Origin and effect of high-order dispersion in ultrashort pulse multiphoton microscopy in the 10 fs regime

Short pulses can induce high nonlinear excitation, and thus they should be favorable for use in multiphoton microscopy. However, the large spectral dispersion can easily destroy the advantages of the ultrashort pulse if there is no compensation. The group delay dispersion (GDD), third-order dispersion, and their effects on the intensity and bandwidth of second-harmonic generation (SHG) signal were analyzed. We found that the prism pair used for compensating the GDD of the two-photon microscope actually introduces significant negative high-order dispersion (HOD), which dramatically narrowed down the two-photon absorption probability for ultrashort pulses. We also investigated the SHG signal after GDD and HOD compensation for different pulse durations. Without HOD compensation, the SHG efficiency dropped significantly for a pulse duration below 20 fs. We experimentally compared the SHG and two-photon excited fluorescence (TPEF) signal intensity for 11 fs versus 50 fs pulses, a pulse duration close to that commonly used in conventional multiphoton microscopy. The result suggested that after adaptive phase compensation, the 11fs pulse can yield a 3.2- to 6.0-fold TPEF intensity and a 5.1-fold SHG intensity, compared to 50 fs pulses.     

Mitigating Chromatic Dispersion with Hybrid Optical Metasurfaces

Metasurfaces control various properties of light via scattering across a large number of subwavelength‐spaced nanostructures. Although metasurfaces appear to be ideal photonic platforms for realizing and designing miniaturized devices, their chromatic aberrations have hindered the large‐scale deployment of this technology in numerous applications. Wavelength‐dependent diffraction and resonant scattering effects usually limit their working operation wavelengths. In refractive optics, chromatic dispersion is a significant problem and is generally treated by cascading multiple lenses into achromatic doublets, triplets, and so on. Recently, broadband achromatic metalenses in the visible have been proposed to circumvent chromatic aberration but their throughput efficiency is still limited. Here, the dispersion of refractive components is corrected by leveraging the inherent dispersion of metasurfaces. Hybrid refractive‐metasurface devices, with nondispersive refraction in the visible, are experimentally demonstrated. The dispersion of this hybrid component, characterized by using a Fourier plane imaging microscopy setup, is essentially achromatic over about 150 nm in the visible. Broadband focusing with composite plano‐convex metasurface lenses is also proposed. These devices could find applications in numerous consumer optics, augmented reality components, and all applications including imaging for which monochromatic performance is not sufficient.     

Quasi‐Continuous Wave Near‐Infrared Excitation of Upconversion Nanoparticles for Optogenetic Manipulation of C. elegans

Optogenetics is an emerging powerful tool to investigate workings of the nervous system. However, the use of low tissue penetrating visible light limits its therapeutic potential. Employing deep penetrating near‐infrared (NIR) light for optogenetics would be beneficial but it cannot be used directly. This issue can be tackled with upconversion nanoparticles (UCNs) acting as nanotransducers emitting at shorter wavelengths extending to the UV range upon NIR light excitation. Although attractive, implementation of such NIR‐optogenetics is hindered by the low UCN emission intensity that necessitates high NIR excitation intensities, resulting in overheating issues. A novel quasi‐continuous wave (quasi‐CW) excitation approach is developed that significantly enhances multiphoton emissions from UCNs, and for the first time NIR light‐triggered optogenetic manipulations are implemented in vitro and in C. elegans. The approach developed here enables the activation of channelrhodopsin‐2 with a significantly lower excitation power and UCN concentration along with negligible phototoxicity as seen with CW excitation, paving the way for therapeutic optogenetics.     

