Illumination Numerical Aperture Research Articles

Simultaneous Multifocal Plane Fourier Ptychographic Microscopy Utilizing a Standard RGB Camera.

Fourier ptychographic microscopy (FPM) is a computational imaging technology that can acquire high-resolution large-area images for applications ranging from biology to microelectronics. In this study, we utilize multifocal plane imaging to enhance the existing FPM technology. Using an RGB light emitting diode (LED) array to illuminate the sample, raw images are captured using a color camera. Then, exploiting the basic optical principle of wavelength-dependent focal length variation, three focal plane images are extracted from the raw image through simple R, G, and B channel separation. Herein, a single aspherical lens with a numerical aperture (NA) of 0.15 was used as the objective lens, and the illumination NA used for FPM image reconstruction was 0.08. Therefore, simultaneous multifocal plane FPM with a synthetic NA of 0.23 was achieved. The multifocal imaging performance of the enhanced FPM system was then evaluated by inspecting a transparent organic light-emitting diode (OLED) sample. The FPM system was able to simultaneously inspect the individual OLED pixels as well as the surface of the encapsulating glass substrate by separating R, G, and B channel images from the raw image, which was taken in one shot.

Systematic and quantitative comparison of axially overlapped multi-focus and Gaussian light sheets

Overlapped multi-focus light sheet (MF-LS) along beam propagation microscope has generated significant interest due to its reportedly superior axial resolving power when compared to conventional Gaussian light sheet (GLS) systems. By axially overlapped multiple light sheets in the illumination path, it would weld the combined field of view (FOV) and be wider than stationary counterparts. However, the effect of diffractive energy entering the focal space of desired beam, impacting the optical sectioning capabilities of the system has not been studied. It introduces the question that does a MF-LS performance better than standard GLS? Here we quantitatively and systematically analyzed the properties between optimized MF-LS and traditional GLS on z-axis resolution, signal to background ratio (SBR), and FOV. We first optimized the MF-LS by analyzing the diffractive energy to acquire the best sectioning capability. Then, we demonstrated a comparable optical sectioning performance between the two types of LS when matching with specific optical parameters including the illumination numerical aperture (NA) and foci numbers. A novel equation has been proposed to describe the equivalence relation. Analysis in the research suggests “putative FOV-extension” characteristics of MF-LS due to energy effects and interference between beams. Finally, we performed simulation and imaging experiments approaches on both fluorescence microspheres and biological samples to validate the viewpoint of this research.

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.

Improved resolution Fourier ptychography scheme using oil‐filled dome‐shaped LED array

Utilising angular-varied illumination in Fourier Ptychography, the resolution of optical microscopes can be increased and the phase of the sample can also be recovered while maintaining a wide field-of-view. In this work, an Fourier Ptychography microscopy imaging setup is demonstrated using a 10X 0.28 NA objective lens and a dome-shaped light-emitting diode array illuminator. By increasing the refractive index of the medium between the sample and light sources using oil filled dome, a synthetic system numerical aperture of 0.68 is achieved using only 37 light-emitting diodes. The measured resolution of 345 nm at a wavelength of 530 nm closely matches the theoretical prediction. Furthermore, the results of the oil-medium illumination are compared to free-space illumination. The oil immersion on the illumination side effectively enhances the illumination numerical aperture as compared to air and reduces the need for far off-axis light-emitting diodes. The result is high-resolution Fourier Ptychography imaging with relatively fewer light-emitting diodes providing both short capture and reconstruction times.

High-speed multi-objective Fourier ptychographic microscopy.

The ability of a microscope to rapidly acquire wide-field, high-resolution images is limited by both the optical performance of the microscope objective and the bandwidth of the detector. The use of multiple detectors can increase electronic-acquisition bandwidth, but the use of multiple parallel objectives is problematic since phase coherence is required across the multiple apertures. We report a new synthetic-aperture microscopy technique based on Fourier ptychography, where both the illumination and image-space numerical apertures are synthesized, using a spherical array of low-power microscope objectives that focus images onto mutually incoherent detectors. Phase coherence across apertures is achieved by capturing diffracted fields during angular illumination and using ptychographic reconstruction to synthesize wide-field, high-resolution, amplitude and phase images. Compared to conventional Fourier ptychography, the use of multiple objectives reduces image acquisition times by increasing the area for sampling the diffracted field. We demonstrate the proposed scaleable architecture with a nine-objective microscope that generates an 89-megapixel, 1.1 µm resolution image nine-times faster than can be achieved with a single-objective Fourier-ptychographic microscope. New calibration procedures and reconstruction algorithms enable the use of low-cost 3D-printed components for longitudinal biological sample imaging. Our technique offers a route to high-speed, gigapixel microscopy, for example, imaging the dynamics of large numbers of cells at scales ranging from sub-micron to centimetre, with an enhanced possibility to capture rare phenomena.

