ABSTRACT
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.
Subject(s)
Brain , Formaldehyde , Neurons , Paraffin Embedding , Tissue Fixation , Animals , Paraffin Embedding/methods , Mice , Tissue Fixation/methods , Neurons/physiology , Fixatives/chemistryABSTRACT
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.
ABSTRACT
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.
ABSTRACT
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.
ABSTRACT
We demonstrated optical bistability in an amorphous silicon Mie resonator with a size of â¼100 nm and Q-factor as low as â¼4 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 â¼100th power over excitation intensity, leading to the potential of advanced optical switching devices and super-resolution microscopy.
ABSTRACT
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 CâC 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.
ABSTRACT
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.
ABSTRACT
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.
Subject(s)
Gold , Metal Nanoparticles , Microscopy, Confocal/methods , Interferometry/methods , Microscopy, Fluorescence/methodsABSTRACT
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 (600-1300â nm) and high spectral energy density (â¼1 nJ/cm-1) enable spectroscopic interrogation across the entire Raman active region (0-4000â 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.
Subject(s)
Microscopy , Spectrum Analysis, Raman , Spectrum Analysis, Raman/methods , Microscopy/methods , Fluorescent Dyes , Nonlinear Optical Microscopy , VibrationABSTRACT
Silicon nanophotonics has attracted significant attention because of its unique optical properties such as efficient light confinement and low non-radiative loss. For practical applications such as all-optical switch, optical nonlinearity is a prerequisite, but the nonlinearity of silicon is intrinsically weak. Recently, we discovered a giant nonlinearity of scattering from a single silicon nanostructure by combining Mie resonance enhanced photo-thermal and thermo-optic effects. Since scattering and absorption are closely linked in Mie theory, we expect that absorption, as well as heating, of the silicon nanostructure shall exhibit similar nonlinear behaviors. In this work, we experimentally measure the temperature rise of a silicon nanoblock by in situ Raman spectroscopy, explicitly demonstrating the connection between nonlinear scattering and nonlinear heating. The results agree well with finite-element simulation based on the photo-thermo-optic effect, manifesting that the nonlinear effect is the coupled consequence of the red shift between scattering and absorption spectra. Our work not only unravels the nonlinear absorption in a silicon Mie-resonator but also offers a quantitative analytic model to better understand the complete photo-thermo-optic properties of silicon nanostructures, providing a new perspective toward practical silicon photonics applications.
ABSTRACT
Studying neural connections and activities in vivo is fundamental to understanding brain functions. Given the cm-size brain and three-dimensional neural circuit dynamics, deep-tissue, high-speed volumetric imaging is highly desirable for brain study. With sub-micrometer spatial resolution, intrinsic optical sectioning, and deep-tissue penetration capability, two-photon microscopy (2PM) has found a niche in neuroscience. However, the current 2PM typically relies on a slow axial scan for volumetric imaging, and the maximal penetration depth is only about 1â mm. Here, we demonstrate that by integrating a gradient-index (GRIN) lens and a tunable acoustic GRIN (TAG) lens into 2PM, both penetration depth and volume-imaging rate can be significantly improved. Specifically, an â¼ 1-cm long GRIN lens allows imaging relay from any target region of a mouse brain, while a TAG lens provides a sub-second volume rate via a 100 kHz â¼ 1â MHz axial scan. This technique enables the study of calcium dynamics in cm-deep brain regions with sub-cellular and sub-second spatiotemporal resolution, paving the way for interrogating deep-brain functional connectome.
ABSTRACT
A unified framework for the analysis of fluorescence data taken by a two-photon imaging system is presented. As in the processing of blood-oxygen-level-dependent signals of functional magnetic resonance imaging, the acquired functional images have to be co-registered with a structural brain atlas before delineating the regions activated by a given stimulus. The voxels whose calcium traces are highly correlated with the predicted responses are demarcated without the need for subjective reasoning. Experimental data acquired while presenting olfactory stimuli are used to demonstrate the efficacy of the proposed schemes. The results indicate that the functional images of a Drosophila individual can be normalized into a standard stereotactic space, and the expected brain regions can be delineated adequately. This framework provides an opportunity to enable the development of a Drosophila functional connectome database.
Subject(s)
Connectome , Drosophila , Animals , Brain/diagnostic imaging , Imaging, Three-Dimensional , Magnetic Resonance ImagingABSTRACT
An amendment to this paper has been published and can be accessed via a link at the top of the paper.
ABSTRACT
Silicon photonics have attracted significant interest because of their potential in integrated photonics components and all-dielectric meta-optics elements. One major challenge is to achieve active control via strong photon-photon interactions, i.e. optical nonlinearity, which is intrinsically weak in silicon. To boost the nonlinear response, practical applications rely on resonant structures such as microring resonators or photonic crystals. Nevertheless, their typical footprints are larger than 10 µm. Here, we show that 100 nm silicon nano-resonators exhibit a giant photothermal nonlinearity, yielding 90% reversible and repeatable modulation from linear scattering response at low excitation intensities. The equivalent nonlinear index is five-orders larger compared with bulk, based on Mie resonance enhanced absorption and high-efficiency heating in thermally isolated nanostructures. Furthermore, the nanoscale thermal relaxation time reaches nanosecond. This large and fast nonlinearity leads to potential applications for GHz all-optical control at the nanoscale and super-resolution imaging of silicon.