Bismuth Ferrite Second Harmonic Nanoparticles for Pulmonary Macrophage Tracking

Recently, second harmonic generation (SHG) nanomaterials have been generated that are efficiently employed in the classical (NIR) and extended (NIR‐II) near infrared windows using a multiphoton microscope. The aim was to test bismuth ferrite harmonic nanoparticles (BFO‐HNPs) for their ability to monitor pulmonary macrophages in mice. BFO‐loaded MH‐S macrophages are given intratracheally to healthy mice or BFO‐HNPs are intranasally instilled in mice with allergic airway inflammation and lung sections of up to 100 μM are prepared. Using a two‐photon‐laser scanning microscope, it is shown that bright BFO‐HNPs signals are detected from superficially localized cells as well as from deep within the lung tissue. BFO‐HNPs are identified with an excellent signal‐to‐noise ratio and virtually no background signal. The SHG from the nanocrystals can be distinguished from the endogenous collagen–derived SHG around the blood vessels and bronchial structures. BFO‐HNPs are primarily taken up by M2 alveolar macrophages in vivo. This SHG imaging approach provides novel information about the interaction of macrophages with cells and the extracellular matrix in lung disease as it is capable of visualizing and tracking NP‐loaded cells at high resolution in thick tissues with minimal background fluorescence.     

Large‐Field‐of‐View Wide‐Spectrum Artificial Reflecting Superposition Compound Eyes

In nature, reflecting superposition compound eyes (RSCEs) found in shrimps, lobsters and some other decapods are extraordinary imaging systems with numerous optical features such as minimum chromatic aberration, wide‐angle field of view (FOV), high sensitivity to light and superb acuity to motion. Here, we present life‐sized, large‐FOV, wide‐spectrum artificial RSCEs as optical imaging devices inspired by the unique designs of their natural counterparts. Our devices can form real, clear images based on reflection rather than refraction, hence avoiding chromatic aberration due to dispersion by the optical materials. Compared to imaging at visible wavelengths using conventional refractive lenses of comparable size, our artificial RSCEs demonstrate minimum chromatic aberration, exceptional FOV up to 165° without distortion, modest aberrations and comparable imaging quality without any post‐image processing. Together with an augmenting cruciform pattern surrounding each focused image, our large‐FOV, wide‐spectrum artificial RSCEs possess enhanced motion‐tracking capability ideal for diverse applications in military, security, medical imaging and astronomy.     

Broadband alignment scheme for a stepper system using combinations of diffractive and refractive lenses

By using broadband illumination, a stepper alignment system is less sensitive to asymmetric coverage of photoresist around alignment targets and realizes high accuracy. However, a projection lens causes chromatic aberrations at the wavelengths in the broadband light. To correct the chromatic aberrations, we employed a Schupmann system composed of a projection lens, an achromat lens, and a refractive-diffractive hybrid lens. With the proposed system the spread of image planes and the magnification difference in the spectral range from 550 to 650 nm can be reduced to 50 μm and 0.05%, whereas without the correction system these values are 11.28 mm and 1.18%, respectively.     

Control of chromatic focal shift through wave-front coding

Control of chromatic aberration through purely optical means is well known. We present a novel, to our knowledge, optical-digital method of controlling chromatic aberration. The optical-digital system, which incorporates a cubic phase-modulation (CPM) plate in the optical system and postprocessing of the detected image, effectively reduces a system's sensitivity to misfocus in general or axial (longitudinal) chromatic aberration, in particular. A fully achromatic imaging system (one that is corrected for a continuous range of wavelengths) can be achieved by initial optimization of the optical system for all aberrations except chromatic aberration. The chromatic aberration is corrected by the inclusion of the CPM plate and postprocessing.     

