Microscope Optics Research Articles

Confocal Microscopy: Principles and Modern Practices.

In light microscopy, illuminating light is passed through the sample as uniformly as possible over the field of view. For thicker samples, where the objective lens does not have sufficient depth of focus, light from sample planes above and below the focal plane will also be detected. The out-of-focus light will add blur to the image, reducing the resolution. In fluorescence microscopy, any dye molecules in the field of view will be stimulated, including those in out-of-focus planes. Confocal microscopy provides a means of rejecting the out-of-focus light from the detector such that it does not contribute blur to the images being collected. This technique allows for high-resolution imaging in thick tissues. In a confocal microscope, the illumination and detection optics are focused on the same diffraction-limited spot in the sample, which is the only spot imaged by the detector during a confocal scan. To generate a complete image, the spot must be moved over the sample and data collected point by point. A significant advantage of the confocal microscope is the optical sectioning provided, which allows for 3D reconstruction of a sample from high-resolution stacks of images. Several types of confocal microscopes have been developed for this purpose, and each has different advantages and disadvantages. This article provides a concise introduction to confocal microscopy. © 2019 by John Wiley & Sons, Inc.

Industrial-grade anti-reflection coatings with extreme scratch resistance.

Anti-reflection coatings are widely used throughout the field of optical technology such as in corrective eyeglasses, camera lenses, and microscope optics, to improve the transmittance and reduce the reflectance of glass and other transparent materials. To date, these coatings have suffered from relatively poor scratch resistance and high scratch visibility compared to standard glasses. This has limited their use in applications requiring high mechanical durability such as on the chemically strengthened glasses widely used in modern touch screen devices. Here extremely scratch-resistant anti-reflection coatings are fabricated using industrially scalable reactive sputtering processes. These coatings provide a combination of surface reflectance below 0.7%, low color shifts, nanoindentation hardness as high as 18 GPa, and levels of scratch resistance which dramatically exceed commercial chemically strengthened glasses. An interdisciplinary opto-mechanical design approach has enabled a significant paradigm shift in the use of high-precision optical coatings for mechanically demanding applications. As a direct outcome of the work reported in this Letter, similar coating designs have been successfully deployed on millions of consumer electronics devices with very robust field performance.

Off-axis parabolic mirror relay microscope for experiments with ultra-cold matter.

A new optical system is introduced for the imaging of Coulomb crystals held in a cryogenic ion trap where there are space limitations preventing the placement of an objective close to the fluorescing ions. The optical system features an off-axis parabolic (OAP) mirror relay microscope that will serve to acquire images of a lattice of fluorescing ions confined within an ultra-high-vacuum vessel operating at temperatures below 10 K. We report that the OAP mirror relay setup can resolve features smaller than the separation between neighboring ions in Coulomb crystals. The setup presented here consists of two 90-degree OAP mirrors arranged into a relay from which standard microscope optics deliver the image to a camera. This design allows the first element in the imaging setup-an OAP mirror-to be located as close as possible to the ion trap, achieving high resolution without the need for a direct line-of-sight to the trap center or for a view port to be located in close proximity to the ion trap. Such an arrangement would not be possible with a standard microscope objective, which is the approach commonly adopted by the field. OAP mirrors represent a novel solution for delivering polychromatic images with micrometer-scale resolution over extended distances.

Variability of Echiniscus tristis Gąsiorek & Kristensen, 2018-is morphology sufficient for taxonomic differentiation of Echiniscidae?

The majority of species in the genus Echiniscus (Heterotardigrada) have been described based on differences in the chaetotaxy or dorsal sculpture. Dorsal sculpture is, in general, considered to be species-specific and not very variable; however, many problems have arisen due to various interpretations of microscope images, which has led to taxonomic confusion in the genus Echiniscus. Conversely, chaetotaxy is generally much easier to interpret, even using low-quality microscope optics. In this study, we emended the description of Madagascan population of Echiniscus tristis Gąsiorek & Kristensen, 2018 that exhibits several different types of chaetotaxy and dorsal sculpture. The analysed specimens were characterised by two types of chaetotaxy, A-C-Dd-E and A-Dd-E, but we also found a wide range of variation in appendage number, shape and length. The observed differences are partly correlated with life stages. Additionally, we analysed DNA sequences of 28S rRNA, ITS-2 and COI of the two main morphotypes, and did not find significant genetic differentiation of the two morphotypes. This highlights the importance of analysing the morphology of both immature stages and adults, as well as of DNA markers in tardigrade species identification.

