Numerical Aperture Objective Lens Research Articles

Using high numerical aperture objective lens in micro-reflectance difference spectrometer

The large convergence angle of high numerical aperture (NA) objective lens is an obstacle to develop a high spatial resolution micro-reflectance difference spectrometer (micro-RDS). Here, we figure out the anisotropic signal mapping of the high NA objective lens induced to micro-RDS using mathematical approaches of vectorial ray-tracing method and Debye–Wolf integral. The spatial symmetry of the RD signal promises a value of zero by the integral over the light beam profile under the condition that the micro-RDS system is perfectly symmetrical. Thus, the RDS system needs to be more strictly symmetrical when a larger NA objective lens is utilized. For demonstration, a micro-RDS based on liquid crystal variable retarder was built. An in-situ calibration method was developed to eliminate the testing errors introduced by the asymmetry of the optical system. As a result, the measurement precision of the setup is better than ±0.002 when a 100× objective (NA=0.9) is used. Finally, to demonstrate the performance of the micro-RDS, we measured the layered black phosphorus using 50× and 100× objective lenses, respectively, and observed a scaled factor tuning the amplitude of the real part of RD signal when a sample is smaller than the light spot. The origins of the scaled factor are discussed.

Changes of intensity distribution of a tightly focused plane-wave pulse induced by lens dispersion

The tightly focusing properties of a linearly polarized plane-wave pulse through a high numerical aperture (NA) lens with dispersion are formulated. The effects of lens dispersion on the tightly focused intensity distribution in the focal region are studied in detail. It is found that, because of the lens dispersion, the intensity of the side lobe of the focused light spot strengthens in the transverse direction and the size of the main lobe of the focused light spot increases in the longitudinal direction as the pulse duration decreases. In addition, we compare the effects of dispersion of lens made by different types of glass on tightly focusing properties of a pulse beam. The results will be helpful to choose suitable high NA objective lens in experiment.

Synthesis of focused beam with controllable arbitrary homogeneous polarization using engineered vectorial optical fields.

The propagation and focusing properties of light beams continue to remain a research interest owning to their promising applications in physics, chemistry and biological sciences. One of the main challenges to these applications is the control of polarization distribution within the focal volume. In this work, we propose and experimentally demonstrate a method for generating a focused beam with arbitrary homogeneous polarization at any transverse plane. The required input field at the pupil plane of a high numerical aperture objective lens can be found analytically by solving an inverse problem with the Richard-Wolf vectorial diffraction method, and can be experimentally created with a vectorial optical field generator. Focused fields with various polarizations are successfully generated and verified using a Stokes parameter measurement to demonstrate the capability and versatility of proposed technique.

SERS optical fiber probe with plasmonic end‐facet

SERS optical fiber probe with plasmonic end‐facet

The spleen dictates platelet destruction, anti-platelet antibody production, and lymphocyte distribution patterns in a murine model of immune thrombocytopenia.

For many years, splenectomy has been used to treat immune thrombocytopenia (ITP), and this procedure benefits approximately two-thirds of the treated patients. Although splenectomy may raise platelet counts, antibody-coated platelets and cytotoxic T lymphocytes appear to persist or can change over time. To better understand how the spleen may affect anti-platelet immune responses, we used a murine model of ITP demonstrating both antibody-mediated and T lymphocyte-mediated thrombocytopenia. Mice with severe combined immunodeficiency (SCID) were either splenectomized or not and transfused with splenocytes from CD61 (GPIIIa) knockout mice immunized against CD61(+) platelets. Platelet counts and anti-platelet antibody levels were performed weekly. After 4weeks, the mice were sacrificed, and lymphoid organs were harvested and examined by flow cytometry to quantify CD4(+)CD25(+)FoxP3(+) Tregs and conventional cross-presenting XCR1(+) and tolerizing SIRPα+ dendritic cells. The results indicate that compared with control non-splenectomized mice, thrombocytopenia was improved and anti-platelet antibody production was significantly diminished in all splenectomized mice that received immune splenocytes. Splenectomized SCID mice also had a marked reduction in Tregs in the thymus together with an increased proportion of both thymic dendritic cell subsets that correlated with increased platelet counts. Of interest, although splenectomy diminished anti-platelet antibody production and raised platelet counts, marrow megakaryocyte densities were still significantly reduced in mice that received immune splenocytes. These results suggest that the spleen in murine ITP not only is the primary site responsible for platelet destruction, but it also controls, to a significant extent, the antibody response against platelets and the migration patterns of lymphocyte subsets.

