Microscope Objective Research Articles

Is the Macro Grounded in the Micro?

Abstract Let a priority micro pluralist be someone who holds that particles or other microscopic objects are fundamental. Rivals to priority micro pluralism include priority monism (the view that the only fundamental concrete object is the entire cosmos) as well as the Aristotelian view that some ordinary macroscopic objects are fundamental. Although priority micro pluralism is popular, I show that it encounters great difficulty in even the most straightforward cases. For example, this tennis ball is spherical; how is this fact to be grounded in facts about the microscopic realm? I consider a number of possible answers to this question. The most promising proposals attempt to exploit the close connection emphasized by Kripke between objects and their original material constituents. I argue that these proposals fail. I conclude that it is worth seriously considering alternatives to this sort of pluralism.

Light-driven microdrones.

When photons interact with matter, forces and torques occur due to the transfer of linear and angular momentum, respectively. The resulting accelerations are small for macroscopic objects but become substantial for microscopic objects with small masses and moments of inertia, rendering photon recoil very attractive to propel micro- and nano-objects. However, until now, using light to control object motion in two or three dimensions in all three or six degrees of freedom has remained an unsolved challenge. Here we demonstrate light-driven microdrones (size roughly 2 μm and mass roughly 2 pg) in an aqueous environment that can be manoeuvred in two dimensions in all three independent degrees of freedom (two translational and one rotational) using two overlapping unfocused light fields of 830 and 980 nm wavelength. To actuate the microdrones independent of their orientation, we use up to four individually addressable chiral plasmonic nanoantennas acting as nanomotors that resonantly scatter the circular polarization components of the driving light into well-defined directions. The microdrones are manoeuvred by only adjusting the optical power for each motor (the power of each circular polarization component of each wavelength). The actuation concept is therefore similar to that of macroscopic multirotor drones. As a result, we demonstrate manual steering of the microdrones along complex paths. Since all degrees of freedom can be addressed independently and directly, feedback control loops may be used to counteract Brownian motion. We posit that the microdrones can find applications in transport and release of cargos, nanomanipulation, and local probing and sensing of nano and mesoscale objects.

Pose Measurement at Small Scale by Spectral Analysis of Periodic Patterns

The retrieval of an observed object’s pose is an essential computer vision problem. The challenge arises in many different fields, among them robotics control, contactless metrology, or augmented reality. When the observed object shrinks from the macroscopic scale to the microscopic, pose estimation is further complicated by the weaker perspective of imaging macroscale lenses down to the quasi-orthographic projection inherent to microscope objectives. This paper tackles this issue of microscale pose estimation in two complementary steps that rely on the use of planar periodic targets. We first consider the orthographic projection case as a means of presenting the theory of the method and showing how the pose of periodic patterns can be directly retrieved from the Fourier frequency spectrum of a given image. We then address the perspective case with long focal lengths, in which the full six-degrees of freedom (6-DOF) pose can be retrieved without ambiguities by following the same theoretical background. In addition to theoretically justifying pose retrieval via Fourier analysis of acquired images, this paper demonstrates the method’s actual performance. Both simulations and experimentation are conducted to validate the method and confirm an experimental resolution lower than \(1/1000{\mathrm{th}}\) of a pixel for translations. For orientation measurement, resolutions below 1 \(\upmu \)rad. for in-plane orientation, and below 100 \(\upmu \)rad. for off-axis orientations can be achieved.

Method of calculation of initial design parameters of microscope objective lenses with a long working distance.

We describe an analytic method of the initial design of microscope objectives with a long working distance. The optical system of a microscope objective is composed of two thin optical elements and a frontal optical element from several thick lenses. The paper describes formulas that make it possible to calculate fundamental parameters of the objective, namely Seidel aberration coefficients, radii of curvature, refractive indices of individual lenses of the microscope objective, etc. The proposed method of calculation of the initial design parameters of the microscope objective, which is based on the theory of aberrations of the third order and chromatic aberrations of the first order, enables us to obtain a good starting design for further optimization using optical design software. The method of the design is presented in an example of the microscope objective with a long working distance.

Far‐Field Parallel Direct Writing of Sub‐Diffraction‐Limit Metallic Nanowires by Spatially Modulated Femtosecond Vector Beam

AbstractMetallic nanowires have exhibited paramount importance in achieving functional electrical, electrochemical, and optical performances due to their unique physicochemical properties. Efficient fabrication of the nanowires with controllable geometry and line width is highly required in related applications. Here a new far‐field laser parallel direct writing approach for fabricating nanowire array far beyond the diffraction limit by a modulated vector beam with dual‐peak intensity distribution is reported. Such a modulated beam can perform selective laser ablation to fabricate nanowires, whose line width can be largely reduced by tuning the dual‐peak orientation relative to the laser scanning direction. As a demonstration, a 157 nm‐wide (less than λ/3) gold nanowire, reaching ≈1/20 of the diffraction limit, has been fabricated in far field by using a low numerical aperture microscope objective (NA = 0.1). The nanowire fabrication efficiency is also largely improved by applying multifocal modulated vector beams for parallel processing.