Asymmetric Morphology Transformation of Azo Molecular Glass Microspheres Induced by Polarized Light.

In this work, photoinduced asymmetric morphology transformation of a type of azo molecular glass microspheres was thoroughly investigated to understand the effects of controlling factors on the process, related mechanism and unique functions. The monodispersed microspheres with their sizes over ten microns were fabricated from an isosorbide-based azo compound (IAC-4) by microfluidics. Under irradiation with linearly polarized light, the ten-micron-scale microspheres were transformed into three-dimensional (3D) asymmetric particles through directional mass transfer. Microscopic observations and optics simulation were employed to investigate the morphology transformations. The results show that the penetration depth of light at different wavelengths plays an extremely important role to affect the asymmetric deformation behavior of the IAC-4 microspheres, which determines deformation region, deformation degree and final shapes of the particles. The light intensity (50-200 mW/cm2) is a less important factor, while the deformation rate of the light-penetrated part linearly increases with the intensity. When the light intensity varies in this range, the deformation degree and the final asymmetric morphology are determined by exposure energy (light intensity × irradiation time). The IAC-4 microspheres with different sizes show distinct morphology transformation behavior and the deformed particles possess different shapes, caused by the variation of volume fraction of the light-penetrated part in the microspheres. The increase in the ratio of the light-penetrated part to the total volume of the microspheres results in larger scale deformations. Based on the above understanding, asymmetric particles with various morphologies can be fabricated through a precisely controllable way. The asymmetric particles loaded on various surfaces show ability to render remarkable wetting anisotropy of water droplets on the substrates.

High-pressure integrated synchrotron infrared spectroscopy system at the Shanghai Synchrotron Radiation Facility.

We describe a new integrated optical spectroscopy facility for high-pressure research in materials research and mineral science located at the beamline BL01B of the Shanghai Synchrotron Radiation Facility. The system combines infrared synchrotron Fourier-Transform spectroscopy with broadband laser visible/near infrared and conventional laser Raman spectroscopy in one instrument. The system utilizes a custom-built microscope optics designed for a variety of diamond anvil cell experiments, which include low-temperature and ultrahigh pressure studies. We demonstrate the capabilities of the facility for studies of a variety of high-pressure phenomena such as phase and electronic transitions and chemical transformations.

Infrared Laser Pulses Excite Action Potentials in Primary Cortex Neurons In Vitro.

Infrared neural modulation (INM) has been well studied in the peripheral nervous system (PNS) for potential clinical applications. However, limited research has been conducted on the central nervous systems (CNS). In this study, we aimed at investigating the feasibility of using pulsed infrared (IR) laser with a wavelength of 1940 nm to excite network activity of cultivated rat cortex neurons.We cultured rat cortex neurons, forming neural networks with spontaneous neural activity, on glass multi-electrode arrays (MEAs). Laser at a power of 600 mW and a pulse rate of 10 Hz were used to stimulate the neural networks using the optics of an inverted microscope. Pulse durations were varied from 200 μs to 1 ms. The spike rate was calculated to evaluate the change of the neural network activity during the IR stimuli and the corresponding frequency components of neural response were calculated to examine whether recorded spikes were evoked by the IR pulse or not. A temperature model was adapted from a previous study to estimate the temperature rise during laser pulsing.We observed that the IR irradiation with a pulse duration of 800 μs and 1 ms could excite neuronal action potentials. The temperature rose 18.5 and 23.9 °C, for pulse durations of 800 μs and 1 ms, respectively. Thus, in addition to previously shown inhibition of IR irradiation with a wavelength of 1550 nm, we demonstrate an optical method that can modulate neural network activity in vitro. The preliminary results from this paper also suggested that MEA recording technology coupled with a laser and microscope systems can be exploited as a new approach for future studies to understand mechanisms and characterize laser parameters of INM for CNS neurons.