Scattering-Based Light-Sheet Microscopy for Rapid Cellular Imaging of Fresh Tissue.

Light-sheet microscopy (LSM) is a novel imaging technology that has been used for imaging fluorescence contrast in basic life science research. In this paper, we have developed a scattering-based LSM (sLSM) for rapidly imaging the cellular morphology of fresh tissues without any exogenous fluorescent dyes. In the sLSM device, a thin light sheet with the central wavelength of 834 nm was incident on the tissue obliquely, 45° relative to the tissue surface. The detection optics was configured to map the light sheet-illuminated area onto a two-dimensional imaging sensor. The illumination numerical aperture (NA) was set as 0.0625, and the detection NA 0.3. The sLSM device achieved a light sheet thickness of less than 6.7 µm over 284 µm along the illumination optical axis. The detection optics of the sLSM device had a resolution of 1.8 µm. The sLSM images of the swine kidney ex vivo visualized tubules with similar sizes and shapes to those observed in histopathologic images. The swine duodenum sLSM images revealed cell nuclei and villi architecture in superficial lesions and glands in deeper regions. The preliminary results suggest that sLSM may have the potential for rapidly examining the freshly-excised tissue ex vivo or intact tissue in vivo at microscopic resolution. Further optimization and performance evaluation of the sLSM technology will be needed in the future. Lasers Surg. Med. © 2020 Wiley Periodicals LLC.

3D nanometrology of transparent objects by phase calibration of a basic bright-field microscope for multiple illumination apertures.

Optical retrieval of the structure of transparent objects at the nanoscale requires adapted techniques capable of probing their interaction with light. Here, we considered a method based on calibration of the defocusing with partially coherent illumination and explored its phase retrieval capability over a wide range of illumination angles. We imaged: (1) commercial dielectric nanospheres to assess the phase calibration when measured along the optical axis, (2) custom-made nano-steps micropatterned in a glass substrate to assess the phase calibration when measured along the transversal axis, and (3) human cancer cells deposited on a glass substrate to assess the results of the calibration on complex transparent 3-dimensional samples. We first verified the model prediction in the spatial frequency domain and subsequently obtained a consistent and linear phase-calibration for illumination numerical apertures ranging from 0.1 to 0.5. Finally, we studied the dependence of the phase retrieval of a complex nanostructured object on the illumination aperture.

Performance tradeoffs for single- and dual-objective open-top light-sheet microscope designs: a simulation-based analysis.

Light-sheet microscopy (LSM) has emerged as a powerful tool for high-speed volumetric imaging of live model organisms and large optically cleared specimens. When designing cleared-tissue LSM systems with certain desired imaging specifications (e.g. resolution, contrast, and working distance), various design parameters must be taken into consideration. In order to elucidate some of the key design tradeoffs for LSM systems, we present a diffraction-based analysis of single- and dual-objective LSM configurations using simulations of LSM point spread functions. We assume Gaussian illumination is utilized. Specifically, we analyze the effects of the illumination and collection numerical aperture (NA), as well as their crossing angle, on spatial resolution and contrast. Assuming an open-top light-sheet (OTLS) architecture, we constrain these parameters based on fundamental geometric considerations as well as those imposed by currently available microscope objectives. In addition to revealing the performance tradeoffs of various single- and dual-objective LSM configurations, our analysis showcases the potential advantages of a novel, non-orthogonal dual-objective (NODO) architecture, especially for moderate-resolution imaging applications (collection NA of 0.5 to 0.8).

Through-focus scanning optical microscopy with the Fourier modal method

We propose a Fourier modal method (FMM) based through-focus scanning optical microscopy (TSOM) featuring sub-nano scale measurement tolerance. TSOM is very recently conceptualized non-destructive optical metrology technique just at the beginning stage of research. Nowadays the reliability and feasibility of TSOM concept is subject to controversy. We experimentally demonstrate stable nano-scale metrology of the FMM-based TSOM for the verification of the TSOM metrology and provide a numerical tool for true nano-meter scale TSOM through devising the FMM based TSOM scheme. By considering the illumination light parameters of incidence angle, polarization, degree of coherence, illumination numerical aperture, and collection numerical apertures in the FMM modeling of TSOM image acquisition, we reach precise agreement between the calculated and experimentally measured TSOM images. The essential elements of the FMM based TSOM for achieving high-level consistency are elucidated.

