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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.)

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.

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 1 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 ∼ 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 ∼ 1 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., Competing Interests: The authors declare that there are no conflicts of interest related to this article., (© 2020 Optical Society of America under the terms of the OSA Open Access Publishing Agreement.)

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.

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 ., Competing Interests: The authors declare that there are no conflicts of interest related to this article.

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.

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.

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.

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.

Insulin-gold nanoclusters exhibit outstanding biocompatibility, photostability, and fluorescence quantum efficiency. However, they have never been used in superresolution microscopy, which requires nonlinear switching or saturation of fluorescence. Here we examine the fluorescence and stimulated emission depletion properties of gold nanoclusters. Their bleaching rate is very slow, demonstrating superior photostability. Surprisingly, however, the best depletion efficiency is less than 70%, whereas the depletion intensity requirement is much higher than the expectation from a simple two-level model. Fluorescence lifetime measurement revealed two distinct lifetime components, which indicate intersystem and reverse intersystem crossing during excitation. Based on population dynamic calculation, excellent agreement of the maximal depletion efficiency is found. Our work not only features the first examination of STED with metallic clusters, but also reveals the significance of molecular transition dynamics when considering a STED labeling.

A new, simple signal processing, low-cost technique for the fabrication of a portable oxygen sensor based on time-resolved fluorescence is described. The sensing film uses the oxygen sensing dye platinum meso-tetra (pentfluorophenyl) porphyrin (PtTFPP) embedded in a polymer matrix. The ratio τ 0 /τ 100 measures sensitivity of the sensing film, where τ 0 and τ 100 represent the detected fluorescence lifetimes from the sensing film exposed to 100% nitrogen and 100% oxygen, respectively. The experimental results reveal that the PtTFPP-doped oxygen sensor has a sensitivity of 2.2 in the 0%-100% range. A preparation procedure for coating the photodiodes with the oxygen sensor film that produces repetitive and reliable sensing devices is proposed. The developed time-resolved optical oxygen sensor is portable, low-cost, has simple signal processing, and lacks optical filter elements. It is a cost-effective alternative to traditional electrochemical-based oxygen sensors and provides a platform for other optical based sensors.

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.

We provide evidence that the chirality of collagen can give rise to strong second-harmonic generation circular dichroism (SHG-CD) responses in nonlinear microscopy. Although chirality is an intrinsic structural property of collagen, most of the previous studies ignore that property. We demonstrate chiral imaging of individual collagen fibers by using a laser scanning microscope and type-I collagen from pig ligaments. 100% contrast level of SHG-CD is achieved with sub-micrometer spatial resolution. As a new contrast mechanism for imaging chiral structures in bio-tissues, this technique provides information about collagen morphology and three-dimensional orientation of collagen molecules.

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.

We have demonstrated coherent anti-Stokes Raman scattering (CARS) microscopy with a single-pass picosecond supercontinuum-seeded optical parametric amplifier (SCOPA). The SCOPA was pumped by a frequency-doubled picosecond passively mode-locked Nd:YVO(4) laser, and was seeded by a supercontinuum light source. Compared with the conventional experimental setups of CARS microscopy, our exposition is substantially simpler because the pump and Stokes lasers are overlapped in the SCOPA automatically and thus steered into a microscope coherently. The feasibility of this novel light source to CARS imaging was illustrated by acquiring the fundamental and overtone CARS images of the aromatic C-H stretching mode of polystyrene beads and an image of the pharynx of a C. elegans of the aliphatic C-H stretching mode.

We have demonstrated a 0.7 microm - 1.9 microm wavelength-tunable light source based on a single-pass optical parametric amplification (OPA) in a multiperiod magnesium oxide-doped periodically poled lithium niobate crystal. The OPA pump was a frequency-doubled ultrafast ytterbium-doped fiber oscillator, and the residual 1040 nm laser power after frequency doubling was recycled to generate a supercontinuum seeding source. Compared with conventional OPAs, this system is free from timing jitter between the pump laser and the seeding source. Over 50% conversion efficiency was obtained with 10 nJ pump energy. Combined with a 50 MHz repetition rate, this versatile source is ideal for biomedical and spectroscopic applications.

We demonstrate detailed simulations and experiments of near-field phase-response in a single silver nanoparticle. The plasmon-photon interaction is directly observed in the vicinity of silver nanoparticles through a near-field scanning optical microscope (NSOM). Our results manifest the correlation of phase-response and size-dependent optical enhancement. Detailed interference behaviors between optical excitation and plasmon mediated re-radiation are revealed on a single particle basis. This observation facilitates nano-applications in controlling the spatial distribution of surface plasmon (SP) modes by means of nanostructures.