ABSTRACT
Featured with a plethora of electric and magnetic Mie resonances, high index dielectric nanostructures offer a versatile platform to concentrate light-matter interactions at the nanoscale. By integrating unique features of far-field scattering control and near-field concentration from radiationless anapole states, here, we demonstrate a giant photothermal nonlinearity in single subwavelength-sized silicon nanodisks. The nanoscale energy concentration and consequent near-field enhancements mediated by the anapole mode yield a reversible nonlinear scattering with a large modulation depth and a broad dynamic range, unveiling a record-high nonlinear index change up to 0.5 at mild incident light intensities on the order of MW/cm2. The observed photothermal nonlinearity showcases three orders of magnitude enhancement compared with that of unstructured bulk silicon, as well as nearly one order of magnitude higher than that through the radiative electric dipolar mode. Such nonlinear scattering can empower distinctive point spread functions in confocal reflectance imaging, offering the potential for far-field localization of nanostructured Si with an accuracy approaching 40 nm. Our findings shed new light on active silicon photonics based on optical anapoles.
ABSTRACT
Nonlinear nanoplasmonics is a largely unexplored research area that paves the way for many exciting applications, such as nanolasers, nanoantennas, and nanomodulators. In the field of nonlinear nanoplasmonics, it is highly desirable to characterize the nonlinearity of the optical absorption and scattering of single nanostructures. Currently, the common method to quantify optical nonlinearity is the z-scan technique, which yields real and imaginary parts of the permittivity by moving a thin sample with a laser beam. However, z-scan typically works with thin films, and thus acquires nonlinear responses from ensembles of nanostructures, not from single ones. In this work, we present an x-scan technique that is based on a confocal laser scanning microscope equipped with forward and backward detectors. The two-channel detection offers the simultaneous quantification for the nonlinear behavior of scattering, absorption and total attenuation by a single nanostructure. At low excitation intensities, both scattering and absorption responses are linear, thus confirming the linearity of the detection system. At high excitation intensities, we found that the nonlinear response can be derived directly from the point spread function of the x-scan images. Exceptionally large nonlinearities of both scattering and absorption are unraveled simultaneously for the first time. The present study not only provides a novel method for characterizing nonlinearity of a single nanostructure, but also reports surprisingly large plasmonic nonlinearities.
ABSTRACT
By coupling dye molecules with a graphene layer and localizing the molecules through quantification of fluorescence lifetime quenching, a novel imaging system offers unprecedented 1-nm resolution with angstrom precision in the axial dimension.
ABSTRACT
All-optical physiology (AOP) manipulates and reports neuronal activities with light, allowing for interrogation of neuronal functional connections with high spatiotemporal resolution. However, contemporary high-speed AOP platforms are limited to single-depth or discrete multi-plane recordings that are not suitable for studying functional connections among densely packed small neurons, such as neurons in Drosophila brains. Here, we constructed a 3D AOP platform by incorporating single-photon point stimulation and two-photon high-speed volumetric recordings with a tunable acoustic gradient-index (TAG) lens. We demonstrated the platform effectiveness by studying the anterior visual pathway (AVP) of Drosophila. We achieved functional observation of spatiotemporal coding and the strengths of calcium-sensitive connections between anterior optic tubercle (AOTU) sub-compartments and >70 tightly assembled 2-µm bulb (BU) microglomeruli in 3D coordinates with a single trial. Our work aids the establishment of in vivo 3D functional connectomes in neuron-dense brain areas.
ABSTRACT
We developed a high-speed two-photon optical ribbon imaging system, which combines galvo-mirrors for an arbitrary curve scan on a lateral plane and a tunable acoustic gradient-index lens for a 100 kHz-1 MHz axial scan. The system provides micrometer/millisecond spatiotemporal resolutions, which enable continuous readout of functional dynamics from small and densely packed neurons in a living adult Drosophila brain. Compared to sparse sampling techniques, the ribbon imaging modality avoids motion artifacts. Combined with a Drosophila anatomical connectome database, which is the most complete among all model animals, this technique paves the way toward establishing whole-brain functional connectome.
ABSTRACT
Drosophila is widely used in connectome studies due to its small brain size, sophisticated genetic tools, and the most complete single-neuron-based anatomical brain map. Surprisingly, even the brain thickness is only 200-µm, common Ti:sapphire-based two-photon excitation cannot penetrate, possibly due to light aberration/scattering of trachea. Here we quantitatively characterized scattering and light distortion of trachea-filled tissues, and found that trachea-induced light distortion dominates at long wavelength by comparing one-photon (488-nm), two-photon (920-nm), and three-photon (1300-nm) excitations. Whole-Drosophila-brain imaging is achieved by reducing tracheal light aberration/scattering via brain-degassing or long-wavelength excitation at 1300-nm. Our work paves the way toward constructing whole-brain connectome in a living Drosophila.