Numerical Aperture Objective Research Articles

Mapping of individual sensory nerve axons from digits to spinal cord with the transparent embedding solvent system.

Achieving uniform optical resolution for a large tissue sample is a major challenge for deep imaging. For conventional tissueclearing methods, loss of resolution and quality in deep regions is inevitable due to limited transparency. Here we describe the Transparent Embedding Solvent System (TESOS) method, which combines tissue clearing, transparent embedding, sectioning and block-face imaging. We used TESOS to acquire volumetric images of uniform resolution for an adult mouse whole-body sample. The TESOS method is highly versatile and can be combined with different microscopy systems to achieve uniformly high resolution. With a light sheet microscope, we imaged the whole body of an adult mouse, including skin, at a uniform 0.8 × 0.8 × 3.5 μm3 voxel resolution within 120 h. With a confocal microscope and a 40×/1.3 numerical aperture objective, we achieved a uniform sub-micron resolution in the whole sample to reveal a complete projection of individual nerve axons within the central or peripheral nervous system. Furthermore, TESOS allowed the first mesoscale connectome mapping of individual sensory neuron axons spanning 5 cm from adult mouse digits to the spinal cord at a uniform sub-micron resolution.

Adaptive optical quantitative phase imaging based on annular illumination Fourier ptychographic microscopy

Quantitative phase imaging (QPI) has emerged as a valuable tool for biomedical research thanks to its unique capabilities for quantifying optical thickness variation of living cells and tissues. Among many QPI methods, Fourier ptychographic microscopy (FPM) allows long-term label-free observation and quantitative analysis of large cell populations without compromising spatial and temporal resolution. However, high spatio-temporal resolution imaging over a long-time scale (from hours to days) remains a critical challenge: optically inhomogeneous structure of biological specimens as well as mechanical perturbations and thermal fluctuations of the microscope body all result in time-varying aberration and focus drifts, significantly degrading the imaging performance for long-term study. Moreover, the aberrations are sample- and environment-dependent, and cannot be compensated by a fixed optical design, thus necessitating rapid dynamic correction in the imaging process. Here, we report an adaptive optical QPI method based on annular illumination FPM. In this method, the annular matched illumination configuration (i.e., the illumination numerical aperture (NA) strictly equals to the objective NA), which is the key for recovering low-frequency phase information, is further utilized for the accurate imaging aberration characterization. By using only 6 low-resolution images captured with 6 different illumination angles matching the NA of a 10x, 0.4 NA objective, we recover high-resolution quantitative phase images (synthetic NA of 0.8) and characterize the aberrations in real time, restoring the optimum resolution of the system adaptively. Applying our method to live-cell imaging, we achieve diffraction-limited performance (full-pitch resolution of 655,nm at a wavelength of 525,nm) across a wide field of view (1.77,mm^2) over an extended period of time.

Single-exposure 3D label-free microscopy based on color-multiplexed intensity diffraction tomography.

We present a 3D label-free refractive index (RI) imaging technique based on single-exposure intensity diffraction tomography (sIDT) using a color-multiplexed illumination scheme. In our method, the chromatic light-emitting diodes (LEDs) corresponding R/G/B channels in an annular programmable ring provide oblique illumination geometry that precisely matches the objective's numerical aperture. A color intensity image encoding the scattering field of the specimen from different directions is captured, and monochromatic intensity images concerning three color channels are separated and then used to recover the 3D RI distribution of the object following the process of IDT. In addition, the axial chromatic dispersion of focal lengths at different wavelengths introduced by the chromatic aberration of the objective lens and the spatial position misalignment of the ring LED source in the imaging system's transfer functions modeling are both corrected to significantly reduce the artifacts in the slice-based deconvolution procedure for the reconstruction of 3D RI distribution. Experimental results on MCF-7, Spirulina algae, and living Caenorhabditis elegans samples demonstrate the reliable performance of the sIDT method in label-free, high-throughput, and real-time (∼24 fps) 3D volumetric biological imaging applications.

