High Numerical Aperture Microscope Objective Research Articles

Shaping an evanescent focus of light for high spatial resolution optogenetic activations in live cells

Confining light illumination in the three dimensions of space is a challenge for various applications. Among these, optogenetic methods developed for live experiments in cell biology would benefit from such a localized illumination as it would improve the spatial resolution of diffusive photosensitive proteins leading to spatially constrained biological responses in specific subcellular organelles. Here, we describe a method to create and move a focused evanescent spot, at the interface between a glass substrate and an aqueous sample, across the field of view of a high numerical aperture microscope objective, using a digital micro-mirror device (DMD). We show that, after correcting the optical aberrations, light is confined within a spot of sub-micron lateral size and ∼100 nm axial depth above the coverslip, resulting in a volume of illumination drastically smaller than the one generated by a standard propagative focus. This evanescent focus is sufficient to induce a more intense and localized recruitment compared to a propagative focus on the optogenetic system CRY2-CIBN, improving the resolution of its pattern of activation.

Optical system design of double-sided telecentric microscope with high numerical aperture and long working distance.

The working distance of the high numerical aperture visible video microscope is extremely short, which greatly limits its application scenarios. To solve this problem, this paper proposes an unobstructed design method of double-sided telecentric microscope with high numerical aperture and long working distance. First, aiming at the obstruction problem of the image-side telecentric catadioptric microscope objective, the structure of the catadioptric optical system is improved. Then, the aspheric design method based on the best aberration compensation is analyzed theoretically to better correct the primary aberration of the high-numerical aperture microscope objective. Finally, a double-sided telecentric microscope optical system with a numerical aperture (NA) of 0.8 and a working distance of 10.0 mm was designed, which is composed of a spherical reflector, a beam splitter plate, a collimating lens group, and an image-side telecentric eyepiece optical system. The design results show that the imaging resolution of this high numerical aperture video microscope is as high as 0.42 µm, and the microscope has a magnification of about 220× for the image with 1080P (1920 × 1080 pixels) resolution. This double-sided telecentric microscope has the advantages of a large field of view, compact structure, good stray light suppression ability, and manufacturability, and has high practical value in the field of high-precision measurement and detection.

High numerical aperture imaging allows chirality measurement in individual collagen fibrils using polarization second harmonic generation microscopy.

Second harmonic generation (SHG) microscopy is a commonly used technique to study the organization of collagen within tissues. However, individual collagen fibrils, which have diameters much smaller than the resolution of most optical systems, have not been extensively investigated. Here we probe the structure of individual collagen fibrils using polarization-resolved SHG (PSHG) microscopy and atomic force microscopy. We find that longitudinally polarized light occurring at the edge of a focal volume of a high numerical aperture microscope objective illuminated with linearly polarized light creates a measurable variation in PSHG signal along the axis orthogonal to an individual collagen fibril. By comparing numerical simulations to experimental data, we are able to estimate parameters related to the structure and chirality of the collagen fibril without tilting the sample out of the image plane, or cutting tissue at different angles, enabling chirality measurements on individual nanostructures to be performed in standard PSHG microscopes. The results presented here are expected to lead to a better understanding of PSHG results from both collagen fibrils and collagenous tissues. Further, the technique presented can be applied to other chiral nanoscale structures such as microtubules, nanowires, and nanoribbons.

Design of Large Field of View and High Numerical Aperture Microscope Objective

Design of Large Field of View and High Numerical Aperture Microscope Objective

Improved two-photon photopolymerisation and optical trapping with aberration-corrected structured light

We demonstrate the effectiveness of phase only aberration corrections of structured light and their application to versatile optical trapping setups. We calculate phase corrections before (ex-situ) and after (in-situ) a high numerical aperture microscope objective using a spatial light modulator (SLM), and investigate how these corrections can be used to improve the efficiency and resolution of micro-structures fabricated through two-photon-photopolymerisation (2PP). We apply a phase retrieval algorithm to correct for distortions in a femtosecond laser that enables the fabrication of 3D structures using as many as 50 simultaneous foci. The inclusion of aberration correction in the fabrication process shows improved confinement of optically trapped particles and more efficient polymerisation while minimising intensity variations at individual foci, which potentially damage the structure during fabrication. We find that phase corrections allow for consistent voxel sizes, increased sharpness, and an expanded effective printing range when using an SLM, while also allowing for closer proximity of individual trap foci, minimising interference effects that hinder fabrication resolution.

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.

Fano Lineshapes and Rabi Splittings: Can They Be Artificially Generated or Obscured by the Numerical Aperture?