We studied the polarization anisotropy of second harmonic generation (SHG) in polyhedral inclusion bodies (PIBs) of nuclear polyhedrosis viruses (NPV). Due to a body-centered-cubic arrangement of polyhedrin trimers, a characteristic SHG polarization property with a mixture of I23 and I3 symmetry was measured from PIBs. With this characteristic SHG anisotropy, it provides an intrinsic nonlinear signal for virus infection studies in living cells. With multimodal harmonic generation microscopy, we also demonstrated 3D imaging on PIBs of NPV in living cells. The distribution and the number of PIBs in intact infected cells can be revealed without the help of fluorescent labeling.

Simultaneous backward and forward second harmonic generations from isolated type-I collagen matrix are observed. Optical interference behaviors of these nonlinear optical signals are studied with accurately determined fibril thickness by an atomic force microscope. The nonlinear emission directions are strongly dependent on the coherent interaction within and between collagen fibrils. A linear relationship is obtained to estimate collagen fibril thickness with nanometer precision noninvasively by evaluating the forward/backward second harmonic generation ratio.

We demonstrate what is to our knowledge the first example of four-photon luminescence microscopy in GaN and apply it to quality mapping of bulk GaN. The simultaneously acquired second- and third-harmonic generation can be used to map the distribution of the piezoelectric field and the band-tail state density, respectively. Through spectrum- and power-dependent studies, the fourth power dependence of the band edge luminescence is confirmed. The superb spatial resolution of the four-photon luminescence modality is also demonstrated. This technique provides a high-resolution, noninvasive monitoring and tool for examining the physical properties of semiconductors.

Because it avoids the in-focus photodamage and phototoxicity problem of two-photon-fluorescence excitation, multiharmonic-generation biopsy based on a 1200-1300-nm light source could provide a truly noninvasive and highly penetrative optical sectioning of skin. We study multiharmonic-generation biopsy of fixed mouse skin. Our preliminary study suggests that this technique could provide submicrometer-resolution deep-tissue noninvasive biopsy images in skin without the use of fluorescence and exogenous markers.

Morphological changes and complex developmental processes inside vertebrate embryos are difficult to observe noninvasively with millimeter-penetration and sub-micrometer-resolution at the same time. By using higher harmonic generation, including second and third harmonics, as the microscopic contrast mechanism, optical noninvasiveness can be achieved due to the virtual-level-transition characteristic. The intrinsic nonlinearity of harmonic generations provides optical sectioning capability while the selected 1230-nm near-infrared light source provides the deeppenetration ability. The complicated development within a ~1.5-mm thick zebrafish (Danio rerio) embryo from initial cell proliferation, gastrulation, to tissue formation can all be observed clearly in vivo without any treatment on the live specimen.

An electric-field-induced second-harmonic-generation signal in a nematic liquid crystal is used to map the electric field in an integrated-circuit-like sample. Since the electric-field-induced second-harmonic-generation signal intensity exhibits a strong dependence on the polarization of the incident laser beam, both the amplitude and the orientation of the electric field vectors can be measured. Combined with scanning second-harmonic-generation microscopy, three-dimensional electric field distribution can be easily visualized with high spatial resolution of the order of 1 microm.

The problem of weak harmonic generation signal intensity limited by photodamage probability in optical microscopy and spectroscopy could be resolved by increasing the repetition rate of the excitation light source. Here we demonstrate the first photomultiplier-based real-time second-harmonic-generation microscopy taking advantage of the strongly enhanced nonlinear signal from a high-repetition-rate Ti:sapphire laser. We also demonstrate that the photodamage possibility in common biological tissues can be efficiently reduced with this high repetition rate laser at a much higher average power level compared to the commonly used ~80- MHz repetition rate lasers.

We demonstrate a novel multimodal nonlinear spectral microscopy based on a femtosecond Cr:forsterite laser at 1230 nm. By acquiring the whole nonlinear spectrum in the visible and near-NIR region, this novel technique allows a combination of different imaging modalities, including second-harmonic generation, third-harmonic generation, and multiple-photon fluorescence. Combined with the selected excitation wavelength, which is located in the IR transparency window, this microscopic technique can provide high penetration depth with reduced damage and is ideal for studying living cells.