Image based evaluation of textured 3DSEM models

Photogrammetric methods enable the construction of 3D SEM models from 2D images. Most software for this purpose is designed for photographic images. The software tries to minimize modelling error but some uncertainty usually remains in the model. In such approaches no ground truth measurement for microscopic objects exists for comparison with the finished model. In the proposed method, a textured model surface is compared to the SEM images to map the error locations on the model. The method is illustrated using two datasets.

Evaluation of Midkine Expression in Oral Epithelial Dysplasia and Oral Squamous Cell Carcinoma: A Cross-Sectional Study

Abstract Introduction Oral cancer accounts for 3% of all malignancies diagnosed worldwide annually. Midkine (MK) plays a role in oncogenesis and tumor progression, including cell migration, mitogenesis, antiapoptosis, and angiogenesis. Aim and Objective To study and compare MK expression in various grades of oral epithelial dysplasia (OED) and oral squamous cell carcinoma (OSCC). Methodology This is a cross-sectional study where 60 archival specimens of OSCC, OED, and control were taken for immunohistochemical examination. Antibody MK (A-9) SC-46701 mouse monoclonal (200µg/mL, Santacruze Biotechnology) was used for immunohistochemical staining. The stained sections were observed under the objective (100 × ) of research microscope (DM 1000 LED) with computer-assisted image analyzer (Leica Application Suit V-4.1). Quantitative analysis was done to count MK reactive cells in all groups. Statistical analysis was performed using Kruskal–Wallis H test with posthoc Bonferroni's test for multiple group comparisons, and p-value < 0.05 was considered as statistically significant. Results MK expression was observed in OSCC and OED and was absent in control. The distribution of average percentage positivity was significantly higher in OED compared with OSCC and control. Statistically significant correlation was found between the size of the tumor and increased MK expression. Conclusion MK protein is overexpressed in OED and OSCC, and can be used as biological marker for early detection.

Mechatronic System Integrating an Electromagnetic Actuator with the Possibility of Moving the Working Microscopic Tool at Submicrometric Resolutions

Abstract The possibility to work by using small objects, under the microscopic view, it is a challenge. The force’s spectrum differs from that of the macroscopic scale, and many unexpected force vectors will appear. A sharp tip must be the ideal pressure vector on different microscopic small object to push, roll, and brake and so on. The positioning of this tip must be a very precise one, comparatively with the working area dimensions and we consider that 10 microns can be a reasonable measuring unit for these moves that are executed into a specific way in these kinds of cases. The mechatronic system designed to actuate the tip, becomes this paper subject. Indisputable we will talk about an actuator with one or more than one freedom degrees. Mechanical aspects are secondary, but necessary to understand the impact of electrical parameters on the minimum possible unit of movement. Electronic control refers to a switching and PWM signal, through a software that develops both the effective part of generating the control signal and the HMI interface through a touch-screen. The aim of this paper was to determine the minimum micrometric travel distance that can be obtained with a 12-bit PWM signal generated by a Raspberry mini-computer

Enhancing near-field optical tweezers by spin-to-orbital angular momentum conversion

Near-field patterns of light provide a way to optically trap, deliver, and sort single nanoscopic particles in a wide variety of applications in nanophotonics, microbiology, and nanotechnology. Using rigorous electromagnetic theory, we investigate the forces and trapping performance of near-field optical tweezers carrying spin and orbital angular momenta. The trapping field is assumed to be generated by a total internal reflection microscope objective at a glass–water interface in conditions where most of the transmitted light is evanescent. We discover aspects of these tweezers, including the possibility to rotate and stably trap nanoscopic beads. More importantly, we show that, under near-field conditions, the contributions of spin and orbital angular momenta to the rotation of small particles are almost equivalent, opening the possibility to cancel each other when they have an opposite sign. We show that these conditions result in optimal optical trapping, giving rise to extremely effective optical tweezers for nanomanipulation, with both circular symmetry and relatively weak rotation.

Fourier lightfield microscopy: a practical design guide.

