Microscope Objective Research Articles

A Systematic View of Microscope Objective Design

The correction of modern microscope objectives is not usually discussed in literature. We have reported a system review and summarized the design principles in a series of papers in 2019 [1-3]. Here we are introducing the systematic view of microscope objective design with an extension of the database till 2021. Furthermore, a systematic synthesis approach aided by AI will also be discussed.

Presença de corpos de Civatte em uma amostra de biópsia incisional de líquen plano oral

ABSTRACT Objective: Civatte bodies are colloid bodies of apoptotic keratinocytes located at the dermoepidermal junction and are common in several dermatoses, including lichen planus lesions. The present study aimed to determine the presence of Civatte bodies in a sample of incisional biopsies obtained from patients diagnosed with oral lichen planus. Methods: A retrospective cross-sectional study, carried out with 34 slides stained with hematoxylin-eosin from the archive of an Oral Pathology Laboratory of a Higher Education Institution. The sample obtained was classified into white and red lesions from the available clinical data. Histological analyses were performed under a light microscope, with 10x and 40x objectives, identifying the presence or absence of Civatte bodies. Results: Colloid bodies were present in 73.5% the total sample studied and in 74.2% white lesions. Conclusion: The Civatte bodies were frequent in all samples analysed and should thus be considered a criterion for histological diagnosis in lesions of Oral Lichen Planus.

Дискретная модель эволюции фазового пространства дробно размерных микросистем

The study of the process of changing information in microscopic systems is a rather complex and urgent scientific problem. The difficulty lies in the fact that, due to the microscopic nature of objects, it is impossible to use well-known thermodynamic methods based on the laws of large numbers. One of the possible methods for studying microscopic systems is the use of discrete Markov models with continuous time. Such models are based on the Heisenberg uncertainty principle and adequately describe the interaction between a macroscopic observer and a microscopic system at a quasi-classical level. At the same time, the use of semiclassical models makes it possible to avoid singularities, including those that cannot be eliminated, arising at small distances and high energies. In this paper, a discrete mathematical model of the phase space of an elementary particle will be used to study microscopic objects with a dimension of n=1. As a result of using the model, an original interpretation of the quark structure of hadrons was obtained. In particular, the dynamics of the functioning of quarks in the proton and neutron, the conditions for the formation of Δ resonances (Δ0, Δ+, Δ+, Δ++) are presented. The problem of the absence of free quarks (the phenomenon of confinement) is also considered. The paper considers only the first generation of quarks and the hadrons based on them. On the basis of known experimental data, numerical calculations have been carried out, showing sufficient adequacy of the model. The mathematical model of the discrete phase space, created to study the evolution of information in microscopic systems, is applicable to solving problems of elementary particle physics and can, in some cases, supplement the existing models of the quark structure of hadrons.

High‐Bandwidth InGaAs Photodetectors Heterogeneously Integrated on Silicon Waveguides Using Optofluidic Assembly

AbstractLight‐induced manipulation techniques have been utilized to transport, trap, or levitate microscopic objects for a wide range of applications in biology, electronics, and photonics. Without making direct physical contact, they can provide simple yet powerful means for high‐precision assembly of microscale functional blocks and components within the integrated circuit platforms, thereby offering a viable alternative to the conventional heterogeneous integration techniques, such as wafer/die bonding and transfer printing. Using a microbubble‐based optofluidic pick‐and‐place assembly process, heterogeneous integration of compact III‐V semiconductor photodetectors on a silicon‐based photonic integrated circuit chip, enabling direct high‐speed vertical electrical contacts for significantly improved photogenerated carrier transit distance/time, is experimentally demonstrated. The microdisk‐shaped InGaAs p‐i‐n photodetector integrated on the silicon waveguide has a 3 dB bandwidth exceeding 50 GHz under the applied bias voltage of −1 V for near‐infrared wavelengths around 1.55 µm. The light‐induced optofluidic assembly will provide a promising route for seamless heterogeneous integration of various optoelectronic components with high‐speed and low‐noise electrical interconnection on the fully processed silicon photonic/electronic integrated circuit platforms.