Spatial sampling effect on data structure and random forest classification of tissue types in High Definition and Standard Definition FT-IR imaging

Infrared radiation imaging (IR) combined with machine learning algorithms is one of the most promising techniques in tissue structures recognition and cancer diagnosis. Pancreatic tissue is heterogeneous and provides variable biochemical composition, as well as exhibits a variety of shapes and sizes having strong impact on physical aspects of light absorption. This research explores the potential of High and Standard Definitions in pancreatic tissue type prediction with random forest classification based on different spectral ranges. IR images spatial resolution depends on wavenumber and objective's numerical aperture. Therefore, data measured in Standard Definition (SD) are affected by under-sampling in the high wavenumbers while High Definition (HD) is free of this limitation for the whole analyzed spectral region. In order to investigate this effect a Random Forest model was created using a set of 56 biopsies measured in both definitions. In terms of signal variance the HD was found to be more spread out (however, with smaller scattering). SD models obtained a higher True Positive Rate than HD, however, spatial detail was better in HD models. Models trained on one definition and predicted on the other suggest that HD model can be used to successfully predict SD data. Finally, models based on only fingerprint and only high wavenumber were created to assess information content in each region. Models based on fingerprint region had very good prediction results across all classes, while for high wavenumber region results were class and definition dependent.

Tailoring Experimental Configurations to Probe Transition Dipoles of Fluorescent Nanoemitters by Polarimetry or Fourier Imaging with Enhanced Sensitivity.

Probing the transition dipoles responsible for the luminescence of a nanoemitter is essential to understanding its physical properties, its interactions with its environment, and its potential applications. Various methods in photoluminescence microscopy, based on polarimetry or Fourier imaging, have been developed to measure an emitter's dipole properties: the number of radiating dipoles, the oscillator strength ratio between them, and their orientation. In this article, we model the most used of these protocols and show that their sensitivity depends crucially on the experimental conditions: substrate material, presence of another lower or upper layer, and objective numerical aperture. We develop guidelines to optimize the measurement sensitivity by tailoring the experimental conditions, depending on the type of protocol used and the dipole property to be measured.

Evaluating structured-illumination patterns in optimizing optical-sectioning of HiLo microscopy

HiLo microscopy is a wide-field, optical-sectioning imaging technique, which is based on computational reconstruction from a uniform-illumination image and a structured-illumination (SI) image. Considering different imaging situations, the parameters of SI patterns should be optimized. However, no such discussion has been presented in grid-pattern (such as sinusoidal pattern and checkerboard pattern) based HiLo microscopy. Here, we analyze the dependence of optical-sectioning performance on parameters of SI pattern via both numerical simulations and imaging experiments. We find that both lower frequency and lower duty cycle of the grid-based pattern would introduce higher modulation depth, which would induce less artifacts in final reconstruction. Besides, at given illumination wavelength and objective numerical aperture, the higher the frequency of the grid-based pattern is, the better the axial resolution is, and thus the better the contrast of the optical-sectioning image is. Specially, the axial resolution is optimal when the frequency of pattern is set to half of cut-off frequency. We expect it as a guide of optimizing parameters of SI patterns for optical-sectioning in HiLo microscopy.

Fluorescence Correlation Spectroscopy Combined with Multiphoton Laser Scanning Microscopy—A Practical Guideline

Multiphoton laser scanning microscopy (MPM) has opened up an optical window into biological tissues; however, imaging is primarily qualitative. Cell morphology and tissue architectures can be clearly visualized but quantitative analysis of actual concentration and fluorophore distribution is indecisive. Fluorescence correlation spectroscopy (FCS) is a highly sensitive photophysical methodology employed to study molecular parameters such as diffusion characteristics on the single molecule level. In combination with laser scanning microscopy, and MPM in particular, FCS has been referred to as a standard and highly useful tool in biomedical research to study diffusion and molecular interaction with subcellular precision. Despite several proof-of-concept reports on the topic, the implementation of MPM-FCS is far from straightforward. This practical guideline aims to clarify the conceptual principles and define experimental operating conditions when implementing MPM-FCS. Validation experiments in Rhodamine solutions were performed on an experimental MPM-FCS platform investigating the effects of objective lens, fluorophore concentration and laser power. An approach based on analysis of time-correlated single photon counting data is presented. It is shown that the requirement of high numerical aperture (NA) objective lenses is a primary limitation that restricts field of view, working distance and concentration range. Within these restrictions the data follows the predicted theory of Poisson distribution. The observed dependence on laser power is understood in the context of perturbation on the effective focal volume. In addition, a novel interpretation of the effect on measured diffusion time is presented. Overall, the challenges and limitations observed in this study reduce the versatility of MPM-FCS targeting biomedical research in complex and deep tissue—being the general strength of MPM in general. However, based on the systematic investigations and fundamental insights this report can serve as a practical guide and inspire future research, potentially overcoming the technical limitations and ultimately allowing MPM-FCS to become a highly useful tool in biomedical research.