All-fiber laser with flattop beam output using a few-mode fiber Bragg grating.

We report a novel method of beam shaping in an all-fiber laser. Flattop beams are first generated from the all-fiber laser using a few-mode fiber Bragg grating by superposing the fundamental mode (LP01) with a solid central beam distribution, and the second-order mode (LP11) gives rise to a donut-shape beam distribution. Two modes are obtained in the fiber laser simultaneously, and the wavelengths of the two modes are 1053.7nm and 1055.4nm, respectively. An experiment has been carried out, and a high-quality flattop profile is obtained for the normalized root-mean-square of the light intensity less than 5% under no numerical aperture illumination, consistent with the theoretical prediction.

Numerically focused optical coherence microscopy with structured illumination aperture

In optical coherence microscopy (OCM) with a given numerical aperture (NA) of the objectives the transverse resolution can be increased by increasing the numerical aperture of illumination (NAi). However, this may also lead to attenuation of the signal with defocus preventing the effective numerically focused 3D imaging of the required sample volume. This paper presents an approach to structuring the illumination aperture, which allows combining the advantages of increased transverse resolution (peculiar to high NAi) with small attenuation of the signal with defocus (peculiar to low NAi) for high-resolution numerically focused 3D imaging in OCM.

Laser ablation caused by geometrically constrained illumination and inventive target design

Modern laser technologies use very sophisticated manipulations with (i) a photon cloud forming an irradiation beam and with (ii) disign of a target. E.g. high numerical aperture illumination at very small, diffraction limited conditions is employed for fabrication of the tiny solitary nanoformations on surface of specially prepared thin films deposited onto usually dielectric or semiconductor substrate. In the paper below we list such cases and consider an example with a free standing gold nanofilm modified by tightly focused femtosecond laser pulse.

Single-fluorophore orientation determination with multiview polarized illumination: modeling and microscope design.

We investigate the use of polarized illumination in multiview microscopes for determining the orientation of single-molecule fluorescence transition dipoles. First, we relate the orientation of single dipoles to measurable intensities in multiview microscopes and develop an information-theoretic metric-the solid-angle uncertainty-to compare the ability of multiview microscopes to estimate the orientation of single dipoles. Next, we compare a broad class of microscopes using this metric-single- and dual-view microscopes with varying illumination polarization, illumination numerical aperture (NA), detection NA, obliquity, asymmetry, and exposure. We find that multi-view microscopes can measure all dipole orientations, while the orientations measurable with single-view microscopes is halved because of symmetries in the detection process. We also find that choosing a small illumination NA and a large detection NA are good design choices, that multiview microscopes can benefit from oblique illumination and detection, and that asymmetric NA microscopes can benefit from exposure asymmetry.

Correcting the limited view in optical-resolution photoacoustic microscopy.

Optical-resolution photoacoustic microscopy (OR-PAM) has proven useful for anatomical and functional imaging with high spatial resolutions. However, the coherent signal generation and the desired reflection-mode detection in OR-PAM can result in a limited detectability of features aligned with the acoustic axis (ie, vertical structures). Here, we investigated the limited-view phenomenon in OR-PAM by simulating the generation and propagation of the acoustic pressure waves and determined the key optical parameters affecting the visibility of vertical structures. Proof-of-concept numerical experiments were performed with different illumination angles, optical foci and numerical apertures (NA) of the objective lens. The results collectively show that an NA of 0.3 can readily improve the visibility of vertical structures in a typical reflection-mode OR-PAM system. This conclusion was confirmed by numerical simulations on the cortical blood vessels in a mouse brain and by experiments in a suture-cross phantom and in a mouse brain in vivo.

Evaluating the effects of modeling errors for isolated finite three-dimensional targets

Optical three-dimensional (3-D) nanostructure metrology utilizes a model-based metrology approach to determine critical dimensions (CDs) that are well below the inspection wavelength. Our project at the National Institute of Standards and Technology is evaluating how to attain key CD and shape parameters from engineered in-die capable metrology targets. More specifically, the quantities of interest are determined by varying the input parameters for a physical model until the simulations agree with the actual measurements within acceptable error bounds. As in most applications, establishing a reasonable balance between model accuracy and time efficiency is a complicated task. A well-established simplification is to model the intrinsically finite 3-D nanostructures as either periodic or infinite in one direction, reducing the computationally expensive 3-D simulations to usually less complex two-dimensional (2-D) problems. Systematic errors caused by this simplified model can directly influence the fitting of the model to the measurement data and are expected to become more apparent with decreasing lengths of the structures. We identify these effects using selected simulation results and present experimental setups, e.g., illumination numerical apertures and focal ranges, that can increase the validity of the 2-D approach.