Pressure of Electromagnetic Radiation on a Linear Vibrator

Nowadays the pressure of electromagnetic radiation in the optical range is widely used in laser traps (so called optical tweezers or single-beam gradient force trap) to control the position of microparticles, biological cells and other microscopic objects. This is possible by focusing the laser radiation into the area of several micrometers in size. The intensity of the radiation in the area is sufficient to hold particles in the beam and manipulate them. We are interested to research similar possibility in the microwave range of wavelengths. However we had faced a number of difficulties in this range: the size of the focal region is much larger, the radiation intensity is less, and to control microscopic objects by means of radiation pressure very high powers are required. And we decided to consider the known effect of a very strong interaction of thin conducting fibers (metal, semiconductor, graphite) with microwave radiation. The efficiency factor of radiation pressure on such objects reaches values of several hundreds and thousands. This can be used to control objects in the form of electrically thin metal conductors by means of radiation pressure. Methods for calculating the pressure of electromagnetic radiation on an infinitely long circular cylinder are known. In this paper we propose a method for calculating the radiation pressure on a circular cylinder (vibrator), the length of which is comparable to the radiation wavelength. We have found out that when the vibrator length is close to half the wavelength, the radiation pressure efficiency factor is much larger than for an infinite cylinder. We have obtained the dependence of the radiation pressure efficiency factor on the length and diameter of an absolutely reflecting and impedance vibrator. It decreases with decreasing conductivity. An infinite cylinder at a certain value of conductivity has a maximum of the radiation pressure efficiency factor.

Low-cost optofluidic add-on enables rapid selective plane illumination microscopy of C. elegans with a conventional wide-field microscope.

.Significance: Selective plane illumination microscopy (SPIM) is an emerging fluorescent imaging technique suitable for noninvasive volumetric imaging of C. elegans. These promising microscopy systems, however, are scarce in academic and research institutions due to their high cost and technical complexities. Simple and low-cost solutions that enable conversion of commonplace wide-field microscopes to rapid SPIM platforms promote widespread adoption of SPIM by biologist for studying neuronal expressions of C. elegans.Aim: We sought to develop a simple and low-cost optofluidic add-on device that enables rapid and immobilization-free volumetric SPIM imaging of C. elegans with conventional fluorescent microscopes.Approach: A polydimethylsiloxane (PDMS)-based device with integrated optical and fluidic elements was developed as a low-cost and miniaturized SPIM add-on for the conventional wide-field microscope. The developed optofluidic chip contained an integrated PDMS cylindrical lens for on-chip generation of the light-sheet across a microchannel. Cross-sectional SPIM images of C. elegans were continuously acquired by the native objective of microscope as worms flowed in an L-shape microchannel and through the light sheet.Results: On-chip SPIM imaging of C. elegans strains demonstrated possibility of visualizing the entire neuronal system in few seconds at single-neuron resolution, with high contrast and without worm immobilization. Volumetric visualization of neuronal system from the acquired cross-sectional two-dimensional images is also demonstrated, enabling the standard microscope to acquire three-dimensional fluorescent images of C. elegans. The full-width at half-maximum width of the point spread function was measured as 1.1 and in the lateral and axial directions, respectively.Conclusion: The developed low-cost optofluidic device is capable of continuous SPIM imaging of C. elegans model organism with a conventional fluorescent microscope, at high speed, and with single neuron resolution.