Efficient positional misalignment correction method for Fourier ptychographic microscopy

Fourier ptychographic microscopy (FPM) is a newly developed super-resolution technique, which employs angularly varying illuminations and a phase retrieval algorithm to surpass the diffraction limit of a low numerical aperture (NA) objective lens. In current FPM imaging platforms, accurate knowledge of LED matrix's position is critical to achieve good recovery quality. Furthermore, considering such a wide field-of-view (FOV) in FPM, different regions in the FOV have different sensitivity of LED positional misalignment. In this work, we introduce an iterative method to correct position errors based on the simulated annealing (SA) algorithm. To improve the efficiency of this correcting process, large number of iterations for several images with low illumination NAs are firstly implemented to estimate the initial values of the global positional misalignment model through non-linear regression. Simulation and experimental results are presented to evaluate the performance of the proposed method and it is demonstrated that this method can both improve the quality of the recovered object image and relax the LED elements' position accuracy requirement while aligning the FPM imaging platforms.

Time-lapse scanning surface plasmon microscopy of living adherent cells with a radially polarized beam.

We report on a fibered high-resolution scanning surface plasmon microscope for long term imaging of living adherent cells. The coupling of a high numerical aperture objective lens and a fibered heterodyne interferometer enhances both the sensitivity and the long term stability of this microscope, allowing for time-lapse recording over several days. The diffraction limit is reached with a radially polarized illumination beam. Adherence and motility of living C2C12 myoblast cells are followed for 50 h, revealing that the dynamics of these cells change after 10 h. This plasmon enhanced evanescent wave microscopy is particularly suited for investigating cell adhesion, since it can not only be performed without staining of the sample but it can also capture in real time the exchange of extracellular matrix elements between the substrate and the cells.

Lensless Imaging and Sensing

High-resolution optical microscopy has traditionally relied on high-magnification and high-numerical aperture objective lenses. In contrast, lensless microscopy can provide high-resolution images without the use of any focusing lenses, offering the advantages of a large field of view, high resolution, cost-effectiveness, portability, and depth-resolved three-dimensional (3D) imaging. Here we review various approaches to lensless imaging, as well as its applications in biosensing, diagnostics, and cytometry. These approaches include shadow imaging, fluorescence, holography, superresolution 3D imaging, iterative phase recovery, and color imaging. These approaches share a reliance on computational techniques, which are typically necessary to reconstruct meaningful images from the raw data captured by digital image sensors. When these approaches are combined with physical innovations in sample preparation and fabrication, lensless imaging can be used to image and sense cells, viruses, nanoparticles, and biomolecules. We conclude by discussing several ways in which lensless imaging and sensing might develop in the near future.

Fluorescence Signal Generation Optimization by Optimal Filling of the High Numerical Aperture Objective Lens for High-Order Deep-Tissue Multiphoton Fluorescence Microscopy

Deep-tissue multiphoton microscopy (MPM) enables noninvasive optical imaging into the deep regions of the tissue in animal models in vivo . High-order MPM techniques, such as three- and four-photon fluorescence microscopy at the 1700-nm window, are emerging as promising imaging techniques for deeper penetration. Currently, signal depletion at large imaging depth sets the depth limit for these imaging techniques. As a result, how to further boost signal level is the key to achieving a larger imaging depth. Contrary to the previous thought that overfilling the back aperture of the objective yields the highest multiphoton fluorescence signal, in this paper, through numerical simulation, we show that, due to the effect of exponential decay of the excitation beam, the signal generation is maximized for certain underfilling of the back aperture of the objective lens. This will provide a simple strategy for signal generation maximization in deep-tissue MPM and potentially enables deeper imaging penetration into the tissue.

Shaping symmetric Airy beam through binary amplitude modulation for ultralong needle focus

Needle-like electromagnetic field has various advantages for the applications in high-resolution imaging, Raman spectroscopy, as well as long-distance optical transportation. The realization of such field often requires high numerical aperture (NA) objective lens and the transmission masks. We demonstrate an ultralong needle-like focus in the optical range produced with an ordinary lens. This is achieved by focusing a symmetric Airy beam (SAB) generated via binary spectral modulation with a digital micromirror device. Such amplitude modulation technique is able to shape traditional Airy beams, SABs, as well as the dynamic transition modes between the one-dimensional and two-dimensional (2D) symmetric Airy modes. The created 2D SAB was characterized through measurement of the propagating fields with one of the four main lobes blocked by an opaque mask. The 2D SAB was verified to exhibit self-healing property against propagation with the obstructed major lobe reconstructed after a certain distance. We further produced an elongated focal line by concentrating the SAB via lenses with different NAs and achieved an ultralong longitudinal needle focus. The produced long needle focus will be applied in optical, chemical, and biological sciences.

Generation of vectorial optical fields with slot-antenna-based metasurface.