In this work, a practical guide for the design of a Fourier lightfield microscope is reported. The fundamentals of the Fourier lightfield are presented and condensed on a set of contour plots from which the user can select the design values of the spatial resolution, the field of view, and the depth of field, as function of the specifications of the hardware of the host microscope. This work guides the reader to select the parameters of the infinity-corrected microscope objective, the optical relay lenses, the aperture stop, the microlens array, and the digital camera. A user-friendly graphic calculator is included to ease the design, even to those who are not familiar with the lightfield technology. The guide is aimed to simplify the design process of a Fourier lightfield microscope, which sometimes could be a daunting task, and in this way, to invite the widespread use of this technology. An example of a design and experimental results on imaging different types of samples is also presented.

Height measurement of microbumps using a white-light triangulation method.

As the electronic interconnection between chips, microbumps are crucial components in advanced packaging for the demand of better performance and higher packaging density. The height and coplanarity of microbumps are critical to ensure the reliability of connections. The poor uniformity in bump height will lead to disconnect or insufficient contact, which will directly result in the failure of the chip's function and a lower yield rate. In this paper, we proposed a height measurement method of microbumps using white-light triangulation combined with geometrical characteristics of bumps. A linear light projection module composed of a lens group and LED light source was set up as well as a high-quality imaging module consisting of a CCD camera and microscope objective group. The projection and imaging model of microbumps illuminated by the light plane at different positions during the scanning process was analyzed. The microbump height is computed from a simple formula based on the geometry of the specimen and the system configuration. The measurement results are compared with that obtained from a commercial optical profiler, and the measurement uncertainty is analyzed in detail.

Optical Frequency Tweezers

We show a concept of optical frequency tweezers for tweezing light in the optical frequency domain with a high resolution, which is the frequency version of the optical tweezers for spatial manipulation of microscopic objects. We report the proof-of-principle experiment via frequency conversion inside a cavity only for the converted light. Thanks to the atypical configuration, the experimental result successfully achieves the tweezing operation in the frequency domain, which picks a light at a target frequency from the frequency-multiplexed input light and converts to a different frequency, without touching any other light sitting in different frequency positions and shaking frequency by the pump light.

Two-Wavelength Computational Holography for Aberration-Corrected Simultaneous Optogenetic Stimulation and Inhibition of In Vitro Biological Samples

Optogenetics is a versatile toolset for the functional investigation of excitable cells such as neurons and cardiomyocytes in vivo and in vitro. While monochromatic illumination of these cells for either stimulation or inhibition already enables a wide range of studies, the combination of activation and silencing in one setup facilitates new experimental interrogation protocols. In this work, we present a setup for the simultaneous holographic stimulation and inhibition of multiple cells in vitro. The system is based on two fast ferroelectric liquid crystal spatial light modulators with frame rates of up to 1.7 kHz. Thereby, we are able to illuminate up to about 50 single spots with better than cellular resolution and without crosstalk, perfectly suited for refined network analysis schemes. System-inherent aberrations are corrected by applying an iterative optimization scheme based on Zernike polynomials. These are superposed on the same spatial light modulators that display the pattern-generating holograms, hence no further adaptive optical elements are needed for aberration correction. A near-diffraction-limited spatial resolution is achieved over the whole field of view, enabling subcellular optogenetic experiments by just choosing an appropriate microscope objective. The setup can pave the way for a multitude of optogenetic experiments, in particular with cardiomyocytes and neural networks.

Elimination of Quadratic Phase Aberration in Digital Holographic Microscopy by Using Transport of Intensity

We propose to reconstruct 3D images by combining the merits of transport of intensity and digital holography. The proposed method solves the transport-of-intensity equation by using digital holographic reconstructed images as inputs. Our simulation and experimental results show that this method can eliminate quadratic phase aberration introduced by the microscope objective in digital holographic microscopy. This proposed phase retrieval method is free of phase unwrapping process. It is thus efficient in removing quadratic phase aberration introduced by the microscope objective.

Snapshot polarimetric imaging in multi-view microscopy.

Polarimetric imaging allows for the vector nature of optical information across a scene to be obtained, with recent applications ranging from remote sensing to microscopy. In polarimetric microscopy in particular, different polarization states are conventionally achieved under time-division multiplexing strategies and are mainly subject to static phenomena. In the present work, we propose a cost-effective technique for polarization sensing with the possibility of real-time imaging microscopy. By modifying a commercial camera and replacing the conventional lens with an optical system that integrates a microscope objective and a lenslet array with a polarization mask, linear Stokes parameters can be obtained in a snapshot. The proposed scheme is robust against misalignment and suitable for handling video sequences of microscopic samples. To the best of our knowledge, this is the first report on combining multi-view sensing and polarization imaging for applications to microscopy.