Dynamically Reconfigurable Bipolar Optical Gradient Force Induced by Mid‐Infrared Graphene Plasmonic Tweezers for Sorting Dispersive Nanoscale Objects

AbstractExisting techniques for optical trapping and manipulation of microscopic objects, such as optical tweezers and plasmonic tweezers, are mostly based on visible and near‐infrared light sources. As it is in general more difficult to confine light to a specific length scale at a longer wavelength, these optical trapping and manipulation techniques have not been extended to the mid‐infrared spectral region or beyond. Here, it is shown that by taking advantage of the fact that many materials have large permittivity dispersions in the mid‐infrared region, optical trapping and manipulation using mid‐infrared excitation can achieve additional functionalities and benefits compared to the existing techniques in the visible and near‐infrared regions. In particular, it is demonstrated that by exploiting the exceedingly high field confinement and large frequency tunability of mid‐infrared graphene plasmonics, high‐performance and versatile mid‐infrared plasmonic tweezers can be realized to selectively trap or repel nanoscale objects of different materials in a dynamically reconfigurable way. This new technique can be utilized for sorting, filtering, and fractionating nanoscale objects in a mixture.

Synthetic aperture interference light (SAIL) microscopy for high-throughput label-free imaging.

Quantitative phase imaging (QPI) is a valuable label-free modality that has gained significant interest due to its wide potentials, from basic biology to clinical applications. Most existing QPI systems measure microscopic objects via interferometry or nonlinear iterative phase reconstructions from intensity measurements. However, all imaging systems compromise spatial resolution for the field of view and vice versa, i.e., suffer from a limited space bandwidth product. Current solutions to this problem involve computational phase retrieval algorithms, which are time-consuming and often suffer from convergence problems. In this article, we presented synthetic aperture interference light (SAIL) microscopy as a solution for high-resolution, wide field of view QPI. The proposed approach employs low-coherence interferometry to directly measure the optical phase delay under different illumination angles and produces large space-bandwidth product label-free imaging. We validate the performance of SAIL on standard samples and illustrate the biomedical applications on various specimens: pathology slides, entire insects, and dynamic live cells in large cultures. The reconstructed images have a synthetic numeric aperture of 0.45 and a field of view of 2.6 × 2.6 mm2. Due to its direct measurement of the phase information, SAIL microscopy does not require long computational time, eliminates data redundancy, and always converges.

Aperture total internal reflection (A-TIR) for contact angle measurement

Recently, aperture total internal reflection (A-TIR) was proposed to characterize the microdroplet patterns, such as the coverage fraction of the droplet, by placing the aperture just in front of the detector in classical total internal reflection (TIR). However, the reflection from the curved liquid-air interface was simulated using simple two-dimensional modeling, causing inaccuracy in A-TIR measurement. In addition, the reflectance dependency on the aperture size and the working distance of the aperture was not investigated, hindering its applications. In this study, the simulation based on three-dimensional (3-D) ray tracing with Fresnel equation modeling was successfully developed and verified to explain the internal reflection from the curved droplet liquid-air interface. With this developed 3-D modeling, A-TIR characteristics were explored using the parameters of the aperture size and the working distance of the aperture as well as the droplet surface coverage fraction, which shows a good agreement between the experiment and the simulation. Furthermore, it was for the first time demonstrated that the droplet contact angle can be effectively determined by obtaining the droplet thickness from the analytic quadratic solution by subtracting the measured reflectance at the two different sized apertures and using the spherical profile relation. Low contact angles in the range of 1∼ 15° were determined experimentally for the micro- and macro-sized droplets with a droplet diameter of 70 ∼ 7000 µm by the measured thickness of 1 ∼ 450 µm using A-TIR and compared with Fizeau interferometry and side-view imaging to show a good agreement. The simulation shows that A-TIR can be a new optical diagnostic tool to measure the contact angles 0 ∼ 90° regardless of the droplet diameter by adjusting the aperture size and the working distance. In addition, A-TIR can effectively determine the small contact angles less than 5°, even ultrasmall contact angles less than 1° for the submicron thickness, not requiring the complicated microscope setup. Thus, we can observe a sessile droplet's drastic contact angle change during wetting phenomena from 90° to 0° on the same A-TIR setup. Additionally, A-TIR can be used for a single or an array of micro or nanodroplets with a microscope objective by reducing the laser beam size and scanning methodology.