Fano resonances and Rabi splittings are routinely reported in the scientific literature. Asymmetric resonance lineshapes are usually associated with Fano resonances, and two split peaks in the spectrum are often attributed to a Rabi splitting. True Fano resonances and Rabi splittings are unequivocal signatures of coherent coupling between subsystems. However, can the same spectral lineshapes characterizing Fano resonances and Rabi splittings arise from a purely incoherent sum of intensities? Here we answer this question through experiments with a tunable Fabry-Pérot cavity containing a CsPbBr3 perovskite crystal. By measuring the transmission and photoluminescence of this system using microscope objectives with different numerical aperture (NA), we find that even a modest NA = 0.4 can artificially generate Fano resonances and Rabi splittings. We furthermore show that this modest NA can obscure the anticrossing of a bona fide strongly coupled light–matter system. Through transfer matrix calculations we confirm that these spectral artifacts are due to the incoherent sum of transmitted intensities at different angles captured by the NA. Our results are relevant to the wide nanophotonics community, characterizing dispersive optical systems with high numerical aperture microscope objectives. We conclude with general guidelines to avoid pitfalls in the characterization of such optical systems.

3D‐Printed high‐NA catadioptric thin lens for suppression of XPM background in Stimulated Raman Scattering microscopy

AbstractStimulated Raman Scattering (SRS) is a fast chemical imaging technique with remarkable bioscience applications. Cross Phase Modulation (XPM) is a ubiquitous nonlinear phenomenon that can create spurious background signals that render difficult a high‐contrast imaging in SRS measurements. The XPM‐induced signal is usually suppressed using high numerical aperture (NA) microscope objectives or condensers to collect the transmitted excitation beam. However, these high NA optics feature short working distances, hence they are not compatible with stage‐top incubators, that are necessary to perform live‐cell time‐lapse experiments in controlled environments. Here, we show a 3D printed high NA compact catadioptric lens that fits inside stage‐top incubators and allows the collection of XPM‐free SRS signals. The lens delivers SRS images and spectra with a quality comparable to a signal collection with a high‐NA microscope objective. We also demonstrate the compatibility of the 3D printed lens with other nonlinear microscopies usually associated with SRS in multimodal microscopes.

Dynamic Light Scattering Measurements for Soft Materials on Solid Substrates: Employing Evanescent-wave Illumination and Dark-field Collection with a High Numerical Aperture Microscope Objective.

We developed an instrument that allows us to measure dynamic light scattering from soft materials on solid substrates by avoiding strong background due to the reflection light from substrates. In the instrument, samples on substrates are illuminated by evanescent-light field and the resultant scattered light from the samples is collected with a dark-field optical configuration by employing a high numerical aperture microscope objective. We applied the instrument to measure the dynamic properties of supported lipid bilayers (SLBs), which have been widely utilized in industries as functional materials such as biosensors. From the time course of the scattered light from the SLBs, the power spectrum with the broad peak ranging from 10 to 20 kHz is observed. The use of the microscope objectives enables us to apply the instrument to future light scattering imaging for dynamic properties of soft materials supported on various substrates by combining with conventional microscope systems.

Correction of spherical surface measurements by confocal microscopy

Refractive microlenses are nowadays widely used in optical systems. Characterizing their surface is essential to ensure their quality and to optimize their fabrication process. This is realized by optical surface profilers thanks to their vertical resolution, short measurement time and areal information. However, when measuring non-flat surfaces, errors appear caused by aberrations of the microscope objective used in such systems, which significantly limit the achievable quality of the manufactured spherical surfaces. Approaches have been proposed to tackle these errors, but none of them demonstrated its validity for measurements of high quality microlenses. In this work, we demonstrate that the surface error depends on the surface position within the field of view of the microscope objective and on the surface slope. We then explain how to record the value of this error experimentally: this can be done by measuring a reference ball placed at different positions in the field of view. We finally use a machine learning algorithm to fit the experimental data in order to correct subsequent measurements. We apply this approach to measurements performed by a 20× numerical aperture 0.6 microscope objective of a confocal microscope. The effectiveness of the proposed method is demonstrated by showing that the surface error corresponds to a RMS wavefront error of λ/7 before correction and of λ/50 after correction for glass microlenses used in the visible range. This method thus allows the use of high numerical aperture microscope objectives for an accurate characterization of microlenses. Likewise, the fabrication capability of microlenses in terms of slope and quality is greatly extended, which is especially important for aspheres or freeforms.

