Fabrication of waveguide directional couplers using 2-photon lithography.

Advances in 2-photon lithography have enabled in-lab production of sub-micron resolution and millimeter scale 3D optical components. The potential complex geometries are well suited to rapid prototyping and production of waveguide structures, interconnects, and waveguide directional couplers, furthering future development and miniaturization of waveguide-based imaging technologies. System alignment is inherent to the 2-photon process, obviating the need for manual assembly and allowing precise micron scale waveguide geometries not possible in traditional fused fiber coupler fabrication. Here we present the use of 2-photon lithography for direct printing of multi-mode waveguide couplers with air cladding and single mode waveguide couplers with uncured liquid photoresin cladding. Experimental results show reproducible coupling which can be modified by selected design parameters.

High-spatial density snapshot imaging spectrometer enabled by 2-photon fabricated custom fiber bundles.

We report on a proof-of-concept snapshot imaging spectrometer developed using an array of optical fibers fabricated with 2-photon polymerization (2PP). The dense input array maps to an output array with engineered void spaces for spectral information. Previously, the development and fabrication of custom fiber arrays for imaging spectrometers have been a complex, time-consuming, and costly process, requiring a semi-manual assembly of commercial components. This work applies an automatic development process based on 2PP additive manufacturing with the Nanoscribe GmbH Quantum X system. The technique allows printing of arbitrary optical quality structures with submicron resolution with less than 5 nm roughness, enabling small core fibers/integrated arrays. Specifically, we developed an array prototype of 40 × 80 with 6-micron pitch at the input and 80-micron pitch at the output. The air-clad fibers had a core diameter of 5 µm. Fabricated optical fiber arrays were incorporated into a prism-based imaging spectrometer system with 48 spectral channels to demonstrate multi-spectral imaging. Imaging of a USAF target and color printed letter C as well as spectral comparisons to a commercial spectrometer were used to validate the performance of the system. These results clearly demonstrate the functionality and potential applications of the 3D-printed fiber-based snapshot imaging spectrometer.

Fabrication of a multifaceted mapping mirror using two-photon polymerization for a snapshot image mapping spectrometer.

A design and fabrication technique for making high-precision and large-format multifaceted mapping mirrors is presented. The method is based on two-photon polymerization, which allows more flexibility in the mapping mirror design. The mirror fabricated in this paper consists of 36 2D tilted square pixels, instead of the continuous facet design used in diamond cutting. The paper presents a detailed discussion of the fabrication parameters and optimization process, with particular emphasis on the optimization of stitching defects by compensating for the overall tilt angle and reducing the printing field of view. The fabricated mirrors were coated with a thin layer of aluminum (93 nm) using sputter coating to enhance the reflection rate over the target wave range. The mapping mirror was characterized using a white light interferometer and a scanning electron microscope, which demonstrates its optical quality surface (with a surface roughness of 12 nm) and high-precision tilt angles (with an average of 2.03% deviation). Finally, the incorporation of one of the 3D printed mapping mirrors into an image mapping spectrometer prototype allowed for the acquisition of high-quality images of the USAF resolution target and bovine pulmonary artery endothelial cells stained with three fluorescent dyes, demonstrating the potential of this technology for practical applications.

Ruggedized, field-ready snapshot light-guide-based imaging spectrometer for environmental and remote sensing applications.

A field-ready, fiber-based high spatial sampling snapshot imaging spectrometer was developed for applications such as environmental monitoring and smart farming. The system achieves video rate frame transfer and exposure times down to a few hundred microseconds in typical daylight conditions with ∼63,000 spatial points and 32 spectral channels across the 470nm to 700nm wavelength range. We designed portable, ruggedized opto-mechanics to allow for imaging from an airborne platform. To ensure successful data collection prior to flight, imaging speed and signal-to-noise ratio was characterized for imaging a variety of land covers from the air. The system was validated by performing a series of observations including: Liriope Muscari plants under a range of water-stress conditions in a controlled laboratory experiment and field observations of sorghum plants in a variety of soil conditions. Finally, we collected data from a series of engineering flights and present reassembled images and spectral sampling of rural and urban landscapes.