A feedback-based manoeuvre planner for nonprehensile magnetic micromanipulation of large microscopic biological objects

This paper reports a feedback-based manoeuvre planning approach for automated nonprehensile selective micromanipulation of large, microscopic biological objects (∼100μm). We employ ferromagnetic micro-particles as microrobots actuated via a global magnetic field produced by electromagnetic coils placed in quadrupole configuration. The microrobot motion is programmed to push the target object to the goal location. We employ a three-step approach comprising: (a) generate a collision-free optimal path between the initial and the commanded goal location, (b) generate a manoeuvre planning algorithm that invokes one of the three motion manoeuvres, namely, ‘approach’, ‘push’, and ‘align’ depending upon the instantaneous locations of the microrobot, target object, and the desired waypoint, and (c) deploy a simple proportional controller that determines the currents required in the electromagnetic coils that can produce a suitable magnetic field for executing the manoeuvre invoked by the manoeuvre planner. This paper reports a number of validation experiments conducted on both zebrafish, i.e., Danio rerio embryos and silica beads as target objects. We envisage that the developed inexpensive approach can be useful in robotic manipulation of biological objects with sizes in hundreds of microns including large biological cells, polyploid giant cancer cells (PGCC), multicellular spheroids, Dictyostelium slug, human oocytes, and autophagy candidates. We also believe that functionalizing the microrobots with living cells or suitable chemicals will make it possible to perform on-chip biological experiments in future.

Geometry dependence of micron-scale NMR signals on NV-diamond chips

Small volume nuclear magnetic resonance spectroscopy (NMR) has recently made considerable progress due to rapid developments in the field of quantum sensing using nitrogen vacancy (NV) centers. These optically active defects in the diamond lattice have been used to probe unprecedented small volumes in the picoliter range with high spectral resolution. However, the NMR signal size depends strongly on both the diamond sensor’s and sample’s geometry. Using Monte-Carlo integration of sample spin dipole moments, the magnetic field projection along the orientation of the NV center for different geometries has been analyzed. We show that the NMR signal strongly depends on the NV-center's orientation with respect to the diamond surface. While the signal of currently used planar diamond sensors converges as a function of the sample volume, more optimal geometries lead to a logarithmically diverging signal. Finally, we simulate the expected signal for spherical, cylindrical and nearly-2D sample volumes, covering relevant geometries for interesting applications in NV-NMR such as single-cell biology or NV-based hyperpolarization. The results provide a guideline for NV-NMR spectroscopy of microscopic objects.

Design of thin-structured-light projection system for small-height measurement.

A thin-structured light projection system with simple setup is proposed and applied to small-height measurement. In this system, the design of structured light pattern is carried out on the computer. The structure, color, geometric dimension, motion mode, projection brightness, contrast, and other attributes of the pattern can be edited easily. The optical system with zoom function is designed, which can output thin-structured-light fringe with a minimum width of 10μm. The camera and microscope are used to construct a vision system, which is used to capture the pattern of structured light. The three-dimensional (3D) shape reconstruction is realized by analyzing the pattern of structured light. The results show that this system can project a small width of thin-structured light pattern, which is very suitable for 3D shape reconstruction of microscopic objects with the small-height of tens of microns to hundreds of microns.

Confocal fluorescence microscopy with high-NA diffractive lens arrays.

Traditionally, there is a trade-off between the numerical aperture and field of view for a microscope objective. Diffractive lens arrays (DLAs) with overlapping apertures are used to overcome such a problem. A spot array with an NA up to 0.83 and a pitch of 75 µm is produced by the proposed DLA at a wavelength of 488 nm. By measurement of the fluorescence beads, the DLA-based confocal setup shows the capability of high-resolution measurement over an area of 3mm×3mm with a 2.5×0.07NA objective. Further, the proposed fluorescence microscope is insensitive to optical aberrations, which has been demonstrated by imaging with a simple doublet lens.