In Vivo NIR Fluorescence Imaging,Biodistribution, and Toxicology of Photoluminescent Carbon Dots Produced from Carbon Nanotubes and Graphite

Oxidization of carbon nanotubes by a mixed acid has been utilized as a standard method to functionalize carbon nanomaterials for years. Here, the products obtained from carbon nanotubes and graphite after a mixed‐acid treatment are carefully studied. Nearly identical carbon dot (Cdot) products with diameters of 3–4 nm are produced using this approach from a variety of carbon starting materials, including single‐walled carbon nanotubes, multiwalled carbon nanotubes, and graphite. These Cdots exhibit strong yellow fluorescence under UV irradiation and shifted emission peaks as the excitation wavelength is changed. In vivo fluorescence imaging with Cdots is then demonstrated in mouse experiments, by using varied excitation wavelengths including some in the near‐infrared (NIR) region. Furthermore, in vivo biodistribution and toxicology of those Cdots in mice over different periods of time are studied; no noticeable signs of toxicity for Cdots to the treated animals are discovered. This work provides a facile method to synthesize Cdots as safe non‐heavy‐metal‐containing fluorescent nanoprobes, promising for applications in biomedical imaging.     

Subwavelength size determination by spatially modulated illumination virtual microscopy

A new approach for determining the sizes of individual, small fluorescent objects with diameters considerably below the optical resolution limit is described in which spatially modulated illumination (SMI) microscopy and 360-647-nm excitation wavelengths are used. The results of SMI virtual microscopy computer simulations indicate that, in this wavelength range, reliable measurements of sizes as small as approximately 20 nm are feasible if the low numbers of fluorescence photons that are usually detected from such small objects are taken into account. This method is based on the well-known fact that the modulation of the diffraction image in a SMI microscope is disturbed by the size of the object. Using appropriately calculated calibration functions, one can use this disturbance of the modulation to determine the size of the original object.     

New modal wave-front sensor: application to adaptive confocal fluorescence microscopy and two-photon excitation fluorescence microscopy

Confocal and multiphoton microscopes are particularly sensitive to specimen- or system-induced aberrations, which result in decreased resolution and signal-to-noise ratio. The inclusion of an adaptive optics correction system could help overcome this limitation and restore diffraction-limited performance, but such a system requires a suitable method of wave-front measurement. By extending the concept of a modal wave-front sensor previously described by Neil et al. [J. Opt. Soc. Am. A 17, 1098-1107 (2000)], we present a new sensor capable of measuring directly the Zernike aberration modes introduced by a specimen. This modal sensor is particularly suited to applications in three-dimensional microscopy because of its inherent axial selectivity; only those wave fronts originating in the focal region contribute to the measured signal. Four wave-front sensor configurations are presented and their input response is characterized. Sensitivity matrices and axial responses are presented.     

Ultrasonic microscope investigations of die attach quality and correlations with thermal resistance

In the semiconductor industry the use of ultrasonic microscopes for evaluating the integrity of the bond between adjacent surfaces has increased dramatically over the last few years. These instruments are rapidly becoming an acceptable diagnostic tool in the QC laboratory and new specifications have been written incorporating them into MIL requirements. Especially in the non-destructive investigation of plastic encapsulated ICs for delaminations, cracks, voids, and die-bonding faults ultrasonic microscopy has great advantages. The main equipment used is SLAM (or through transmission mode acoustic microscope) with a frequency range of 10–200 MHz and C-SAM (or reflective mode acoustic microscope) working in the range 5–100 MHz. In this paper, standard measurements of die attach quality and thermal resistance are correlated with C-SAM results. A general correlation between the amount of delamination and the measured thermal resistance was found.     

Optical manipulation in combination with multiphoton microscopy for single-cell studies

We demonstrate how optical tweezers can be incorporated into a multiphoton microscope to achieve three-dimensional imaging of trapped cells. The optical tweezers, formed by a cw 1064 nm Nd:YVO4 laser, were used to trap live yeast cells in suspension while the 4',6-diamidino-2-phenylindole-stained nucleus was imaged in three dimensions by use of a pulsed femtosecond laser. The trapped cell was moved in the axial direction by changing the position of an external lens, which was used to control the divergence of the trapping laser beam. This gives us a simple method to use optical tweezers in the laser scanning of confocal and multiphoton microscopes. It is further shown that the same femtosecond laser as used for the multiphoton imaging could also be used as laser scissors, allowing us to drill holes in the membrane of trapped spermatozoa.     