Realistic, Vectorial Modeling of the Detection Point Spread Function for Single Molecule and Brightfield Microscopy

In previous work, we presented a rigorous, vectorial theory for the illumination point spread function (PSF). Here, we extend this approach to model the detection PSF, which is responsible for optical image formation in virtually all microscopy applications, such as conventional widefield microscopy, confocal scanning microscopy, fluorescence correlation spectroscopy, single molecule imaging, and super-resolution microscopy. We cover the most general case of a single dipolar scatterer or emitter, such as a single fluorescent molecule, which is imaged by a microscope objective-tube lens combination. The dipole radiation passes through stratified media composed of up to three layers (sample medium, glass coverslip, and immersion medium). The theoretical model accounts for a high numerical aperture tube lens and microscope objective (corrected for refractive indices of coverslip/immersion medium and coverslip thickness), a finite detector or detection pinhole, and an optional Babinet-Soleil compensator and polarization analyzer in the detection path. The resulting equations are readily evaluated, and multiple application examples will be discussed, such as the influence of dipole orientation and polarization detection on the detection PSF.

Long focal length high repetition rate femtosecond laser glass welding.

A long focal length focusing device is proposed for the process of glass welding by femtosecond laser pulses at high repetition rate and we report on the significant advantages. The study is performed using a 100 mm focusing length F-theta lens. The results are compared to those obtained with a high numerical aperture microscope objective. The long focal length with the associated Rayleigh length method allows a robust high process speed: welding at 1000 mm/s has been achieved, several orders of magnitude larger compared to what was reported until now. Moreover, for one pulse, the same energy on a larger laser spot leads to a lower thermic gradient increase. As a result, with the proposed parameters, the heat accumulation process reduces the residual stress in the welding seams, preventing the formation of fractures inside the seams: mechanical resistance at 30 MPa has been measured.

Development of a confocal scanning microscope for fluorescence imaging and spectroscopy at variable temperatures.

We developed and tested a confocal scanning optical microscope that fits into a thermally controlled, commercial research cryostat designed for operation from ambient temperature down to below 4 K. The home-built microscope is a fiber-coupled, self-contained instrument based on readily available mechanical and optical components. Its sample module is sealed in a protective stainless steel tube that minimizes vibrations caused by the flow of cryogenic gas. A high numerical aperture microscope objective specifically designed for cryogenic and high-vacuum applications focuses the excitation light onto the sample, while the core of an optical fiber attached to an avalanche photodiode acts as the confocal detection pinhole. The sample is displaced using a piezotube scanner mounted on top of a three-axis, low-temperature nanopositioner assembly for coarse sample positioning. A broadband polarizing cube beam splitter in the emission path allows for polarization-resolved imaging and spectroscopy. Fluorescence excitation scans are acquired with custom-written software that correlates fluorescence photon counts with the output from a high precision wavelength meter, which is part of a narrow-band, tunable dye laser setup. The imaging and spectral data acquisition capabilities of the microscope were confirmed using a variety of samples and excitation wavelengths at temperatures ranging from 5 K to room temperature.

Synchrotron macro ATR-FTIR microspectroscopy for high-resolution chemical mapping of single cells.

Attenuated total reflection Fourier transform infrared (ATR-FTIR) spectroscopy has been used widely for probing the molecular properties of materials. Coupling a synchrotron infrared (IR) beam to an ATR element using a high numerical aperture (NA) microscope objective enhances the spatial resolution, relative to transmission or transflectance microspectroscopy, by a factor proportional to the refractive index (n) of the ATR element. This work presents the development of the synchrotron macro ATR-FTIR microspectroscopy at Australian Synchrotron Infrared Microspectroscopy (IRM) Beamline, and demonstrates that high quality FTIR chemical maps of single cells and tissues can be achieved at an enhanced spatial resolution. The so-called "hybrid" macro ATR-FTIR device was developed by modifying the cantilever arm of a standard Bruker macro ATR-FTIR unit to accept germanium (Ge) ATR elements with different facet sizes (i.e. 1 mm, 250 μm and 100 μm in diameter) suitable for different types of sample surfaces. We demonstrated the capability of the technique for high-resolution single cell analysis of malaria-infected red blood cells, individual neurons in a brain tissue and cellular structures of a Eucalyptus leaf. The ability to measure a range of samples from soft membranes to hard cell wall structures demonstrates the potential of the technique for high-resolution chemical mapping across a broad range of applications in biology, medicine and environmental science.