Errors in the Vickers diagonal length measurement caused by different designs of microscopes

The errors in the Vickers diagonal length measurement are discussed from the point of view of the design of microscope. Effects of the numerical aperture (NA) of an objective lens are evaluated through the experiment. With smaller NA, the resolving power of the microscope gets worse and the depth of focus gets deeper as predicted Rayleigh’s criterion. The relationship between the repeatability of diagonal length and the objective NA follows the theory. In addition, the variation of NA relates the measured diagonal length. Those effects are much significant than the image resolution or the minimum count of a measuring system.

Laser-driven Marangoni flow and vortex formation in a liquid droplet

We present a systematic study of the laser-driven Marangoni flow and curvature induced vortex formation in a copper sulfate pentahydrate solution, visualized by dispersed carbon nanotube (CNT) bundles. The experiments are carried out using different objectives of numerical aperture (NA) in the range of 0.1–0.6 to investigate the effect of focusing on the flow dynamics. The flow velocities measured (for 0.1 NA) are in the range of 2 mm/s–5 mm/s depending on the size of CNTs. Both primary and secondary vortices are observed in our experiment. In the primary vortex, with a sixfold increase in NA, a tenfold increase in the angular velocity of CNTs is measured. We also discuss the important role played by the curvature of the droplet in the vortex formation. The numerical simulations carried out for flow velocity are in agreement with the experimental values.

Photonic nanojet mediated Raman enhancement: Vertical Raman mapping and simple ray matrix analysis

AbstractA new method for enhancing the Raman scattering signal has emerged recently, based on dielectric enhancement. Especially promising is the dielectric method based on microspheres and photonic nanojet. In this paper, geometrical aspects and the influence of the incident beam parameters on Raman enhancement by silica microspheres were systematically investigated in three steps: by characterizing the incident beam using knife‐edge method, performing horizontal and vertical Raman mapping imaging, and analyzing the results using ray transfer matrix analysis. Maps show a distinct enhancement (hotspot) area caused by the microsphere photonic nanojet and lens effect compared to a plain silicon substrate. Enhancement value on maps was the highest (5.7×) for 0.50 numerical aperture objective, when the incident beam size matched the microsphere diameter, and the focus of the incident beam was below the top of the sphere, so that the output beam focus was at the microsphere–substrate contact area. This geometrical configuration was confirmed as ideal by performing simple ray transfer matrix analysis. The ideal ranges of incident and output beam parameters match with the measured hotspot area. This three‐step process and the usage of vertical Raman mapping have been, for the best of our knowledge, performed for the first time in such configuration. This research introduces a new way of investigating microsphere‐assisted Raman enhancement, offers different approach to microsphere optics research, and improves current knowledge of the influence of the incident beam on the enhancement.

Polarization properties of a single polygon of the cuticle of the scarab beetle C. mutabilis studied by micropolarimetry

Abstract The polarimetric response of a single polygon of the cuticle of the scarab beetle C. mutabilis is studied by micropolarimetry with a high numerical aperture (NA) objective lens. The imaging Mueller matrix and its scalar metrics were determined under a reflection configuration. The images of the Mueller-matrix resemble to some images reported for conoscopy measurements of anisotropic materials. The cuticle of the C. mutabilis was also illuminated with unconventional polarized light using the same experimental setup. Radial and azimuthal polarization states provide additional information to conventional polarized light showing two-fold helix symmetry.

High-speed Fourier ptychographic microscopy based on programmable annular illuminations

High-throughput quantitative phase imaging (QPI) is essential to cellular phenotypes characterization as it allows high-content cell analysis and avoids adverse effects of staining reagents on cellular viability and cell signaling. Among different approaches, Fourier ptychographic microscopy (FPM) is probably the most promising technique to realize high-throughput QPI by synthesizing a wide-field, high-resolution complex image from multiple angle-variably illuminated, low-resolution images. However, the large dataset requirement in conventional FPM significantly limits its imaging speed, resulting in low temporal throughput. Moreover, the underlying theoretical mechanism as well as optimum illumination scheme for high-accuracy phase imaging in FPM remains unclear. Herein, we report a high-speed FPM technique based on programmable annular illuminations (AIFPM). The optical-transfer-function (OTF) analysis of FPM reveals that the low-frequency phase information can only be correctly recovered if the LEDs are precisely located at the edge of the objective numerical aperture (NA) in the frequency space. By using only 4 low-resolution images corresponding to 4 tilted illuminations matching a 10×, 0.4 NA objective, we present the high-speed imaging results of in vitro Hela cells mitosis and apoptosis at a frame rate of 25 Hz with a full-pitch resolution of 655 nm at a wavelength of 525 nm (effective NA = 0.8) across a wide field-of-view (FOV) of 1.77 mm2, corresponding to a space–bandwidth–time product of 411 megapixels per second. Our work reveals an important capability of FPM towards high-speed high-throughput imaging of in vitro live cells, achieving video-rate QPI performance across a wide range of scales, both spatial and temporal.

