problem, on using novel structured illumination, near-field scanning, and nonlinear optics approaches, and on designing superlenses based on nanoplasmonics, metamaterials, transformation optics, and microsphere-assisted approaches. To this end, this Roadmap brings under the same umbrella researchers from the physics and biomedical optics communities in which such studies have often been developing separately. The ultimate intent of this paper is to create a vision for the current and future developments of LFSR imaging based on its physical mechanisms and to create a great opening for the series of articles in this field.

The canonical studies on Mie scattering unravel strong electric/magnetic optical responses in nanostructures, laying foundation for emerging meta-photonic applications. Conventionally, the morphology-sensitive resonances hinge on the normalized frequency, i.e. particle size over wavelength, but non-paraxial incidence symmetry is overlooked. Here, through confocal reflection microscopy with a tight focus scanning over silicon nanostructures, the scattering point spread functions unveil distinctive spatial patterns featuring that linear scattering efficiency is maximal when the focus is misaligned. The underlying physical mechanism is the excitation of higher-order multipolar modes, not accessible by plane wave irradiation, via displacement resonance, which showcases a significant reduction of nonlinear response threshold, sign flip in all-optical switching, and spatial resolution enhancement. Our result fundamentally extends the century-old light scattering theory, and suggests new dimensions to tailor Mie resonances., (© 2023. The Author(s).)

In this paper, a fuzzy hierarchical optimization framework is proposed for identifying potential antiviral targets for treating severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection in the heart. The proposed framework comprises four objectives for evaluating the elimination of viral biomass growth and the minimization of side effects during treatment. In the application of the framework, Dulbecco's modified eagle medium (DMEM) and Ham's medium were used as uptake nutrients on an antiviral target discovery platform. The prediction results from the framework reveal that most of the antiviral enzymes in the aforementioned media are involved in fatty acid metabolism and amino acid metabolism. However, six enzymes involved in cholesterol biosynthesis in Ham's medium and three enzymes involved in glycolysis in DMEM are unable to eliminate the growth of the SARS-CoV-2 biomass. Three enzymes involved in glycolysis, namely BPGM, GAPDH, and ENO1, in DMEM combine with the supplemental uptake of L-cysteine to increase the cell viability grade and metabolic deviation grade. Moreover, six enzymes involved in cholesterol biosynthesis reduce and fail to reduce viral biomass growth in a culture medium if a cholesterol uptake reaction does not occur and occurs in this medium, respectively., (© 2023. The Author(s).)

Stimulated Raman scattering (SRS) spectromicroscopy is a powerful technique that enables label-free detection of chemical bonds with high specificity. However, the low Raman cross section due to typical far-electronic resonance excitation seriously restricts the sensitivity and undermines its application to bio-imaging. To address this bottleneck, the electronic preresonance (EPR) SRS technique has been developed to enhance the Raman signals by shifting the excitation frequency toward the molecular absorption. A fundamental weakness of the previous demonstration is the lack of dual-wavelength tunability, making EPR-SRS only applicable to a limited number of species in the proof-of-concept experiment. Here, we demonstrate the EPR-SRS spectromicroscopy using a multiple-plate continuum (MPC) light source able to examine a single vibration mode with independently adjustable pump and Stokes wavelengths. In our experiments, the C═C vibration mode of Alexa 635 is interrogated by continuously scanning the pump-to-absorption frequency detuning throughout the entire EPR region enabled by MPC. The results exhibit 150-fold SRS signal enhancement and good agreement with the Albrecht A-term preresonance model. Signal enhancement is also observed in EPR-SRS images of the whole Drosophila brain stained with Alexa 635. With the improved sensitivity and potential to implement hyperspectral measurement, we envision that MPC-EPR-SRS spectromicroscopy can bring the Raman techniques closer to a routine in bio-imaging.

In this paper, we theoretically and experimentally demonstrated photothermal nonlinearities of both forward and backward scattering intensities from quasi-perfect absorbing silicon-based metasurface with only λ /7 thickness. The metasurface is efficiently heated up by photothermal effect under laser irradiation, which in turn modulates the scattering spectra via thermo-optical effect. Under a few milliwatt continuous-wave excitation at the resonance wavelength of the metasurface, backward scattering cross-section doubles, and forward scattering cross-section reduces to half. Our study opens up the all-optical dynamical control of the scattering directionality, which would be applicable to silicon photonic devices., (© 2022 the author(s), published by De Gruyter, Berlin/Boston.)