Design of Microscope Optics for the Acquisition of Multiple Wavelength Band Image.

We design combinations of filters that lead to make outputs of multiple wavelength bands simultaneously. These bands are either 1) Infrared, RGB, Ultraviolet-3ranges 2) IR, Red, Green, Blue, UV-5ranges 3) IR+ Red, Green, Blue+UV-3ranges. Each is mostly devised by the arrangement of normal cold mirror, UV cold mirror, Philips(RGB) prism, UV filter and color filter. For the selection 1), UV cold mirror reflects UV and transmit VIS + IR. VIS and IR are sorted by normal cold mirror, which passes IR and reflects VIS. Each sorted light goes to each sensor to capture the images. The sorting of VIS and UV also can be done by UV filter. For the selection 2), have a same process in 1) but make VIS pass Philips(RGB) prism which sorts VIS into R, G, B. For the selection 3), the light passes green color filter first, Green color is only transmitted and the others are reflected. And then reflected light goes through UV cold mirror so that UV and Blue (wavelength <; 500nm) are reflected. And the others(wavelength>550nm, but green color zone was already sorted roughly wavelength>600nm) are transmitted, which is Red and IR. This sorting can be done by another design. After sorting all ranges by the selection 2), we can arrange UV goes a same pass with blue, and IR with red. We need band pass filters (in here, color filters) to revise the passes.

A 37-actuator polyimide deformable mirror with electrostatic actuation for adaptive optics microscopy

Widespread use of adaptive optics in life-science microscopy is predominantly hindered by the high cost of conventional MEMS deformable mirrors. We present here a potentially low-cost and compact alternative in the form of an electrostatic deformable mirror developed particularly for applications in adaptive optics microscopy. The device is fabricated with standard micro-machining processes using widely available materials to minimize cost, but still offers exceptional post-assembly mirror quality. With an aperture size of 10 mm, the device has a post-fabrication root-mean-square mirror flatness error of <250 nm and an active best-flat of <15 nm. The membrane is made of an aluminum-coated 3.5 m thick polyimide membrane that has high compliance, allowing a maximum peak-to-valley stroke of 15 m at 375 V. Driven using a custom-developed optimization-based open-loop control scheme, we successfully reproduced Zernike modes of up to fifth radial order with high shape fidelity.

MyoRobot 2.0: An advanced biomechatronics platform for automated, environmentally controlled skeletal muscle single fiber biomechanics assessment employing inbuilt real-time optical imaging

We present an enhanced version of our previously engineered MyoRobot system for reliable, versatile and automated investigations of skeletal muscle or linear polymer material (bio)mechanics. That previous version already replaced strenuous manual protocols to characterize muscle biomechanics properties and offered automated data analysis. Here, the system was further improved for precise control over experimental temperature and muscle single fiber sarcomere length. Moreover, it also now features the calculation of fiber cross-sectional area via on-the-fly optical diameter measurements using custom-engineered microscope optics. With this optical systems integration, the MyoRobot 2.0 allows to tailor a wealth of recordings for relevant physiological parameters to be sequentially executed in living single myofibers. Research questions include assessing temperature-dependent performance of active or passive biomechanics, or automated control over length-tension or length-velocity relations. The automatically obtained passive stress-strain relationships and elasticity modules are important parameters in (bio)material science. From the plethora of possible applications, we validated the improved MyoRobot 2.0 by assessing temperature-dependent myofibrillar Ca2+ sensitivity, passive axial compliance and Young's modulus. We report a Ca2+ desensitization and a narrowed dynamic range at higher temperatures in murine M. extensor digitorum longus single fibers. In addition, an increased axial mechanical compliance in single muscle fibers with Young's moduli between 40 - 60 kPa was found, compatible with reported physiological ranges. These applications demonstrate the robustness of our MyoRobot 2.0 for facilitated single muscle fiber biomechanics assessment.