A transmission-type metasurface composed of carefully designed rectangular slot antennas for the generation of vectorial optical fields is proposed and demonstrated. Acting as local linear polarizers, these slot antennas enable the spatial modulation of optical fields in amplitude, phase, and polarization for the cross-polarized component of the scattered field. As an illustration, a metasurface capable of forming a radially polarized scattered field with specific vectorial beam patterns with appropriate excitation at normal incidence is designed, fabricated, and tested. The radially polarized scattered field is designed to be further tightly focused by a high numerical aperture objective lens in order to obtain a uniform longitudinally polarized optical needle field along the propagation direction. Characterization experiments demonstrate that its overall extinction ratio satisfies the amplitude modulation requirement, and a corresponding π phase modulation is realized as proposed.

Solid immersion lenses for enhancing the optical resolution of thermal and electroluminescence mapping of GaN-on-SiC transistors

Solid immersion lenses (SILs) are shown to greatly enhance optical spatial resolution when measuring AlGaN/GaN High Electron Mobility Transistors (HEMTs), taking advantage of the high refractive index of the SiC substrates commonly used for these devices. Solid immersion lenses can be applied to techniques such as electroluminescence emission microscopy and Raman thermography, aiding the development device physics models. Focused ion beam milling is used to fabricate solid immersion lenses in SiC substrates with a numerical aperture of 1.3. A lateral spatial resolution of 300 nm is demonstrated at an emission wavelength of 700 nm, and an axial spatial resolution of 1.7 ± 0.3 μm at a laser wavelength of 532 nm is demonstrated; this is an improvement of 2.5× and 5×, respectively, when compared with a conventional 0.5 numerical aperture objective lens without a SIL. These results highlight the benefit of applying the solid immersion lenses technique to the optical characterization of GaN HEMTs. Further improvements may be gained through aberration compensation and increasing the SIL numerical aperture.

Creation of identical multiple focal spots with prescribed axial distribution.

We present a scheme for the construction of coaxially equidistant multiple focal spots with identical intensity profiles for each individual focus and a predetermined number and spacing. To achieve this, the radiation field from an antenna is reversed and then gathered by high numerical aperture objective lenses. Radiation patterns from three types of line sources, i.e., the electric current, magnetic current and electromagnetic current distributions, with cosine-squared taper are respectively employed to generate predominately longitudinally polarized bright spots, azimuthally polarized doughnuts, and focal spots with a perfect spherically symmetric intensity distribution. The required illuminations at the pupil plane of a 4Pi focusing configuration for the creation of these identical multiple focal spots can be easily derived by solving the inverse problem of the antenna radiation field. These unique focal field distributions may find potential applications in laser direct writing and optical microscopy, as well as multiple-particle trapping, alignment, and acceleration along the optical axis.

Spinning-Spot Shadowless TIRF Microscopy.

Total internal reflection fluorescence (TIRF) microscopy is a powerful tool for visualizing near-membrane cellular structures and processes, including imaging of local Ca2+ transients with single-channel resolution. TIRF is most commonly implemented in epi-fluorescence mode, whereby laser excitation light is introduced at a spot near the periphery of the back focal plane of a high numerical aperture objective lens. However, this approach results in an irregular illumination field, owing to interference fringes and scattering and shadowing by cellular structures. We describe a simple system to circumvent these limitations, utilizing a pair of galvanometer-driven mirrors to rapidly spin the laser spot in a circle at the back focal plane of the objective lens, so that irregularities average out during each camera exposure to produce an effectively uniform field. Computer control of the mirrors enables precise scanning at 200 Hz (5ms camera exposure times) or faster, and the scan radius can be altered on a frame-by-frame basis to achieve near-simultaneous imaging in TIRF, widefield and ‘skimming plane’ imaging modes. We demonstrate the utility of the system for dynamic recording of local inositol trisphosphate-mediated Ca2+ signals and for imaging the redistribution of STIM and Orai proteins during store-operated Ca2+ entry. We further anticipate that it will be readily applicable for numerous other near-membrane studies, especially those involving fast dynamic processes.

Comparative study of iterative reconstruction algorithms for missing cone problems in optical diffraction tomography.

In optical tomography, there exist certain spatial frequency components that cannot be measured due to the limited projection angles imposed by the numerical aperture of objective lenses. This limitation, often called as the missing cone problem, causes the under-estimation of refractive index (RI) values in tomograms and results in severe elongations of RI distributions along the optical axis. To address this missing cone problem, several iterative reconstruction algorithms have been introduced exploiting prior knowledge such as positivity in RI differences or edges of samples. In this paper, various existing iterative reconstruction algorithms are systematically compared for mitigating the missing cone problem in optical diffraction tomography. In particular, three representative regularization schemes, edge preserving, total variation regularization, and the Gerchberg-Papoulis algorithm, were numerically and experimentally evaluated using spherical beads as well as real biological samples; human red blood cells and hepatocyte cells. Our work will provide important guidelines for choosing the appropriate regularization in ODT.