Interferometric scattering (iSCAT) microscopy is a highly sensitive imaging technique that uses common-path interferometry to detect the linear scattering fields associated with samples. However, when measuring a complex sample, such as a biological cell, the superposition of the scattering signals from various sources, particularly those along the optical axis of the microscope objective, considerably complicates the data interpretation. Herein, we demonstrate high-speed, wide-field iSCAT microscopy in conjunction with confocal optical sectioning. Utilizing the multibeam scanning strategy of spinning disk confocal microscopy, our iSCAT confocal microscope acquires images at a rate of 1,000 frames per second (fps). The configurations of the spinning disk and the background correction procedures are described. The iSCAT confocal microscope is highly sensitive-individual 10 nm gold nanoparticles are successfully detected. Using high-speed iSCAT confocal imaging, we captured the rapid movements of single nanoparticles on the model membrane and single native vesicles in the living cells. Label-free iSCAT confocal imaging enables the detailed visualization of nanoscopic cell dynamics in their most native forms. This holds promise to unveil cell activities that are previously undescribed by fluorescence-based microscopy.

Stimulated Raman scattering (SRS) has attracted increasing attention in bio-imaging because of the ability toward background-free molecular-specific acquisitions without fluorescence labeling. Nevertheless, the corresponding sensitivity and specificity remain far behind those of fluorescence techniques. Here, we demonstrate SRS spectro-microscopy driven by a multiple-plate continuum (MPC), whose octave-spanning bandwidth (600-1300 nm) and high spectral energy density (∼1 nJ/cm -1 ) enable spectroscopic interrogation across the entire Raman active region (0-4000 cm -1 ), SRS imaging of a Drosophila brain, and electronic pre-resonance (EPR) detection of a fluorescent dye. We envision that utilizing MPC light source will substantially enhance the sensitivity and specificity of SRS by implementing EPR mode and spectral multiplexing via accessing three or more coherent wavelengths.

The purpose of the study is to investigate how much of variance in Parkinson's Disease (PD) individuals' speech intelligibility could be predicted by seven speech fluency indicators (i.e., repetition , omission , distortion , correction , unfilled pauses , filled pauses, and speaking rate ). Speech data were retrieved from a database containing a reading task produced by a group of 16 English-speaking individuals with PD (Jaeger, Trivedi & Stadtchnitzer, 2019). The results from a multiple regression indicated that an addition of 54% of variance in the speech intelligibility scores among individuals with PD could be accounted for after the speakers' PD severity level measured based on Hoehn and Yahr's (1967) disease stage was included as a covariate. In addition, omission and correction were the two fluency indicators that contributed to the general intelligibility score in a statistically significant way. Specifically, for every one-unit gain in the number of correction and omission , speech intelligibility scores would decline by 0.687 and 0.131 point (out of a 7-point scale), respectively. The current study hence supported Magee, Copland, and Vogel's (2019) view that the language production abilities and quantified dysarthria measures among individuals with PD should be explored together. Additionally, the clinical implications based on the current findings were discussed., Competing Interests: The authors declare that there is no conflict of interest regarding the publication of this paper., (Copyright © 2022 Chin-Ting Liu et al.)