荧光薄膜材料荧光特性对荧光显微镜分析测量效果的影响

荧光显微镜测量结果的准确性,受仪器的线性动态范围、光场均匀性及光源稳定性等因素影响。运用荧光薄膜材料对荧光显微镜进行实验验证,发现荧光线性、均匀性及稳定性等荧光特性对于荧光显微镜分析测量效果十分重要。对荧光薄膜材料的光谱特性、线性、均匀性及光稳定性等方面进行研究,建立了荧光薄膜材料荧光特性的测量方法,对荧光显微镜校准方法进行了初步探索,为荧光显微镜的校准奠定了基础。     

Low Voltage Transmission Electron Microscopy of Graphene

The initial isolation of graphene in 2004 spawned massive interest in this two‐dimensional pure sp² carbon structure due to its incredible electrical, optical, mechanical, and thermal effects. This in turn led to the rapid development of various characterization tools for graphene. Examples include Raman spectroscopy and scanning tunneling microscopy. However, the one tool with the greatest prowess for characterizing and studying graphene is the transmission electron microscope. State‐of‐the‐art (scanning) transmission electron microscopes enable one to image graphene with atomic resolution, and also to conduct various other characterizations simultaneously. The advent of aberration correctors was timely in that it allowed transmission electron microscopes to operate with reduced acceleration voltages, so that damage to graphene is avoided while still providing atomic resolution. In this comprehensive review, a brief introduction is provided to the technical aspects of transmission electron microscopes relevant to graphene. The reader is then introduced to different specimen preparation techniques for graphene. The different characterization approaches in both transmission electron microscopy and scanning transmission electron microscopy are then discussed, along with the different aspects of electron diffraction and electron energy loss spectroscopy. The use of graphene for other electron microscopy approaches such as in‐situ investigations is also presented.     

Theory for double beam interference microscopes with coherence effects and verification using the linnik microscope

Abstract

A theoretical model for the interferogram from double beam interference microscopes, which takes into account the coherence effects, is presented. The model is based on the general imaging theory of a lens in defocus. For the case of zero relative lateral displacements between the reference and object beams a simplified expression is found for the defocus and path length dependence of the interferogram. Based on this expression the characteristics of the interferogram are studied and special attention is devoted to explaining the dependence of the fringe size on the objective numerical aperture, and the effect of the spatial and temporal coherence. For the Linnik microscope in which two objectives and a beam splitter cube are used, the effect of the mismatch between chromatic aberrations of the two objectives, and the effect of glass dispersions and misalignment of the beam splitter cube are investigated. Experimental results using the Linnik microscope are presented and they confirm the proposed model.     

Quantitative phase imaging of live cells using fast Fourier phase microscopy

Using the decomposition of an image field in two spatial components that can be controllably shifted in phase with respect to each other, a new quantitative-phase microscope has been developed. The new instrument, referred to as the fast Fourier phase microscope (f-FPM), provides a factor of 100 higher acquisition rate compared with our previously reported Fourier phase microscope. The resulting quantitative-phase images are characterized by diffraction limited transverse resolution and path-length stability better than 2 nm at acquisition rates of 10 frames/s or more. These features make the f-FPM particularly appealing for investigating the structure and dynamics of live cells over a broad range of time scales. In addition, we demonstrate the possibility of examining subcellular structures by digitally processing the amplitude and phase information provided by the instrument. Thus we developed software that can emulate phase contrast and differential interference contrast microscopy images by numerically processing FPM images. This approach adds the flexibility of digitally varying the phase shift between the two interfering beams. The images obtained appear as if they were recorded by variable phase contrast or differential interference contrast microscopes that deliver an enhanced view to the subcellular structure when compared with the typical commercial microscope.     