Radiometric and design model for the tunable light-guide image processing snapshot spectrometer (TuLIPSS).

The tunable light-guide image processing snapshot spectrometer (TuLIPSS) is a novel remote sensing instrument that can capture a spectral image cube in a single snapshot. The optical modelling application for the absolute signal intensity on a single pixel of the sensor in TuLIPSS has been developed through a numerical simulation of the integral performance of each optical element in the TuLIPSS system. The absolute spectral intensity of TuLIPSS can be determined either from the absolute irradiance of the observed surface or from the tabulated spectral reflectance of various land covers and by the application of a global irradiance approach. The model is validated through direct comparison of the simulated results with observations. Based on tabulated spectral reflectance, the deviation between the simulated results and the measured observations is less than 5% of the spectral light flux across most of the detection bandwidth for a Lambertian-like surface such as concrete. Additionally, the deviation between the simulated results and the measured observations using global irradiance information is less than 10% of the spectral light flux across most of the detection bandwidth for all surfaces tested. This optical modelling application of TuLIPSS can be used to assist the optimal design of the instrument and explore potential applications. The influence of the optical components on the light throughput is discussed with the optimal design being a compromise among the light throughput, spectral resolution, and cube size required by the specific application under consideration. The TuLIPSS modelling predicts that, for the current optimal low-cost configuration, the signal to noise ratio can exceed 10 at 10 ms exposure time, even for land covers with weak reflectance such as asphalt and water. Overall, this paper describes the process by which the optimal design is achieved for particular applications and directly connects the parameters of the optical components to the TuLIPSS performance.

Fabrication of optical components using a consumer-grade lithographic printer.

The ability to 3D print optical elements will greatly expand the accessibility of optical fabrication. Here, we report on two fabrication techniques for plano-convex lens files using a consumer-grade lithographic printer. Lenses were post-processed using a simple spin coating technique with the resin used in the printing process or by curing directly on glass concave lenses. Average RMS roughness values were between 13 and 28 nm and RMS wavefront deviations were between 0.297 and 0.374 wave for spin-coated lenses. The average roughness RMS for the glass-cured lenses was 6 nm and the average form RMS was 0.048 wave.

High performance image mapping spectrometer (IMS) for snapshot hyperspectral imaging applications.

A high performance, snapshot Image Mapping Spectrometer was developed that provides fast image acquisition (100 Hz) of 16 bit hyperspectral data cubes (210x210x46) over a spectral range of 515-842 nm. Essential details of the opto-mechanical design are presented. Spectral accuracy, precision, and image reconstruction metrics such as resolution are discussed. Fluorescently stained cell samples were used to directly compare the data obtained using newly developed and the reference image mapping spectrometer. Additional experimental results are provided to demonstrate the abilities of the new spectrometer to acquire highly-resolved, motion-artifact-free hyperspectral images at high temporal sampling rates.

Light-guide snapshot imaging spectrometer for remote sensing applications.

A fiber-based snapshot imaging spectrometer was developed with a maximum of 31853 (~188 x 170) spatial sampling and 61 spectral channels in the 450nm-750nm range. A compact, custom-fabricated fiber bundle was used to sample the object image at the input and create void spaces between rows at the output for dispersion. The bundle was built using multicore 6x6 fiber block ribbons. To avoid overlap between the cores in the direction of dispersion, we selected a subset of cores using two alternative approaches; a lenslet array and a photomask. To calibrate the >30000 spatial samples of the system, a rapid spatial calibration method was developed based on phase-shifting interferometry (PSI). System crosstalk and spectral resolution were also characterized. Preliminary hyperspectral imaging results of the Rice University campus landscape, obtained with the spectrometer, are presented to demonstrate the system's spectral imaging capability for distant scenes. The spectrum of different plant species with different health conditions, obtained with the spectrometer, was in accordance with reference instrument measurements. We also imaged Houston traffic to demonstrate the system's snapshot hyperspectral imaging capability. Potential applications of the system include terrestrial monitoring, land use, air pollution, water resources, and lightning spectroscopy. The fiber-based system design potentially allows tuning between spatial and spectral sampling to meet specific imaging requirements.