Absolute characterization of high numerical aperture microscope objectives utilizing a dipole scatterer

Measuring the aberrations of optical systems is an essential step in the fabrication of high precision optical components. Such a characterization is usually based on comparing the device under investigation with a calibrated reference object. However, when working at the cutting-edge of technology, it is increasingly difficult to provide an even better or well-known reference device. In this manuscript we present a method for the characterization of high numerical aperture microscope objectives, functioning without the need of calibrated reference optics. The technique constitutes a nanoparticle, acting as a dipole-like scatterer, that is placed in the focal volume of the microscope objective. The light that is scattered by the particle can be measured individually and serves as the reference wave in our system. Utilizing the well-characterized scattered light as nearly perfect reference wave is the main idea behind this manuscript.

Adaptive coherence volume in full-field optical coherence tomography

Optical sectioning is instrumental for the observation of extended biological samples. It allows the observation of only a slice of the sample while rejecting contributions from out of focus depths. The acquisition of the whole volume then requires an axial displacement of the sample or the focus. To satisfy Nyquist sampling, this axial displacement has to be equal to half the axial resolution. As lateral and axial resolutions are coupled by the numerical aperture of the microscope objective in most imaging techniques, high-resolution imaging of a volume is a time-consuming task, especially caused by the slow axial scanning. Here, we propose to adapt the axial resolution, or axial extent of the coherence volume, by filtering the spectrum of the illumination of an interferometric imaging technique. We applied our approach on full-field optical coherence tomography and show a tuning of this axial extent from 1.5 to 15 μm, allowing to adapt both the acquisition time and the amount of data. We finally demonstrate that the method is especially suited to image large biological samples such as millimetric engineered tissues.

Optically Accessible Microfluidic Flow Channels for Noninvasive High-Resolution Biofilm Imaging Using Lattice Light Sheet Microscopy.

Imaging platforms that enable long-term, high-resolution imaging of biofilms are required to study cellular level dynamics within bacterial biofilms. By combining high spatial and temporal resolution and low phototoxicity, lattice light sheet microscopy (LLSM) has made critical contributions to the study of cellular dynamics. However, the power of LLSM has not yet been leveraged for biofilm research because the open-on-top imaging geometry using water-immersion objective lenses is not compatible with living bacterial specimens; bacterial growth on the microscope’s objective lenses makes long-term time-lapse imaging impossible and raises considerable safety concerns for microscope users. To make LLSM compatible with pathogenic bacterial specimens, we developed hermetically sealed, but optically accessible, microfluidic flow channels that can sustain bacterial biofilm growth for multiple days under precisely controllable physical and chemical conditions. To generate a liquid- and gas-tight seal, we glued a thin polymer film across a 3D-printed channel, where the top wall had been omitted. We achieved negligible optical aberrations by using polymer films that precisely match the refractive index of water. Bacteria do not adhere to the polymer film itself, so that the polymer window provides unobstructed optical access to the channel interior. Inside the flow channels, biofilms can be grown on arbitrary, even nontransparent, surfaces. By integrating this flow channel with LLSM, we were able to record the growth of S. oneidensis MR-1 biofilms over several days at cellular resolution without any observable phototoxicity or photodamage.

Simultaneous microscopic imaging of thickness and refractive index of thin layers by heterodyne interferometric reflectometry (HiRef)

The detection of spatial or temporal variations in very thin samples has important applications in the biological sciences. For example, cellular membranes exhibit changes in lipid composition and order, which in turn modulate their function in space and time. Simultaneous measurement of thickness and refractive index would be one way to observe these variations, yet doing it noninvasively remains an elusive goal. Here we present a microscopic-imaging technique to simultaneously measure the thickness and refractive index of thin layers in a spatially resolved manner using reflectometry. A coherent laser beam, focussed by a high-numerical-aperture microscope objective and reflected by the sample, is measured using its heterodyne interference with a reference in order to determine the amplitude and phase of the reflected field and thus the complex reflection coefficient. Comparing the results with the theoretically calculated reflection of a thin layer under coherent illumination of high numerical aperture by the microscope objective, the refractive index and thickness of the layer are determined. We present results on a layer of polyvinylacetate with a thickness of approximately 80 nm. These results have a precision better than 10% in the thickness and better than 1% in the refractive index. The measured refractive index is consistent with literature values, and the measured thickness is close to measurements of the same sample by quantitative differential interference contrast. We discuss the significance of these results and the possibility of performing accurate measurements on nanometric layers. Notably, the shot-noise limit of the technique is below 0.5 nm in thickness and 0.0005 in refractive index for millisecond measurement times.

