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.

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.

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.

Evaluation of the LeukoScope for Point-of-Care Measurement of White Blood Cell and Neutrophil Counts in Malawi.

White blood cell (WBC) and neutrophil counts are important laboratory tests used by clinicians to assess a variety of conditions. However, current methods to measure WBC and neutrophil counts are difficult to perform at the point of care, being either cost or labor prohibitive. To meet this need, we developed the LeukoScope: a portable, imaging-based system to measure WBC and neutrophil counts from a drop of blood. Here, we present the performance of the LeukoScope in 136 pediatric and 164 neonatal subjects at a central hospital in Malawi. For pediatric patients, 95.4, 66.7, and 80.0% of samples with normal, low, and high WBC counts, respectively, were correctly identified, and 88.6, 100.0, and 89.3% of samples with normal, low, and high neutrophil counts, respectively, were correctly identified. Accuracy was lower overall for neonatal samples; 92.1, 64.3, and 26.7% of samples with normal, low, and high WBC counts, respectively, were correctly identified, and 73.2 and 78.6% of samples with normal and high neutrophil counts, respectively, were correctly identified. Results of this study show that the LeukoScope can help meet need for point-of-care measurement of WBC counts in pediatric patients and highlight the challenges of point-of-care assessment of WBC counts in neonatal patients.

Toward development of a large field-of-view cancer screening patch (CASP) to detect cervical intraepithelial neoplasia.

Cervical cancers are primarily diagnosed via colposcopy, in which the tissue is visually assessed by a clinician for abnormalities, followed by directed biopsies and histologic analysis of excised tissue. Optical biopsy technologies offer a less invasive method of imaging such that subcellular features can be resolved without removing tissue. These techniques, however, are limited in field-of-view by the distal end of the probe. We present a prototype that incorporates a rigid, machinable waveguide that is in direct contact with a fluorescently-labeled sample paired with a scanning fluorescent microscope. The system is capable of imaging large areas of tissue without the need to re-position the tissue-probe interface. A mosaicing algorithm was developed to quantify scanning shifts and stitch neighboring frames together to increase the field-of-view. Our prototype can yield a maximum axial resolution of <5 µm for individual frames and can produce mosaiced images with a field-of-view greater than 15 mm x 15 mm without sacrificing resolution. We validated the system with a 1951 USAF resolution target, fluorescent in vitro standards, and a patient study where ex vivo conization samples of squamous cervical epithelium were imaged. The results of the patient study indicate that architectural features of subcellular components could be detected and differentiated between normal tissue and precancerous lesions.

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.

A Dye-Free Analog to Retinal Angiography Using Hyperspectral Unmixing to Retrieve Oxyhemoglobin Abundance.

PURPOSE: Retinal angiography evaluates retinal and choroidal perfusion and vascular integrity and is used to manage many ophthalmic diseases, such as age-related macular degeneration. The most common method, fluorescein angiography (FA), is invasive and can lead to untoward effects. As an emerging replacement, noninvasive OCT angiography (OCTA) is used regularly as a dye-free substitute with superior resolution and additional depth-sectioning abilities; however, general trends in FA as signified by varying intensity in images are not always reproducible in the fine structural detail in an OCTA image stack because of the source of their respective signals, OCT speckle decorrelation versus fluorescein emission. METHODS: We present a noninvasive/dye-free analog to angiography imaging using retinal hyperspectral imaging with a nonscanning spectral imager, the image mapping spectrometer (IMS), to reproduce perfusion-related data based on the abundance of oxyhemoglobin (HbO2) in the retina. With a new unmixing procedure of the IMS-acquired spectral data cubes (350 × 350 × 43), we produced noninvasive HbO2 maps unmixed from reflectance spectra. RESULTS: Here, we present 15 HbO2 maps from seven healthy and eight diseased retinas and compare these maps with corresponding FA and OCTA results with a discussion of each technique. CONCLUSIONS: Our maps showed visual agreement with hypo- and hyperfluorescence trends in venous phase FA images, suggesting that our method provides a new use for hyperspectral imaging as a noninvasive angiography-analog technique and as a complementary technique to OCTA. TRANSLATIONAL RELEVANCE: The application of hyperspectral imaging and spectral analysis can potentially improve/broaden retinal disease screening and enable a noninvasive technique, which complements OCTA.

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.

Simple ultraviolet microscope using off-the-shelf components for point-of-care diagnostics.