3D printed fiber optic faceplates by custom controlled fused deposition modeling.

A 3D printing technique for manufacturing air-clad coherent fiber optic faceplates is presented. The custom G-code programming is implemented on a fused deposition modeling (FDM) desktop printer to additively draw optical fibers using high-transparency thermoplastic filaments. The 3D printed faceplate consists of 20000 fibers and achieves spatial resolution 1.78 LP/mm. Transmission loss and crosstalk are characterized and compared among the faceplates printed from four kinds of transparent filaments as well as different faceplate thicknesses. The printing temperature is verified by testing the transmission of the faceplates printed under different temperatures. Compared with the conventional stack-and-draw fabrication, the FDM 3D printing technique simplifies the fabrication procedure. The ability to draw fibers with arbitrary organization, structure and overall shape provides additional degree of freedom to opto-mechanical design. Our results indicate a promising capability of 3D printing as the manufacturing technology for fiber optical devices.

Quantitative evaluation of performance of 3D printed lenses.

We present an analysis of the shape, surface quality, and imaging capabilities of custom 3D printed lenses. 3D printing technology enables lens prototypes to be fabricated without restrictions on surface geometry. Thus, spherical, aspherical and rotationally non-symmetric lenses can be manufactured in an integrated production process. This technique serves as a noteworthy alternative to multistage, labor-intensive, abrasive processes such as grinding, polishing and diamond turning. Here, we evaluate the quality of lenses fabricated by Luxexcel using patented Printoptical© technology that is based on an inkjet printing technique by comparing them to lenses made with traditional glass processing technologies (grinding, polishing etc.). The surface geometry and roughness of the lenses were evaluated using white-light and Fizeau interferometers. We have compared peak-to-valley wavefront deviation, root-mean-squared wavefront error, radii of curvature and the arithmetic average of the roughness profile (Ra) of plastic and glass lenses. Additionally, the imaging performance of selected pairs of lenses was tested using 1951 USAF resolution target. The results indicate performance of 3D printed optics that could be manufactured with surface roughness comparable to that of injection molded lenses (Ra < 20 nm). The RMS wavefront error of 3D printed prototypes was at a minimum 18.8 times larger than equivalent glass prototypes for a lens with a 12.7 mm clear aperture, but when measured within 63% of its clear aperture, 3D printed components' RMS wavefront error was comparable to glass lenses.

High spatial sampling light-guide snapshot spectrometer.

A prototype fiber-based imaging spectrometer was developed to provide snapshot hyperspectral imaging tuned for biomedical applications. The system is designed for imaging in the visible spectral range from 400 to 700 nm for compatibility with molecular imaging applications as well as satellite and remote sensing. An 81 × 96 pixel spatial sampling density is achieved by using a custom-made fiber-optic bundle. The design considerations and fabrication aspects of the fiber bundle and imaging spectrometer are described in detail. Through the custom fiber bundle, the image of a scene of interest is collected and divided into discrete spatial groups, with spaces generated in between groups for spectral dispersion. This reorganized image is scaled down by an image taper for compatibility with following optical elements, dispersed by a prism, and is finally acquired by a CCD camera. To obtain an (x, y, λ) datacube from the snapshot measurement, a spectral calibration algorithm is executed for reconstruction of the spatial-spectral signatures of the observed scene. System characterization of throughput, resolution, and crosstalk was performed. Preliminary results illustrating changes in oxygen-saturation in an occluded human finger are presented to demonstrate the system's capabilities.