Achieving superresolution with illumination-enhanced sparsity.

Recent advances in superresolution fluorescence microscopy have been limited by a belief that surpassing two-fold resolution enhancement of the Rayleigh resolution limit requires stimulated emission or the fluorophore to undergo state transitions. Here we demonstrate a new superresolution method that requires only image acquisitions with a focused illumination spot and computational post-processing. The proposed method utilizes the focused illumination spot to effectively reduce the object size and enhance the object sparsity and consequently increases the resolution and accuracy through nonlinear image post-processing. This method clearly resolves 70nm resolution test objects emitting ~530nm light with a 1.4 numerical aperture (NA) objective, and, when imaging through a 0.5NA objective, exhibits high spatial frequencies comparable to a 1.4NA widefield image, both demonstrating a resolution enhancement above two-fold of the Rayleigh resolution limit. More importantly, we examine how the resolution increases with photon numbers, and show that the more-than-two-fold enhancement is achievable with realistic photon budgets.

LITE microscopy: Tilted light-sheet excitation of model organisms offers high resolution and low photobleaching.

Fluorescence microscopy is a powerful approach for studying subcellular dynamics at high spatiotemporal resolution; however, conventional fluorescence microscopy techniques are light-intensive and introduce unnecessary photodamage. Light-sheet fluorescence microscopy (LSFM) mitigates these problems by selectively illuminating the focal plane of the detection objective by using orthogonal excitation. Orthogonal excitation requires geometries that physically limit the detection objective numerical aperture (NA), thereby limiting both light-gathering efficiency (brightness) and native spatial resolution. We present a novel live-cell LSFM method, lateral interference tilted excitation (LITE), in which a tilted light sheet illuminates the detection objective focal plane without a sterically limiting illumination scheme. LITE is thus compatible with any detection objective, including oil immersion, without an upper NA limit. LITE combines the low photodamage of LSFM with high resolution, high brightness, and coverslip-based objectives. We demonstrate the utility of LITE for imaging animal, fungal, and plant model organisms over many hours at high spatiotemporal resolution.

Determining the number of layers in few‐layer graphene by combining Raman spectroscopy and optical contrast

Raman spectroscopy is commonly used to determine the number of layers of few‐layer graphene (FLG) samples. In this work, we focus on the criteria based on the G‐band integrated intensity and on the laser optical contrast. Limitations due to stacking order are discussed and lead to the conclusion that it is necessary to combine Raman and optical contrast to avoid misinterpretation. Both methods enable to distinguish unambiguously between single layer graphene and multilayer graphene. However, neither each method separately nor the combination of the two enable a determination of the number of layers for all possible stacking orientations. Importantly, because the two methods always significantly disagree when they fail, the comparison of the values deduced by each method allows to discriminate if the determined number of layers can be specified or not. Other important parameters (substrate, laser wavelength, objective numerical aperture) are discussed to define a reliable method to determine the number of graphene layers in FLG and its domain of validity. The proposed method that combines Raman and optical contrast measurements, carried out with a 532 nm laser and using a 100× objective with a numerical aperture of 0.9, allows the determination of the number of layers for (up to 5) FLG on the following substrates: (1) glass (soda lime glass or similar with refractive index between 1.50 and 1.55) and (2) oxidized silicon (SiO2 on silicon, with a SiO2 thickness of 90 ± 5 nm). The method is however limited to high quality graphene and FLG with small defect density and low residue. Copyright © 2017 John Wiley & Sons, Ltd.