Fluorescence microscopy for visualizing single-molecule protein dynamics

BackgroundSingle-molecule fluorescence imaging (smFI) has evolved into a valuable method used in biophysical and biochemical studies as it can observe the real-time behavior of individual protein molecules, enabling understanding of their detailed dynamic features. smFI is also closely related to other state-of-the-art microscopic methods, optics, and nanomaterials in that smFI and these technologies have developed synergistically. Scope of reviewThis paper provides an overview of the recently developed single-molecule fluorescence microscopy methods, focusing on critical techniques employed in higher-precision measurements in vitro and fluorescent nanodiamond, an emerging promising fluorophore that will improve single-molecule fluorescence microscopy. Major conclusionssmFI will continue to improve regarding the photostability of fluorophores and will develop via combination with other techniques based on nanofabrication, single-molecule manipulation, and so on. General significanceQuantitative, high-resolution single-molecule studies will help establish an understanding of protein dynamics and complex biomolecular systems.

A Simple μ-PTV Setup to Estimate Single-Particle Charge of Triboelectrically Charged Particles.

Triboelectric separation is a useful phenomenon that can be used to separate fine powders. To design technical devices or evaluate the potential of powders to be triboelectrically separated, knowledge about the charge distribution on a single-particle level has to be obtained. To estimate the single-particle charge distribution in an application-oriented way, a simple μ-PTV system was developed. The designed setup consists of a dispersing and a charging unit using a Venturi nozzle and a tube, respectively, followed by a separation chamber. In the separation chamber, a homogenous electrical field leads to a deflection of the particles according to their individual charge. The trajectories of the particles are captured on single frames using microscope optics and a high-speed camera with a defined exposure time. The particles are illuminated using a laser beam combined with a cylindrical lens. The captured images enable simultaneous measurement of positively and negatively charged particles. The charge is calculated assuming a mean particle mass derived from the mean particle size. Initial experiments were carried out using starch of different botanical origins and protein powder. Single-component experiments with starch powders show very different charge distributions for positively and negatively charged particles, whereas protein powder shows bipolar charging. Different starch-protein mixtures show similar patterns for positive and negative charge distributions.

Axicon-based beam shaping for low-loss nonlinear microscopic optics

Low-loss and broadband optics is important in optical measurement and imaging. Here, we design an axicon-based beam shaper for low-loss and broadband microscopic optics by numerically calculating the transmittance and focusing performance, assuming an application to stimulated Raman scattering (SRS) microscopy. Furthermore, we demonstrate the beam shaper equipped in an SRS microscope and confirm that the axicon-based optics increases transmittance without sacrificing spatial resolution or signal intensity.

A light scattering model for total internal reflection microscopy of geometrically anisotropic particles

ABSTRACTIn this paper, a light scattering model for Total Internal Reflection Microscopy (TIRM) is described. The model handles the scattering by an axisymmetric particle of arbitrary orientation situated in the evanescent field near a plane surface, and the imaging of the scattered light via microscope optics. The scattering problem is solved by using the -matrix method and the rotation addition theorem for spherical vector wave functions, while the image of the scattered field is computed by using the Debye diffraction integral. The numerical simulations provide evidence of two working regimes for TIRM: the first regime, corresponding to an incident angle less than the critical angle of total internal reflection, provides information on the size and the orientation of the particle, while the second regime, corresponding to an incident angle larger than the critical angle of total internal reflection, is recommended for measuring the distance between the particle and plane surface.

Automated sensorless single-shot closed-loop adaptive optics microscopy with feedback from computational adaptive optics.

Traditional wavefront-sensor-based adaptive optics (AO) techniques face numerous challenges that cause poor performance in scattering samples. Sensorless closed-loop AO techniques overcome these challenges by optimizing an image metric at different states of a deformable mirror (DM). This requires acquisition of a series of images continuously for optimization - an arduous task in dynamic in vivo samples. We present a technique where the different states of the DM are instead simulated using computational adaptive optics (CAO). The optimal wavefront is estimated by performing CAO on an initial volume to minimize an image metric, and then the pattern is translated to the DM. In this paper, we have demonstrated this technique on a spectral-domain optical coherence microscope for three applications: real-time depth-wise aberration correction, single-shot volumetric aberration correction, and extension of depth-of-focus. Our technique overcomes the disadvantages of sensor-based AO, reduces the number of image acquisitions compared to traditional sensorless AO, and retains the advantages of both computational and hardware-based AO.