Snapshot Hyperspectral Light-Sheet Imaging of Signal Transduction in Live Pancreatic Islets.

The observation of ionic signaling dynamics in intact pancreatic islets has contributed greatly to our understanding of both α- and ß-cell function. Insulin secretion from ß-cells depends on the firing of action potentials and consequent rises of intracellular calcium activity ([Ca(2+)]i). Zinc (Zn(2+)) is cosecreted with insulin, and has been postulated to play a role in cell-to-cell cross talk within an islet, in particular inhibiting glucagon secretion from α-cells. Thus, measuring [Ca(2+)]i and Zn(2+) dynamics from both α- and ß-cells will elucidate mechanisms underlying islet hormone secretion. [Ca(2+)]i and intracellular Zn(2+) can be measured using fluorescent biosensors, but the most efficient sensors have overlapping spectra that complicate their discrimination. Hyperspectral imaging can be used to distinguish signals from multiple fluorophores, but available hyperspectral implementations are either too slow to measure the dynamics of ionic signals or not suitable for thick samples. We have developed a five-dimensional (x,y,z,t,λ) imaging system that leverages a snapshot hyperspectral imaging method, image mapping spectrometry, and light-sheet microscopy. This system provides subsecond temporal resolution from deep within multicellular structures. Using a single excitation wavelength (488 nm) we acquired images from triply labeled samples with two biosensors and a genetically expressing fluorescent protein (spectrally overlapping with one of the biosensors) with high temporal resolution. Measurements of [Ca(2+)]i and Zn(2+) within both α- and ß-cells as a function of glucose concentration show heterogeneous uptake of Zn(2+) into α-cells that correlates to the known heterogeneities in [Ca(2+)]i. These differences in intracellular Zn(2+) among α-cells may contribute to the inhibition in glucagon secretion observed at elevated glucose levels.

Development of tunable miniature piezoelectric-based scanners validated by the combination of two scanners in a direct image relay technique.

Miniature piezoelectric actuators are commonly used as a compact means to relay images for numerous endoscopic applications. These scanners normally consist of an electrically driven lead zirconate titanate (PZT) tube that oscillates an optical fiber at its resonant frequency. The diameter and length of the PZT and fiber, the attachment of the fiber to the PZT, as well as the driving signal determine the main characteristics of the scan-frequency and amplitude of vibration. We present a new, robust, and repeatable method for producing miniature PZT actuators. The described technology allows for continuous tuning of the scanner mechanical properties during the assembly stage, enabling adjustment of resonant frequency and subsequent amplitude of vibration without a priori knowledge of the fiber's mechanical properties. The method consists of manufacturing high-precision fiber-holding plastic inserts with diamond turning lathes that allow for the fiber length to be quickly varied and locked during operation in order to meet the preferred performance. This concept of tuned PZTs was demonstrated with an imaging technique that combined two scanners oscillating in unison at the ends of a single optical fiber to relay images without the need to correlate the driving signal with a detector.

Quantitative sectioning and noise analysis for structured illumination microscopy: erratum.

We correct an error in the original manuscript, where an unrecognized assumption was made about the relationship between the out-of-focus light and the in-focus light. We summarize the condition under which the assumption may still hold, and mention alternative methods researchers can use to obtain accurate quantitative sectioning.

Real-time hyperspectral fluorescence imaging of pancreatic ß-cell dynamics with the image mapping spectrometer.