Optical path difference microscopy with a Shack-Hartmann wavefront sensor.

In this Letter, we show that a Shack-Hartmann wavefront sensor can be used for the quantitative measurement of the specimen optical path difference (OPD) in an ordinary incoherent optical microscope, if the spatial coherence of the illumination light in the plane of the specimen is larger than the microscope resolution. To satisfy this condition, the illumination numerical aperture should be smaller than the numerical aperture of the imaging lens. This principle has been successfully applied to build a high-resolution reference-free instrument for the characterization of the OPD of micro-optical components and microscopic biological samples.

Optical diffraction tomography microscopy with transport of intensity equation using a light-emitting diode array

Optical diffraction tomography (ODT) is an effective label-free technique for quantitatively refractive index imaging, which enables long-term monitoring of the internal three-dimensional (3D) structures and molecular composition of biological cells with minimal perturbation. However, existing optical tomographic methods generally rely on interferometric configuration for phase measurement and sophisticated mechanical systems for sample rotation or beam scanning. Thereby, the measurement is suspect to phase error coming from the coherent speckle, environmental vibrations, and mechanical error during data acquisition process. To overcome these limitations, we present a new ODT technique based on non-interferometric phase retrieval and programmable illumination emitting from a light-emitting diode (LED) array. The experimental system is built based on a traditional bright field microscope, with the light source replaced by a programmable LED array, which provides angle-variable quasi-monochromatic illumination with an angular coverage of ±37 degrees in both x and y directions (corresponding to an illumination numerical aperture of ∼0.6). Transport of intensity equation (TIE) is utilized to recover the phase at different illumination angles, and the refractive index distribution is reconstructed based on the ODT framework under first Rytov approximation. The missing-cone problem in ODT is addressed by using the iterative non-negative constraint algorithm, and the misalignment of the LED array is further numerically corrected to improve the accuracy of refractive index quantification. Experiments on polystyrene beads and thick biological specimens show that the proposed approach allows accurate refractive index reconstruction while greatly reduced the system complexity and environmental sensitivity compared to conventional interferometric ODT approaches.

Parameter optimization for through-focus scanning optical microscopy.

It is important to economically and non-destructively analyze three-dimensional (3-D) shapes of nanometer to micrometer scale objects with sub-nanometer measurement resolution for emerging high-volume nanomanufacturing, especially for process control. High-throughput through-focus scanning optical microscopy (TSOM) demonstrates promise for such applications. TSOM uses a conventional optical microscope for 3-D shape metrology by making use of the complete set of through-focus, four-dimensional optical data. However, a systematic study showing the effect of various parameters on the TSOM method is lacking. Here we present the optimization of the basic parameters such as illumination numerical aperture (NA), collection NA, focus step height, digital camera pixel size, illumination polarization, and illumination wavelength to achieve peak performance of the TSOM method.

Reflective Fourier ptychography.

The Fourier ptychography technique in reflection mode has great potential applications in tissue imaging and optical inspection, but the current configuration either has a limitation on cut-off frequency or is not practical. By placing the imaging aperture stop outside the illumination path, the illumination numerical aperture (NA) can be greater than the imaging NA of the objective lens. Thus, the cut-off frequency achieved in the proposed optical system is greater than twice the objective's NA divided by the wavelength (2NAobj/λ ), which is the diffraction limit for the cut-off frequency in an incoherent epi-illumination configuration. We experimentally demonstrated that the synthesized NA is increased by a factor of 4.5 using the proposed optical concept. The key advantage of the proposed system is that it can achieve high-resolution imaging over a large field of view with a simple objective. It will have a great potential for applications in endoscopy, biomedical imaging, surface metrology, and industrial inspection.

Active limited-angle tomographic phase microscope

We demonstrate an active, holographic tomography system, working with limited angle of projections, realized by optical-only, diffraction-based beam steering. The system created for this purpose is a Mach–Zehnder interferometer modified to serve as a digital holographic microscope with a high numerical aperture illumination module and a spatial light modulator (SLM). Such a solution is fast and robust. Apart from providing an elegant solution to viewing angle shifting, it also adds new capabilities of the holographic microscope system. SLM, being an active optical element, allows wavefront correction in order to improve measurement accuracy. Integrated phase data captured with different illumination scenarios within a highly limited angular range are processed by a new tomographic reconstruction algorithm based on the compressed sensing technique: total variation minimization, which is applied here to reconstruct nonpiecewise constant samples. Finally, the accuracy of full measurement and the proposed processing path is tested for a calibrated three-dimensional micro-object as well as a biological object--C2C12 myoblast cell.