Wavefront imaging by using an inline holographic microscopy system based on a double-sideband filter

In this letter, we propose an inline holographic microscopy (ILHM) system, based on the double-sideband technique (DST), for wavefront imaging. The presented optical system shows all the benefits of the previously reported DST (instantaneous removing of ghost images in an inline scheme) but adapted to a microscopic system. The double-sideband filter is implemented by using a transparent liquid crystal (LC) bi-pixel device. Specifically, by addressing the proper phase values to each half of the LC bi-panel, which is located at the Fourier plane of the holographic system, the conjugate image is removed. What is more, by using a high numerical aperture microscope objective, we achieved the microscopic wavefront holography imaging. Finally, the feasibility of the proposed system is testified by obtaining holographic wavefront images of different objects.

Demonstration of longitudinally polarized optical needles.

Longitudinally polarized optical needles are beams that exhibit ultra-long depth of field, subwavelength transverse confinement, and polarization oriented along the longitudinal direction. Although several techniques have been proposed to generate such needles, their scarce experimental observations have been indirect and incomplete. Here, we demonstrate the creation and full three-dimensional verification of a longitudinally polarized optical needle. This needle is produced by generating a radially polarized Bessel-Gauss beam at the focus of a high numerical aperture microscope objective. Using three-dimensional spatial mapping of second-harmonic generation from a single vertically aligned GaAs nanowire, we directly verify such a longitudinally polarized optical needle's properties, which are formed at the focus. The needle exhibits a dominant polarization, which is oriented along the longitudinal direction, an ultra-long depth of field (30 λ), and high spatial homogeneity. These are in agreement with corresponding focal field calculations that use vector diffraction theory. Our findings open new opportunities for manipulation and utilization of longitudinally polarized optical needles.

Three-dimensional polarization algebra for all polarization sensitive optical systems.

Using three-dimensional (3D) coherency vector (9 × 1), we develop a new 3D polarization algebra to calculate the polarization properties of all polarization sensitive optical systems, especially when the incident optical field is partially polarized or un-polarized. The polarization properties of a high numerical aperture (NA) microscope objective (NA = 1.25 immersed in oil) are analyzed based on the proposed 3D polarization algebra. Correspondingly, the polarization simulation of this high NA optical system is performed by the commercial software VirtualLAB Fusion. By comparing the theoretical calculations with polarization simulations, a perfect matching relation is obtained, which demonstrates that this 3D polarization algebra is valid to quantify the 3D polarization properties for all polarization sensitive optical systems.

Fabrication and characterization of machined multi-core fiber tweezers for single cell manipulation

Optical tweezing is a non-invasive technique that can enable a variety of single cell experiments; however, it tends to be based on a high numerical aperture (NA) microscope objective to both deliver the tweezing laser light and image the sample. This introduces restrictions in system flexibility when both trapping and imaging. Here, we demonstrate a novel, high NA tweezing system based on micro-machined multicore optical fibers. Using the machined, multicore fiber tweezer, cells are optically manipulated under a variety of microscopes, without requiring a high NA objective lens. The maximum NA of the fiber-based tweezer demonstrated is 1.039. A stable trap with a maximum total power 30 mW has been characterized to exert a maximum optical force of 26.4 pN, on a trapped, 7 μm diameter yeast cell. Single cells are held 15-35 μm from the fiber end and can be manipulated in the x, y and z directions throughout the sample. In this way, single cells are controllably trapped under a Raman microscope to categorize the yeast cells as live or dead, demonstrating trapping by the machined multicore fiber-based tweezer decoupled from the imaging or excitation objective lens.

Label-Free Single-Molecule Imaging with Numerical-Aperture-Shaped Interferometric Scattering Microscopy.

Our ability to optically interrogate nanoscopic objects is controlled by the difference between their extinction cross sections and the diffraction-limited area to which light can be confined in the far field. We show that a partially transmissive spatial mask placed near the back focal plane of a high numerical aperture microscope objective enhances the extinction contrast of a scatterer near an interface by approximately T–1/2, where T is the transmissivity of the mask. Numerical-aperture-based differentiation of background from scattered light represents a general approach to increasing extinction contrast and enables routine label-free imaging down to the single-molecule level.

Surface and bulk second-harmonic responses from a glass slide using tightly focused radially polarized light.

The radially polarized beam is tightly focused by a high numerical aperture microscope objective with adjustable cover glass correction. The intensities of a second-harmonic (SH) signal as a function of the focal position inside a glass slide for different settings of cover glass correction are measured. As compared to linear polarization illumination, the surface responses at the two surfaces of the glass slide are more easily observed in the case of radial polarization. For the first time, to the best of our knowledge, we present the experimental demonstration that surface and bulk SH responses from solid centrosymmetric materials can be separately addressed by adjusting the cover glass correction thickness and the position of the focal exciting field.