P41 Endoscopy in cerebellopontine angle lesions: feasibility and technical considerations

ObjectivesTo define selection criteria and describe technique nuances for the use of endoscopy in surgery for cerebello-pontine angle (CPA) lesions.DesignProspective observational study.SubjectsPatients undergoing CPA lesion resection were selected for endoscope-assisted and/or endoscope-guided lesion resection.MethodsCPA interventions without mass lesions were excluded. 10 CPA lesions were identified pre-operatively for intra-operative endoscopic use across two neurosurgical centres. We describe equipment and technique selection.Results10 cases were selected over a one year period. Histology revealed 3 vestibular schwannomas (VS) (30%), 1 cyst (10%), 3 epidermoids (30%), 3 meningiomata (30%). Three cases were planned and carried out fully endoscopically (including two VS and the cyst) based on patient factors and favourable anatomy. Four cases were carried out with endoscopic assistance. The endoscope was used in three cases for anatomical orientation ‘around the corner’ only. Techniques and equipment vary depending on surgical aims, surgical anatomy and working area. Tips and pitfalls are identified and described.ConclusionsEndoscopy can be applied safely in CPA pathologies, as an alternative to the operating microscope for highly selected cases, or as an adjunct for specific surgical steps, especially when microscope optics do not allow angled visualisation. It is particularly useful in identifying lesion residuum and ‘working around the corner’. High level training is required before applying endoscopy to the CPA.

Evaluation of wound healing potential of pomegranate (Punica granatum) whole fruit extract on skin burn wound in rats (Rattus norvegicus).

Objective:This research was conducted to study the wound healing process of whole fruit pomegranate extract (punica granatum) standardized with 40% ellagic acid ointment for deep second-degree burn wound of skin in the rat (Rattus norvegicus).Materials and Methods:Powder of standardized pomegranate extract (SPE) with 40% ellagic acid was processed to become ointments. Twenty-five male rats, weighed 150–180 gm at 3 months of age, were randomly divided into five groups. After anesthetized, stainless circle plate with 1 cm of diameter in 85°C was contacted firmly toward right gluteal of rat skins for 5 sec in order to create deep second-degree burn wound. Control groups consist of (T0) cream base and (T1) 1% silver sulfadiazine. Treatment groups consist of (T2) 2.5% SPE, (T3) 5% SPE, and (T4) 10% SPE. Histopathological preparation used hematoxylin-eosin stained skin samples. Histological observations were performed using the optics microscope against collagen, the number of polymorphonuclear cell (PMN) infiltration, the degree of angiogenesis, and re-epithelization. The results were statistically compared between groups.Results:Microscopic observation on the wound healing process on the collagen, PMN infiltration, angiogenesis, and re-epithelization showed that topical administration of 10% SPE in burns gives the best result. This is characterized by a high density of collagen with a good arrangement, which is accompanied by a complete and mature epithelium, low number of inflammatory cells, and angiogenesis. This may be caused by the compounds in the pomegranate extract, which have the antioxidant, anti-inflammatory, and anti-bacterial effects.Conclusion:This study reveals that 10% SPE accelerates the healing of deep second-degree burn wound. Thus, pomegranate standardized with 40% ellagic acid is a promising herb for the healing of burn wound of skin.