Three-dimensional particle tracking in microfluidic channel flow using in and out of focus diffraction

Three-dimensional particle tracking is important to accurately understand the motion of particles within complex flow fields. We show that three-dimensional trajectories of particles within microfluidic flow can be extracted from two-dimensional bright field video microscopy. The method utilizes the defocusing that occurs as particles move out of the objective focal plane when viewed through a high numerical aperture objective lens. A fast and simple algorithm based on cross correlation to a set of reference images taken at prescribed amounts of defocus is used to extract out-of-plane particle position. In-plane particle position is determined through center point detection and therefore the particle position in all three dimensions can be constructed at each time point. Particle trajectories at high flow velocity of greater than 2mm/s can be tracked by utilizing a high speed camera to obtain unblurred images. Three dimensional computational fluid simulations are used to validate the particle tracking methods.

Separation of three-dimensional scattering effects in tilt-series Fourier ptychography

We investigate a strategy for separating the influence of three-dimensional scattering effects in tilt-series reconstruction, a method for computationally increasing the resolution of a transmission microscope with an objective lens of small numerical aperture, as occurs in the transmission electron microscope (TEM). Recent work with visible light refers to the method as Fourier ptychography. To date, reconstruction methods presume that the object is thin enough so that the beam tilt induces only a shift of the diffraction pattern in the back focal plane. In fact, it is well known that the diffraction pattern changes as a function of beam tilt when the object is thick. In this paper, we use a simple visible light model to demonstrate a proof-of-principle of a new reconstruction algorithm that can cope with this difficulty and compare it with the aperture-scanning method. Although the experiment uses a model specimen with just two distinct layers separated along the optic axis, it should in principle be extendable to continuous objects.

Label-free imaging and quantitative chemical analysis of Alzheimer's disease brain samples with multimodal multiphoton nonlinear optical microspectroscopy.

We developed multimodal multiphoton microspectroscopy using a small-diameter probe with gradient-index lenses and applied it to unstained Alzheimer's disease (AD) brain samples. Our system maintained the image quality and spatial resolution of images obtained using an objective lens of similar numerical aperture. Multicolor images of AD brain samples were obtained simultaneously by integrating two-photon excited fluorescence and second-harmonic generation on a coherent anti-Stokes Raman scattering (CARS) microendoscope platform. Measurements of two hippocampal regions, the cornus ammonis-1 and dentate gyrus, revealed more lipids, amyloid fibers, and collagen in the AD samples than in the normal samples. Normal and AD brains were clearly distinguished by a large spectral difference and quantitative analysis of the CH mode using CARS microendoscope spectroscopy. We expect this system to be an important diagnosis tool in AD research

Precise Three-Dimensional Scan-Free Multiple-Particle Tracking over Large Axial Ranges with Tetrapod Point Spread Functions

We employ a novel framework for information-optimal microscopy to design a family of point spread functions (PSFs), the Tetrapod PSFs, which enable high-precision localization of nanoscale emitters in three dimensions over customizable axial (z) ranges of up to 20 μm with a high numerical aperture objective lens. To illustrate, we perform flow profiling in a microfluidic channel and show scan-free tracking of single quantum-dot-labeled phospholipid molecules on the surface of living, thick mammalian cells.

Rapid Thiol‐Yne‐Mediated Fabrication and Dual Postfunctionalization of Micro‐Resolved 3D Mesostructures

3D mesostructures with a height of up to 1 mm and micrometer feature size are fabricated employing a writing speed of 1 cm s−1 via direct laser writing utilizing a novel functional photoresist based on the radical coupling reaction of thiols and alkynes. The refractive index of the resist—consisting of a tetrafunctional thiol, a tetrafunctional alkyne and a photoinitiator—is tailored to be compatible with the employed high numerical aperture (NA) objective lens, thus enabling a Dip‐in configuration. Mesostructures are characterized by scanning electron microscopy, optical photography, and nondestructive 3D time‐of‐flight secondary ion mass spectrometry. Woodpile photonic crystals are fabricated as benchmark structures in order to investigate the axial resolution. Verification of the chemical fabrication mechanism is achieved via transmission Fourier transform infrared (FTIR) spectroscopy of fabricated cuboid structures by monitoring the decrease of corresponding thiol and alkyne absorption peaks. Postmodification reactions, namely the thiol‐Michael addition and the copper‐catalyzed azide alkyne cycloaddition, are conducted employing residual thiols and alkynes throughout the cuboid structures. Successful dual and orthogonal modification throughout the structure and on the surface is achieved and verified utilizing transmission FTIR spectroscopy and time‐of‐flight secondary ion mass spectrometry.