A hybrid coupler for directing quantum light emission with high radiative Purcell enhancement to a dielectric metasurface lens

Quantum photonic technologies such as quantum sensing, metrology, and simulation could be transformatively enabled by the availability of integrated single photon sources with high radiative rates and photon collection efficiencies. We address these challenges for quantum emitters formed from color center defect sites such as those in hexagonal boron nitride, which are promising candidates as single photon sources due to their bright, stable, polarized, and room temperature emission. We report design of a nanophotonic coupler from color center quantum emitters to a dielectric metasurface lens. The coupler is comprised of a hybrid plasmonic–dielectric resonator that achieves a large radiative Purcell enhancement and partial control of far-field radiation. We report radiative Purcell factors up to 285 and photon collection efficiencies up to 89% for a lossless metasurface, applying a continuous hyperboloidal phase-front. Our hybrid plasmonic–dielectric coupler interfacing two nanophotonic elements is a compound optical element, analogous to those found in microscope objective lenses, which combine multiple optical functions into a single component for improved performance.

Effect of inoculum size and antibiotics on bacterial traveling bands in a thin microchannel defined by optical adhesive

Phenotypic diversity in bacterial flagella-induced motility leads to complex collective swimming patterns, appearing as traveling bands with transient locally enhanced cell densities. Traveling bands are known to be a bacterial chemotactic response to self-generated nutrient gradients during growth in resource-limited microenvironments. In this work, we studied different parameters of Escherichia coli (E. coli) collective migration, in particular the quantity of bacteria introduced initially in a microfluidic chip (inoculum size) and their exposure to antibiotics (ampicillin, ciprofloxacin, and gentamicin). We developed a hybrid polymer-glass chip with an intermediate optical adhesive layer featuring the microfluidic channel, enabling high-content imaging of the migration dynamics in a single bacterial layer, i.e., bacteria are confined in a quasi-2D space that is fully observable with a high-magnification microscope objective. On-chip bacterial motility and traveling band analysis was performed based on individual bacterial trajectories by means of custom-developed algorithms. Quantifications of swimming speed, tumble bias and effective diffusion properties allowed the assessment of phenotypic heterogeneity, resulting in variations in transient cell density distributions and swimming performance. We found that incubation of isogeneic E. coli with different inoculum sizes eventually generated different swimming phenotype distributions. Interestingly, incubation with antimicrobials promoted bacterial chemotaxis in specific cases, despite growth inhibition. Moreover, E. coli filamentation in the presence of antibiotics was assessed, and the impact on motility was evaluated. We propose that the observation of traveling bands can be explored as an alternative for fast antimicrobial susceptibility testing.

Multidirectional focus measure for accurate three‐dimensional shape recovery of microscopic objects

Shape from focus (SFF) is a technique to recover the shape of an object from multiple images taken at various focus settings. Most of conventional SFF techniques compute focus value of a pixel by applying one of focus measure operators on neighboring pixels on the same image frame. However, in the optics with limited depth of field, neighboring pixels of an image have different degree of focus for curved objects, thus the computed focus value does not reflect the accurate focus level of the pixel. Ideally, an accurate focus value of a pixel needs to be measured from the neighboring pixels lying on tangential plane of the pixel in image space. In this article, a tangential plane on each pixel location (i, j) in image sensor is searched by selecting one of five candidate planes based on the assumption that the maximum variance of focus values along the optical axis is achieved from the neighborhood lying on tangential plane of the pixel (i, j). Then, a focus measure operator is applied on neighboring pixels lying on the searched plane. The experimental results on both the synthetic and real microscopic objects show the proposed method produces more accurate three-dimensional shape in comparison to conventional SFF method that applies focus measures on original image planes.