Probing Temperature-Induced Plasmonic Nonlinearity: Unveiling Opto-Thermal Effects on Light Absorption and Near-Field Enhancement.

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.

Optical Bistability in Nanosilicon with Record Low Q-Factor.

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.

Spinning disk interferometric scattering confocal microscopy captures millisecond timescale dynamics of living cells.

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.

Towards stimulated Raman scattering spectro-microscopy across the entire Raman active region using a multiple-plate continuum.

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.

Nonlinear heating and scattering in a single crystalline silicon nanostructure.

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.

Millisecond two-photon optical ribbon imaging for small-animal functional connectome study.

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.

Optimizing depth-of-field extension in optical sectioning microscopy techniques using a fast focus-tunable lens.

Volume imaging based on a fast focus-tunable lens (FTL) allows three-dimensional (3D) observation within milliseconds by extending the depth-of-field (DOF) with sub-micrometer transverse resolution on optical sectioning microscopes. However, the previously published DOF extensions were neither axially uniform nor fit with theoretical prediction. In this work, complete theoretical treatments of focus extension with confocal and various multiphoton microscopes are established to correctly explain the previous results. Moreover, by correctly placing the FTL and properly adjusting incident beam diameter, a uniform DOF is achieved in which the actual extension nicely agrees with the theory. Our work not only provides a theoretical platform for volumetric imaging with FTL but also demonstrates the optimized imaging condition.

3D resolution enhancement of deep-tissue imaging based on virtual spatial overlap modulation microscopy.

During the last decades, several resolution enhancement methods for optical microscopy beyond diffraction limit have been developed. Nevertheless, those hardware-based techniques typically require strong illumination, and fail to improve resolution in deep tissue. Here we develop a high-speed computational approach, three-dimensional virtual spatial overlap modulation microscopy (3D-vSPOM), which immediately solves the strong-illumination issue. By amplifying only the spatial frequency component corresponding to the un-scattered point-spread-function at focus, plus 3D nonlinear value selection, 3D-vSPOM shows significant resolution enhancement in deep tissue. Since no iteration is required, 3D-vSPOM is much faster than iterative deconvolution. Compared to non-iterative deconvolution, 3D-vSPOM does not need a priori information of point-spread-function at deep tissue, and provides much better resolution enhancement plus greatly improved noise-immune response. This method is ready to be amalgamated with two-photon microscopy or other laser scanning microscopy to enhance deep-tissue resolution.

Temperature- and roughness- dependent permittivity of annealed/unannealed gold films.

Intrinsic absorption and subsequent heat generation have long been issues for metal-based plasmonics. Recently, thermo-plasmonics, which takes the advantage of such a thermal effect, is emerging as an important branch of plasmonics. However, although significant temperature increase is involved, characterization of metal permittivity at different temperatures and corresponding thermo-derivative are lacking. Here we measure gold permittivity from 300K to 570K, which the latter is enough for gold annealing. More than one order difference in thermo-derivative is revealed between annealed and unannealed films, resulting in a large variation of plasmonic properties. In addition, an unusual increase of imaginary permittivity after annealing is found. Both these effects can be attributed to the increased surface roughness incurred by annealing. Our results are valuable for characterizing extensively used unannealed nanoparticles, or annealed nanostructures, as building blocks in future thermo-nano-plasmonic systems.

Unusual imaging properties of superresolution microspheres.

We employ a self-assembly method to fabricate dielectric microsphere arrays that can be transferred to any desired positions. The arrays not only enable far-field, broad-band, high-speed, large-area, and wide-angle field of views but also achieve superresolution reaching λ/6.4. We also find that many proposed theories are insufficient to explain the imaging properties; including the achieved superresolution, effects of immersion, and unusual size-dependent magnification. The half-immersed microspheres certainly do not behave like any ordinary solid immersion lenses and new mechanisms must be incorporated to explain their unusual imaging properties.

Multiphoton imaging to identify grana, stroma thylakoid, and starch inside an intact leaf.