The development of multi-colored fluorescent proteins, nanocrystals and organic fluorophores, along with the resulting engineered biosensors, has revolutionized the study of protein localization and dynamics in living cells. Hyperspectral imaging has proven to be a useful approach for such studies, but this technique is often limited by low signal and insufficient temporal resolution. Here, we present an implementation of a snapshot hyperspectral imaging device, the image mapping spectrometer (IMS), which acquires full spectral information simultaneously from each pixel in the field without scanning. The IMS is capable of real-time signal capture from multiple fluorophores with high collection efficiency (∼65%) and image acquisition rate (up to 7.2 fps). To demonstrate the capabilities of the IMS in cellular applications, we have combined fluorescent protein (FP)-FRET and [Ca(2+)](i) biosensors to measure simultaneously intracellular cAMP and [Ca(2+)](i) signaling in pancreatic ß-cells. Additionally, we have compared quantitatively the IMS detection efficiency with a laser-scanning confocal microscope.

Rapid fabrication of miniature lens arrays by four-axis single point diamond machining.

A novel method for fabricating lens arrays and other non-rotationally symmetric free-form optics is presented. This is a diamond machining technique using 4 controlled axes of motion - X, Y, Z, and C. As in 3-axis diamond micro-milling, a diamond ball endmill is mounted to the work spindle of a 4-axis ultra-precision computer numerical control (CNC) machine. Unlike 3-axis micro-milling, the C-axis is used to hold the cutting edge of the tool in contact with the lens surface for the entire cut. This allows the feed rates to be doubled compared to the current state of the art of micro-milling while producing an optically smooth surface with very low surface form error and exceptionally low radius error.

Snapshot 3D optical coherence tomography system using image mapping spectrometry.

A snapshot 3-Dimensional Optical Coherence Tomography system was developed using Image Mapping Spectrometry. This system can give depth information (Z) at different spatial positions (XY) within one camera integration time to potentially reduce motion artifact and enhance throughput. The current (x,y,λ) datacube of (85×356×117) provides a 3D visualization of sample with 400 µm depth and 13.4 µm in transverse resolution. Axial resolution of 16.0 µm can also be achieved in this proof-of-concept system. We present an analysis of the theoretical constraints which will guide development of future systems with increased imaging depth and improved axial and lateral resolutions.

Quantitative sectioning and noise analysis for structured illumination microscopy.

Structured illumination (SI) has long been regarded as a nonquantitative technique for obtaining sectioned microscopic images. Its lack of quantitative results has restricted the use of SI sectioning to qualitative imaging experiments, and has also limited researchers' ability to compare SI against competing sectioning methods such as confocal microscopy. We show how to modify the standard SI sectioning algorithm to make the technique quantitative, and provide formulas for calculating the noise in the sectioned images. The results indicate that, for an illumination source providing the same spatially-integrated photon flux at the object plane, and for the same effective slice thicknesses, SI sectioning can provide higher SNR images than confocal microscopy for an equivalent setup when the modulation contrast exceeds about 0.09.

Correction of vignetting and distortion errors induced by two-axis light beam steering.

A mirror facet's angle correction approach is presented for eliminating pupil plane distortions and sub-field image vignetting in the image mapping spectrometry (IMS). The two-axis light reflection problem on the image mapper is solved and a rigorous analytical solution is provided. The cellular fluorescence imaging experiment demonstrates that, with an angle-corrected image mapper, the acquired image quality of spectral channels has been significantly improved compared to previous IMS images. The proposed mathematical model can also be used in solving general two-axis beam steering problems for instruments with active optical mirrors.

Image mapping spectrometry: calibration and characterization.

Image mapping spectrometry (IMS) is a hyperspectral imaging technique that simultaneously captures spatial and spectral information about an object in real-time. We present a new calibration procedure for the IMS as well as the first detailed evaluation of system performance. We correlate optical components and device calibration to performance metrics such as light throughput, scattered light, distortion, spectral image coregistration, and spatial/spectral resolution. Spectral sensitivity and motion artifacts are also evaluated with a dynamic biological experiment. The presented methodology of evaluation is useful in assessment of a variety of hyperspectral and multi-spectral modalities. Results are important to any potential users/developers of an IMS instrument and to anyone who may wish to compare the IMS to other imaging spectrometers.