Phase effects owing to multilayer coatings in a two-mirror extreme-ultraviolet schwarzschild objective

The aberrations of a multilayer-coated reflective Schwarzschild objective, which are influenced both by mirror surface profiles and by multilayer coatings, are evaluated with a phase-shifting point diffraction interferometer operating in the extreme ultraviolet. Using wave-front measurements at multiple wavelengths near 13.4 nm, we observed chromatic aberrations and wavelength-dependent transmission changes that were due to molybdenum-silicon multilayer coatings. The effects of chromatic vignetting due to limited multilayer reflection passbands on the imaging performance of the Schwarzschild optic are considered. The coating characteristics extracted from the interferometry data on the two-mirror optical system are compared with previously reported coating properties measured on individual mirror substrates.     

Second‐Harmonic Generation from a Single Core/Shell Quantum Dot

Nanoparticles emitting two‐photon luminescence are broadly used as photostable emitters for nonlinear microscopy. Second‐harmonic generation (SHG) as another two‐photon mechanism offers complementary optical properties but the reported sizes of nanoparticles are still large, of a few tens of nanometers. Herein, coherent SHG from single core/shell CdTe/CdS nanocrystals with a diameter of 10 to 15 nm is reported. The nanocrystal excitation spectrum reveals resonances in the nonlinear efficiency with an overall maximum at about 970 nm. Polarization analysis of the second‐harmonic emission confirms the expected zinc blende symmetry, and allows extraction of the three‐dimensional nanocrystal orientation. The small size of these nonlinearly active quantum dots, together with the intrinsic coherence and orientation sensitivity of the SHG process, are well adapted for ultrafast probing of optical near‐fields with high resolution as well as for orientation tracking for bioimaging applications.     

Amorphous Quantum Nanomaterials

In quantum materials, macroscopic behavior is governed in nontrivial ways by quantum phenomena. This is usually achieved by exquisite control over atomic positions in crystalline solids. Here, it is demonstrated that the use of disordered glassy materials provides unique opportunities to tailor quantum material properties. By borrowing ideas from single‐molecule spectroscopy, single delocalized π‐electron dye systems are isolated in relatively rigid ultrasmall (<10 nm diameter) amorphous silica nanoparticles. It is demonstrated that chemically tuning the local amorphous silica environment around the dye over a range of compositions enables exquisite control over dye quantum behavior, leading to efficient probes for photodynamic therapy (PDT) and stochastic optical reconstruction microscopy (STORM). The results suggest that efficient fine‐tuning of light‐induced quantum behavior mediated via effects like spin‐orbit coupling can be effectively achieved by systematically varying averaged local environments in glassy amorphous materials as opposed to tailoring well‐defined neighboring atomic lattice positions in crystalline solids. The resulting nanoprobes exhibit features proven to enable clinical translation.     

3D‐Printed Scanning‐Probe Microscopes with Integrated Optical Actuation and Read‐Out

Scanning‐probe microscopy (SPM) is the method of choice for high‐resolution imaging of surfaces in science and industry. However, SPM systems are still considered as rather complex and costly scientific instruments, realized by delicate combinations of microscopic cantilevers, nanoscopic tips, and macroscopic read‐out units that require high‐precision alignment prior to use. This study introduces a concept of ultra‐compact SPM engines that combine cantilevers, tips, and a wide variety of actuator and read‐out elements into one single monolithic structure. The devices are fabricated by multiphoton laser lithography as it is a particularly flexible and accurate additive nanofabrication technique. The resulting SPM engines are operated by optical actuation and read‐out without manual alignment of individual components. The viability of the concept is demonstrated in a series of experiments that range from atomic‐force microscopy engines offering atomic step height resolution, their operation in fluids, and to 3D printed scanning near‐field optical microscopy. The presented approach is amenable to wafer‐scale mass fabrication of SPM arrays and capable to unlock a wide range of novel applications that are inaccessible by current approaches to build SPMs.