BACKGROUND: Grana and starch are major functional structures for photosynthesis and energy storage of plant, respectively. Both exhibit highly ordered molecular structures and appear as micrometer-sized granules inside chloroplasts. In order to distinguish grana and starch, we used multiphoton microscopy, with simultaneous acquisition of two-photon fluorescence (2PF) and second harmonic generation (SHG) signals. SHG is sensitive to crystallized structures while 2PF selectively reveals the distribution of chlorophyll. RESULT: Three distinct microstructures with different contrasts were observed, i.e. "SHG dominates", "2PF dominates", and "SHG collocated with 2PF". It is known that starch and grana both emit SHG due to their highly crystallized structures, and no autofluorescence is emitted from starch, so the "SHG dominates" contrast should correspond to starch. The contrast of "SHG collocated with 2PF" is assigned to be grana, which exhibit crystallized structure with autofluorescent chlorophyll. The "2PF dominates" contrast should correspond to stroma thylakoid, which is a non-packed membrane structure with chrolophyll. The contrast assignment is further supported by fluorescence lifetime measurement. CONCLUSION: We have demonstrated a straightforward and noninvasive method to identify the distribution of grana and starch within an intact leaf. By merging the 2PF and SHG images, grana, starch and stroma thylakoid can be visually distinguished. This approach can be extended to the observation of 3D grana distribution and their dynamics in living plants.

Point spread function analysis with saturable and reverse saturable scattering.

Nonlinear plasmonics has attracted a lot of interests due to its wide applications. Recently, we demonstrated saturation and reverse saturation of scattering from a single plasmonic nanoparticle, which exhibits extremely narrow side lobes and central peaks in scattering images [ACS Photonics 1(1), 32 (2014)]. It is desirable to extract the reversed saturated part to further enhance optical resolution. However, such separation is not possible with conventional confocal microscope. Here we combine reverse saturable scattering and saturated excitation (SAX) microscopy. With quantitative analyses of amplitude and phase of SAX signals, unexpectedly high-order nonlinearities are revealed. Our result provides greatly reduced width in point spread function of scattering-based optical microscopy. It will find applications in not only nonlinear material analysis, but also high-resolution biomedical microscopy.

Measurement of a saturated emission of optical radiation from gold nanoparticles: application to an ultrahigh resolution microscope.

We show that scattering from a single gold nanoparticle is saturable for the first time. Wavelength-dependent study reveals that the saturation behavior is governed by depletion of surface plasmon resonance, not the thermal effect. We observed interesting flattening of the point spread function of scattering from a single nanoparticle due to saturation. By extracting the saturated part of scattering via temporal modulation, we achieve λ/8 point spread function in far-field imaging with unambiguous separation of adjacent particles.

Multiplane differential saturated excitation microscopy using varifocal lenses.

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.

TAG-SPARK: Empowering High-Speed Volumetric Imaging With Deep Learning and Spatial Redundancy.

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.

Revealing intact neuronal circuitry in centimeter-sized formalin-fixed paraffin-embedded brain.

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.

Electronic Preresonance Stimulated Raman Scattering Spectromicroscopy Using Multiple-Plate Continuum.

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.

Multipole engineering by displacement resonance: a new degree of freedom of Mie resonance.

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.

Roadmap on Label-Free Super-Resolution Imaging.

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.

Observation of spontaneous polarization misalignments in periodically poled crystals using second-harmonic generation microscopy.

Periodically poled crystal (PPC) is a key component for nonlinear optical applications. Its poling quality relies largely on successful domain inversion and the alignment of spontaneous polarization (SP) vectors in each domain. Here we report the unexpected observation of bulk second harmonic generation (SHG) in PPC when excitation propagating along its optical axis. Based on its tensorial nature, SHG is highly sensitive to the orientation of SP, and therefore the misalignment of SP in each domain of PPC can be revealed noninvasively by SHG microscopy. This nonlinear imaging modality provides optical sectioning capability with 3D sub-micrometer resolution, so it will be useful for in situ investigation of poling quality in PPC.