Raman mapping of the S3− chromophore in degraded ultramarine blue paints

AbstractA specific case of degradation was observed in synthetic ultramarine paint within 3 paintings from the early 20th century, manifesting as an intricate pattern of white lines criss‐crossing the blue paint surface. Raman spectroscopy can be performed directly on untreated sample surfaces and is sensitive to the colour components in ultramarine (S3− and S2− chromophores). This method was chosen to map the chromophore intensity distribution and to relate it to the surface degradation pattern. Raman signal intensity, however, decreases when the laser is out of focus, providing a challenge when mapping signal intensity across rough or undulating surfaces. To account for this, a series of experiments was conducted to determine the laser and objective combination least sensitive to changes in surface topography. The optimal settings were found to be the 785‐nm excitation wavelength with the 50× long working distance objective (numerical aperture 0.50). This combination gave the smallest focus‐dependent signal decrease on test samples: When shifted 5 μm out of focus above or below the sample surface, the signal from the same spot showed a decrease of 7% only. Maps of the blue (S3−) chromophore signal at 548 cm−1 taken from 3 samples showed a clear decrease in intensity on the degraded white lines. Patterns of signal intensity distribution matched well with the optical degradation pattern. From this observation, in conjunction with previous surface characterisation reported elsewhere, it was concluded that the surface phenomenon was indeed a discolouration of the ultramarine pigment, caused by a reduction in the concentration of the chromophore.

Focused-Laguerre-Gaussian 3D-Trapping and Spanner at Low Numerical Aperture

Stable 3D micromanipulation by light requires that gradient force overcome axial scattering force introduced by an objective lens. Although high numerical aperture (NA) objective lenses in conventional optical tweezers could match the requirement, the dramatic limited axial working range and a narrow view field draw back the application seriously. With purpose to improve the application of micromanipulation, we succeed in the three dimensional (3D) trapping of polystyrene microspheres with a low-numerical-aperture (NA=0.40) objective releasing a long working distance (WD=5.89mm) by utilizing the Laguerre-Gaussian beams. A series of rotating manipulation through modulating the asymmetry of Laguerre-Gaussian beams are presented. This work offers an extended axial trapping range for 3D manipulation and a delicate hand-actuated rotating system for optical manipulation.

Nonlinear optical corneal collagen crosslinking of ex vivo rabbit eyes

To determine whether riboflavin-induced collagen crosslinking (CXL) could be precisely achieved in the corneal stroma of exvivo rabbit eyes using nonlinear optical excitation with a low numerical aperture lens and enlarged focal volume. Gavin Herbert Eye Institute, University of California Irvine, Irvine, California, USA. Experimental study. The corneal epithelium was removed and the corneas were soaked in 0.5% riboflavin solution. Using a 0.1 numerical aperture objective, a theoretical excitation volume of 150μm×3μm was generated using 1W of 760nmfemtosecond laser light and raster scanned with 4.4μm line separation at varying effective speeds over a 4.50mm×2.25mm area. Corneal sections were examined for collagen autofluorescence. Collagen autofluorescence was enhanced 2.9 times compared with ultraviolet-A (UVA) CXL. Also, increasing speed was linearly associated with decreasing autofluorescence intensity. The slowest speed of 2.69mm/s showed a mean of 182.97μm±52.35 (SD) long autofluorescent scan lines axially in the central cornea compared with 147.84±4.35μm for UVA CXL. Decreasing dwell time was linearly associated with decreasing autofluorescence intensity, approaching that of UVA CXL at a speed of 8.9mm/s. Using an effective speed of 8.9mm/s, nonlinear optical CXL could be achieved over a 3.0mm diameter area in fewer than 4minutes.Further development of nonlinear optical CXL might result in safer, faster, and more effective CXL treatments. None of the authors has a financial or proprietary interest in any material or method mentioned.

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

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

How Confocal Is Confocal Raman Microspectroscopy on the Skin? Impact of Microscope Configuration and Sample Preparation on Penetration Depth Profiles

Background/Aims: The aim of the study was to elucidate the effect of sample preparation and microscope configuration on the results of confocal Raman microspectroscopic evaluation of the penetration of a pharmaceutical active into the skin (depth profiling). Methods: Pig ear skin and a hydrophilic formulation containing procaine HCl were used as a model system. The formulation was either left on the skin during the measurement, or was wiped off or washed off prior to the analysis. The microscope configuration was varied with respect to objectives and pinholes used. Results: Sample preparation and microscope configuration had a tremendous effect on the results of depth profiling. Regarding sample preparation, the best results could be observed when the formulation was washed off the skin prior to the analysis. Concerning microscope configuration, the use of a 40 × 0.6 numerical aperture (NA) objective in combination with a 25-µm pinhole or a 100 × 1.25 NA objective in combination with a 50-µm pinhole was found to be advantageous. Conclusion: Complete removal of the sample from the skin before the analysis was found to be crucial. A thorough analysis of the suitability of the chosen microscope configuration should be performed before acquiring concentration depth profiles.