At the primary care setting, where there are often no or minimal laboratories, examinations often consist of self-testing and rapid diagnostics. Because of this, medical devices must be simple, robust, and easy to operate. To address these concerns, an alternate fluorescence microscope design uses ultraviolet (UV) excitation, since fluorescent dyes that are excitable in the visible region are also excitable by UV. This may allow for the removal of typical excitation, emission, and dichroic filters as optical components absorb UV wavelengths and UV is not detected by silicon based detectors. Additionally, UV has a very low penetration into samples, which may allow for controlling the depth of excitation, and thus the imaging volume. Based on these ideas, we developed a simple fluorescence microscope built completely from off-the-shelf components that uses UV to image fluorescently stained samples. The simple opto-mechanical design of the system may allow it to be more compact and easy to use, as well as decrease the overall cost of the diagnostic device. For biological validation, we imaged whole blood stained with acridine orange and performed a two-part white blood cell differential count.

High-resolution endomicroscopy with a spectrally encoded miniature objective.

Fiber bundle endomicroscopy techniques have been used for numerous minimally invasive imaging applications. However, these techniques may provide limited spatial sampling due to the limited number of imaging cores inside the fiber bundle. Here, we present a custom-fabricated miniature objective that can be coupled to a fiber bundle and can overcome the fiber bundle's sampling threshold by utilizing the spectral encoding concept. The objective has an NA of 0.3 and an outer diameter of 2.4 mm, and can yield a maximum spatial resolution of 2 µm. The objective has been validated against a USAF resolution target and ex vivo tissue samples, and as a result yielded images with higher resolution and more details after the spectral encoding concept was employed.

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.

Clinical training and validation of the LeukoScope: a low-cost, point-of-care device to perform white blood cell and neutrophil counts.

A white blood cell (WBC) count with partial differential is an important clinical laboratory test. However, current methods to perform a WBC count and differential are difficult to use at the point of care or too expensive for use in low-resource settings. To meet this need, we developed the LeukoScope: a low-cost system to measure a WBC and neutrophil count from a single drop of blood at the point of care. The LeukoScope is battery powered and has a sample-to-answer time of <5 minutes. A drop of blood from a finger stick is added to a LeukoScope sample cartridge where pre-dried acridine orange fluorescently stains WBCs. The cartridge is then inserted into the LeukoScope reader where a portable fluorescence microscope captures a color image of the sample, which is analyzed to report results to the user. The LeukoScope system was tested at the point of care using fingerprick samples collected from 105 general oncology patients in Houston, TX. Performance of the LeukoScope was compared to that of a HemoCue WBC DIFF performed using the same fingerprick sample; clinical laboratory analysis of a venous blood draw was used as the gold standard in all cases. Bland-Altman analysis showed that the LeukoScope and HemoCue WBC DIFF had similar accuracy for measurement of WBC and neutrophil counts as compared to the gold standard. Seven out of eight patients with abnormal WBC count values were correctly identified using the LeukoScope, while six out of eight were correctly identified using the HemoCue WBC DIFF. Five out of six patients with abnormal neutrophil counts were correctly identified using the LeukoScope, while six of six were correctly identified using the HemoCue WBC DIFF.

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.

Development of a universal, tunable, miniature fluorescence microscope for use at the point of care.

Fluorescence microscopy can be a powerful tool for cell-based diagnostic assays; however, imaging can be time consuming and labor intensive to perform. Tunable systems give the ability to electronically focus at user selected depths inside an object volume and may simplify the opto-mechanical design of the imaging system. We present a prototype of a universal, tunable, miniature fluorescence microscope built from poly(methyl methacrylate) singlets that incorporates miniature, electrowetted lenses for electronic focusing. We demonstrate the ability of this system to perform clinically relevant differential white blood cell counts using single use custom cartridges pre-loaded with the fluorescent dye acridine orange.

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.

Development of a multimodal foveated endomicroscope for the detection of oral cancer.

A multimodal endomicroscope was developed for cancer detection that combines hyperspectral and confocal imaging through a single foveated objective and a vibrating optical fiber bundle. Standard clinical examination has a limited ability to identify early stage oral cancer. Optical detection methods are typically restricted by either achievable resolution or a small field-of-view. By combining high resolution and widefield spectral imaging into a single probe, a device was developed that provides spectral and spatial information over a 5 mm field to locate suspicious lesions that can then be inspected in high resolution mode. The device was evaluated on ex vivo biopsies of human oral tumors.

Lenslet array tunable snapshot imaging spectrometer (LATIS) for hyperspectral fluorescence microscopy.

Snapshot hyperspectral imaging augments pixel dwell time and acquisition speeds over existing scanning systems, making it a powerful tool for fluorescence microscopy. While most snapshot systems contain fixed datacube parameters (x,y,λ), our novel snapshot system, called the lenslet array tunable snapshot imaging spectrometer (LATIS), demonstrates tuning its average spectral resolution from 22.66 nm (80x80x22) to 13.94 nm (88x88x46) over 485 to 660 nm. We also describe a fixed LATIS with a datacube of 200x200x27 for larger field-of-view (FOV) imaging. We report <1 sec exposure times and high resolution fluorescence imaging with minimal artifacts.