Inferring the solution space of microscope objective lenses using deep learning

Lens design extrapolation (LDE) is a data-driven approach to optical design that aims to generate new optical systems inspired by reference designs. Here, we build on a deep learning-enabled LDE framework with the aim of generating a significant variety of microscope objective lenses (MOLs) that are similar in structure to the reference MOLs, but with varied sequences-defined as a particular arrangement of glass elements, air gaps, and aperture stop placement. We first formulate LDE as a one-to-many problem-specifically, generating varied lenses for any set of specifications and lens sequence. Next, by quantifying the structure of a MOL from the slopes of its marginal ray, we improve the training objective to capture the structures of the reference MOLs (e.g., Double-Gauss, Lister, retrofocus, etc.). From only 34 reference MOLs, we generate designs across 7432 lens sequences and show that the inferred designs accurately capture the structural diversity and performance of the dataset. Our contribution answers two current challenges of the LDE framework: incorporating a meaningful one-to-many mapping, and successfully extrapolating to lens sequences unseen in the dataset-a problem much harder than the one of extrapolating to new specifications.

Optimal trap velocity in a dynamic holographic optical trap using a nematic liquid crystal spatial light modulator

A dynamic holographic optical trap uses a dynamic diffractive optical element such as a liquid crystal spatial light modulator to realize one or more optical traps with independent controls. Such holographic optical traps provide a number of flexibilities and conveniences useful in various applications. One key requirement for such a trap is the ability to move the trapped microscopic object from one point to the other with the optimal velocity. In this paper we develop a nematic liquid crystal spatial light modulator based holographic optical trap and experimentally investigate the optimal velocity feasible for trapped beads of different sizes, in such a trap. Our results show that the achievable velocity of the trapped bead is a function of size of the bead, step size, interval between two steps and power carried by the laser beam. We observe that the refresh rate of a nematic liquid crystal spatial light modulator is sufficient to achieve an optimal velocity approaching the theoretical limit in the respective holographic trap for beads with radius larger than the wavelength of light.

Optically accessible microfluidic flow channels for non-invasive high-resolution biofilm imaging using lattice light sheet microscopy

An imaging system that can achieve long-term, high-resolution imaging of biofilms is essential for studying cellular level dynamics within bacterial biofilms. Combining high spatial-temporal resolution and low phototoxicity, lattice light sheet microscopy (LLSM) has proven powerful for non-invasive 3D imaging of living cells and tissues. However, its application in biofilm research is limited because the open-on-top imaging geometry using water-immersion objective lenses is not compatible with living bacterial specimens; bacterial growth in the media basin and on the microscope's objective lenses makes long-term time-lapse imaging impossible.

Approach for patch‐clamping using an upright microscope with z‐axis movable stage

We describe an approach for studying the physiology of single live cells using the conceptionally novel upright microscope/patch-clamp configuration. Electrophysiology experiments typically require a microscope with the fixed stage position and the motion control of the microscope objective. Here, we demonstrate that a microscope with a z-axis movable stage and a fixed objective can also be efficiently used in combination with the patch-clamp technique. We define a set of underlying principles governing the operation of this microscope/patch-clamp configuration and demonstrate its performance in practice using cultured astrocytes, microglia, and oligodendrocytes. Experimental results show that our custom configuration provides stable recordings, has a high success rate of the whole-cell patch-clamp trials, can be effectively applied to study cellular physiology of glial cells, and provides comparable performance and usability to the commercially available systems. Our system can be easily replicated or adapted to suit the needs of the research groups and can be cost-effective in reducing the investments in purchasing additional equipment. We provide step-by-step instructions on implementing an upright microscope with z-axis movable stage as a routine workhorse for patch-clamping. RESEARCH HIGHLIGHTS: Approach for efficient patch-clamping using an upright microscope with a z-axis movable stage. Routine study of live cell physiology. Custom microscope/patch-clamp configuration that is cost-effective and overcomes equipment limitations.

Scan-less machine-learning-enabled incoherent microscopy for minimally-invasive deep-brain imaging.

Deep-brain microscopy is strongly limited by the size of the imaging probe, both in terms of achievable resolution and potential trauma due to surgery. Here, we show that a segment of an ultra-thin multi-mode fiber (cannula) can replace the bulky microscope objective inside the brain. By creating a self-consistent deep neural network that is trained to reconstruct anthropocentric images from the raw signal transported by the cannula, we demonstrate a single-cell resolution (< 10μm), depth sectioning resolution of 40 μm, and field of view of 200 μm, all with green-fluorescent-protein labelled neurons imaged at depths as large as 1.4 mm from the brain surface. Since ground-truth images at these depths are challenging to obtain in vivo, we propose a novel ensemble method that averages the reconstructed images from disparate deep-neural-network architectures. Finally, we demonstrate dynamic imaging of moving GCaMp-labelled C. elegans worms. Our approach dramatically simplifies deep-brain microscopy.