Purpose: To determine the effect of valproic acid (VPA) on bleb morphology and scar characteristics in a rabbit model of minimally invasive glaucoma surgery (MIGS)., Methods: Nine New Zealand white rabbits were subjected to MIGS with intraoperative implantation of the PreserFlo MicroShunt. Rabbits were then administered with subconjunctival injections of phosphate buffered saline (PBS) (n=4) or with VPA (n=5). Bleb morphology was examined by slit-lamp biomicroscopy and in vivo confocal microscopy. Postoperative day 28 tissues were examined by immunohistochemical evaluation and label-free multiphoton microscopy to visualise the collagen matrix, by terminal deoxynucleotidyl transferase dUTP nick-end labelling assay and immunofluorescent labelling for Ki67 expression to detect apoptosis and cell growth, and by real-time quantitative PCR to measure Col1a1 , Fn , and Smad6 transcript expression., Results: VPA-treated blebs were detectable on day 28, while the PBS-treated blebs were not detectable by day 14. VPA-treated blebs were diffuse, extended posteriorly with near normal conjunctival vascularity and featured a combination of reticular/blurred stromal pattern with evidence of relatively large stromal cysts. Instead of the deposition of thick, disorganised collagen fibres characteristic of the PBS bleb, the VPA bleb contained conspicuously thinner collagen fibres which were associated with similarly thinner fibronectin fibres. In corroboration, Col1a1 and Fn mRNA expression was reduced in the VPA blebs, while increased Smad6 expression implicated the disruption of the transforming growth factor beta pathway. Apoptosis and cell growth profiles appeared similar with both treatments., Conclusions: The results support the application of VPA to enhance bleb morphology associated with good bleb function in MIGS with no apparent cytotoxicity., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2022. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Valproic acid (VPA), widely used for the treatment of neurological disorders, has anti-fibrotic activity by reducing collagen production in the postoperative conjunctiva. In this study, we investigated the capacity of VPA to modulate the postoperative collagen architecture. Histochemical examination revealed that VPA treatment was associated with the formation of thinner collagen fibers in the postoperative days 7 and 14 scars. At the micrometer scale, measurements by quantitative multiphoton microscopy indicated that VPA reduced mean collagen fiber thickness by 1.25-fold. At the nanometer scale, collagen fibril thickness and diameter measured by transmission electron microscopy were decreased by 1.08- and 1.20-fold, respectively. Moreover, delicate filamentous structures in random aggregates or closely associated with collagen fibrils were frequently observed in VPA-treated tissue. At the molecular level, VPA reduced Col1a1 but induced Matn2, Matn3, and Matn4 in the postoperative day 7 conjunctival tissue. Elevation of matrilin protein expression induced by VPA was sustained till at least postoperative day 14. In primary conjunctival fibroblasts, Matn2 expression was resistant to both VPA and TGF-β2, Matn3 was sensitive to both VPA and TGF-β2 individually and synergistically, while Matn4 was modulable by VPA but not TGF-β2. MATN2, MATN3, and MATN4 localized in close association with COL1A1 in the postoperative conjunctiva. These data indicate that VPA has the capacity to reduce collagen fiber thickness and potentially collagen assembly, in association with matrilin upregulation. These properties suggest potential VPA application for the prevention of fibrotic progression in the postoperative conjunctiva. KEY MESSAGES: VPA reduces collagen fiber and fibril thickness in the postoperative scar. VPA disrupts collagen fiber assembly in conjunctival wound healing. VPA induces MATN2, MATN3, and MATN4 in the postoperative scar., (© 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Plasmonic structural color, in which vivid colors are generated via resonant nanostructures made of common plasmonic materials, such as noble metals have fueled worldwide interest in backlight-free displays. However, plasmonic colors that were withstanding ultrahigh temperatures without damage remain an unmet challenge due to the low melting point of noble metals. Here, we report the refractory hafnium nitride (HfN) plasmonic crystals that can generate full-visible color with a high image resolution of ∼63,500 dpi while withstanding a high temperature (900 °C). Plasmonic colors that reflect visible light could be attributed to the unique features in plasmonic HfN, a high bulk plasmon frequency of 3.1 eV, whichcould support localized surface plasmon resonance (LSPR) in the visible range. By tuning the wavelength of the LSPR, the reflective optical response can be controlled to generate the colors from blue to red across a wide gamut. The novel refractory plasmonic colors pave the way for emerging applications ranging from reflective displays to solar energy harvesting systems., (© 2022 Zong-Yi Chiao et al., published by De Gruyter, Berlin/Boston.)

Background: Coronavirus disease 2019 (COVID-19) has caused a substantial increase in mortality and economic and social disruption. The absence of US Food and Drug Administration-approved drugs for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) highlights the need for new therapeutic drugs to combat COVID-19., Methods: The present study proposed a fuzzy hierarchical optimization framework for identifying potential antiviral targets for COVID-19. The objectives in the decision-making problem were not only to evaluate the elimination of the virus growth, but also to minimize side effects causing treatment. The identified candidate targets could promote processes of drug discovery and development., Significant Findings: Our gene-centric method revealed that dihydroorotate dehydrogenase (DHODH) inhibition could reduce viral biomass growth and metabolic deviation by 99.4% and 65.6%, respectively, and increase cell viability by 70.4%. We also identified two-target combinations that could completely block viral biomass growth and more effectively prevent metabolic deviation. We also discovered that the inhibition of two antiviral metabolites, cytidine triphosphate (CTP) and uridine-5'-triphosphate (UTP), exhibits effects similar to those of molnupiravir, which is undergoing phase III clinical trials. Our predictions also indicate that CTP and UTP inhibition blocks viral RNA replication through a similar mechanism to that of molnupiravir., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2022 Taiwan Institute of Chemical Engineers. Published by Elsevier B.V. All rights reserved.)