35. Laser in neurosurgery: Critical issues in security management

Purpose Safe use of LASER in Neurosurgery. Methods The Neurosurgery Department of Niguarda Hospital has recently acquired a class 4 LASER, with a wavelength of 2010 nm, for the resection of brain tumors. This high-technological device represents a great innovation for neurosurgeons with remarkable improvements in the operating mechanisms but it requires, at the same time, a particular attention in the choice of the other tools, such as endoscopes and microscopes. The protection of the operator’s eyes during surgery is particularly critical. Protection glasses, provided to the whole staff, interfere with the usual procedure of the neurosurgeon who keeps his eye in contact with the eyepiece. A possible option could be the introduction of a protective lens within the microscope optics, as in ophthalmology LASERs. We tried to solve the problem on our own. Results The introduction of this LASER brought to the prohibition of use with some particular tools and to the need of modify other devices. The microscope in Niguarda’s Neurosurgery Department is the mod. M530 produced by Leica. The LASER attenuation required to the lens for a safe use has been calculated, taking into account the need to visualize the pointing laser and the markers, necessary for the identification of the tumor mass. Since the microscope optics does not guarantee this protection, a system has been designed to hold an additional lens with the required protection on the microscope. Conclusions In order to provide a safe use of the new LASER, the staff has been equipped with specific protective glasses and the use of the disposable endoscope has been forbidden. The designed system is put into production and the use will be evaluated by the neurosurgeons. Safe use of LASER in Neurosurgery. The Neurosurgery Department of Niguarda Hospital has recently acquired a class 4 LASER, with a wavelength of 2010 nm, for the resection of brain tumors. This high-technological device represents a great innovation for neurosurgeons with remarkable improvements in the operating mechanisms but it requires, at the same time, a particular attention in the choice of the other tools, such as endoscopes and microscopes. The protection of the operator’s eyes during surgery is particularly critical. Protection glasses, provided to the whole staff, interfere with the usual procedure of the neurosurgeon who keeps his eye in contact with the eyepiece. A possible option could be the introduction of a protective lens within the microscope optics, as in ophthalmology LASERs. We tried to solve the problem on our own. The introduction of this LASER brought to the prohibition of use with some particular tools and to the need of modify other devices. The microscope in Niguarda’s Neurosurgery Department is the mod. M530 produced by Leica. The LASER attenuation required to the lens for a safe use has been calculated, taking into account the need to visualize the pointing laser and the markers, necessary for the identification of the tumor mass. Since the microscope optics does not guarantee this protection, a system has been designed to hold an additional lens with the required protection on the microscope. In order to provide a safe use of the new LASER, the staff has been equipped with specific protective glasses and the use of the disposable endoscope has been forbidden. The designed system is put into production and the use will be evaluated by the neurosurgeons.

Demonstration of Triband Multi-Focal Imaging with Optical Coherence Tomography.

We demonstrate an extended depth of focus optical coherence tomography (OCT) system based on the use of chromatic aberration to create displaced focal planes in the sample. The system uses a wavelength-swept source tuning over three spectral bands and three separate interferometers, each of which interfaces to a single illumination/collection fiber. The resulting three imaged volumes are merged in post-processing to generate an image with a larger depth of focus than is obtained from each band individually. The improvements are demonstrated in structural imaging of a porous phantom and a lipid-cleared murine brain, and by angiographic imaging of human skin. By using a coaxial approach with Gaussian beams, this approach enables an extended focus with relatively simple microscope optics and data-merging algorithms.

Clearing for Deep Tissue Imaging.

Biologic tissues are generally opaque due to optical properties that result in scattering and absorption of light. Preparation of tissues for optical microscopy often involves sectioning to a thickness of 50-100 µm, the practical limits of light penetration and recovery. A researcher who wishes to image a whole tissue must acquire potentially hundreds of individual sections before rendering them into a three-dimensional volume. Clearing removes strongly light-scattering and light-absorbing components of a tissue and equalizes the refractive index of the imaging medium to that of the tissue. After clearing, the maximum depth of imaging is often defined by the microscope optics rather than the tissue. Such visibility enables the interrogation of whole tissues and even animals without the need to section. Researchers can study a biological process in the context of its three-dimensional environment, identify rare events in large volumes of tissues, and trace cells and cell-cell interactions over large distances. This article describes four popular clearing protocols that are relevant to a wide variety of scenarios across biologic disciplines: CUBIC, CLARITY, 3DISCO, and SeeDB. © 2018 by John Wiley & Sons, Inc.