Multifaceted mirror array illuminator for fluorescence excitation-scanning spectral imaging microscopy.

Significance: Hyperspectral imaging (HSI) technologies offer great potential in fluorescence microscopy for multiplexed imaging, autofluorescence removal, and analysis of autofluorescent molecules. However, there are also associated trade-offs when implementing HSI in fluorescence microscopy systems, such as decreased acquisition speed, resolution, or field-of-view due to the need to acquire spectral information in addition to spatial information. The vast majority of HSI fluorescence microscopy systems provide spectral discrimination by filtering or dispersing the fluorescence emission, which may result in loss of emitted fluorescence signal due to optical filters, dispersive optics, or supporting optics, such as slits and collimators. Technologies that scan the fluorescence excitation spectrum may offer an approach to mitigate some of these trade-offs by decreasing the complexity of the emission light path. Aim: We describe the development of an optical technique for hyperspectral imaging fluorescence excitation-scanning (HIFEX) on a microscope system. Approach: The approach is based on the design of an array of wavelength-dependent light emitting diodes (LEDs) and a unique beam combining system that uses a multifurcated mirror. The system was modeled and optimized using optical ray trace simulations, and a prototype was built and coupled to an inverted microscope platform. The prototype system was calibrated, and initial feasibility testing was performed by imaging multilabel slide preparations. Results: We present results from optical ray trace simulations, prototyping, calibration, and feasibility testing of the system. Results indicate that the system can discriminate between at least six fluorescent labels and autofluorescence and that the approach can provide decreased wavelength switching times, in comparison with mechanically tuned filters. Conclusions: We anticipate that LED-based HIFEX microscopy may provide improved performance for time-dependent and photosensitive assays.

Microscopy is better in color: development of a streamlined spectral light path for real-time multiplex fluorescence microscopy.

Spectroscopic image data has provided molecular discrimination for numerous fields including: remote sensing, food safety and biomedical imaging. Despite the various technologies for acquiring spectral data, there remains a trade-off when acquiring data. Typically, spectral imaging either requires long acquisition times to collect an image stack with high spectral specificity or acquisition times are shortened at the expense of fewer spectral bands or reduced spatial sampling. Hence, new spectral imaging microscope platforms are needed to help mitigate these limitations. Fluorescence excitation-scanning spectral imaging is one such new technology, which allows more of the emitted signal to be detected than comparable emission-scanning spectral imaging systems. Here, we have developed a new optical geometry that provides spectral illumination for use in excitation-scanning spectral imaging microscope systems. This was accomplished using a wavelength-specific LED array to acquire spectral image data. Feasibility of the LED-based spectral illuminator was evaluated through simulation and benchtop testing and assessment of imaging performance when integrated with a widefield fluorescence microscope. Ray tracing simulations (TracePro) were used to determine optimal optical component selection and geometry. Spectral imaging feasibility was evaluated using a series of 6-label fluorescent slides. The LED-based system response was compared to a previously tested thin-film tunable filter (TFTF)-based system. Spectral unmixing successfully discriminated all fluorescent components in spectral image data acquired from both the LED and TFTF systems. Therefore, the LED-based spectral illuminator provided spectral image data sets with comparable information content so as to allow identification of each fluorescent component. These results provide proof-of-principle demonstration of the ability to combine output from many discrete wavelength LED sources using a double-mirror (Cassegrain style) optical configuration that can be further modified to allow for high speed, video-rate spectral image acquisition. Real-time spectral fluorescence microscopy would allow monitoring of rapid cell signaling processes (i.e., Ca2+ and other second messenger signaling) and has potential to be translated to clinical imaging platforms.

Validation of Excitation-Scan Hyperspectral Multi-faceted Mirror Array Prototype System Advancements to Hyperspectral Imaging Applications.

Hyperspectral imaging technologies (HSI) have undergone rapid development since their beginning stages. While original applications were in remote sensing, other uses include agriculture, food safety and medicine. HSI has shown great utility in fluorescence microscopy for detecting signatures from many fluorescent molecules; however, acquisitions speeds have been slow due to light losses associated with spectral filtering. Therefore, we designed a novel light emitting diode (LED)-based rapid excitation scanning hyperspectral imaging platform allowing users to obtain simultaneous measurements of fluorescent labels without compromising acquisition speeds. Previously, we reported our results of the optical ray trace simulations and the geometrical capability of designing a multifaceted mirror imaging system as an initial approach to combine light at many wavelengths. The design utilized LEDs and a multifaceted mirror array to combine light sources into a liquid light guide. The computational model was constructed using Monte Carlo optical ray software (TracePro, Lambda Research Corp.). Recent prototype validation results show that when compared to a commercial emission scanning spectral confocal microscope (Zeiss-LSM-980), the novel LED-based excitation scanning HSI prototype successfully detected and separated six fluorescent labels from a custom 6-label African green monkey kidney epithelial cells. We report on the prototype's ability to overcome limitations of acquisition speeds, sensitivity, and specificity present in conventional systems. Future work will evaluate prototype's light losses to determine latent design modifications needed to demonstrate the system's feasibility as a promising solution for overcoming HSI acquisition speeds. This work was supported by NSF award MRI1725937.

Optical simulations for determining efficacy of new light source designs for excitation-scanning high-speed hyperspectral imaging systems.

Positive outcomes for colorectal cancer treatment have been linked to early detection. The difficulty in detecting early lesions is the limited contrast with surrounding mucosa and minimal definitive markers to distinguish between hyperplastic and carcinoma lesions. Colorectal cancer is the 3rd leading cancer for incidence and mortality rates which is potentially linked to missed early lesions which allow for increased growth and metastatic potential. One potential technology for early-stage lesion detection is hyperspectral imaging. Traditionally, hyperspectral imaging uses reflectance spectroscopic data to provide a component analysis, per pixel, of an image in fields such as remote sensing, agriculture, food processing and archaeology. This work aims to acquire higher signal-to-noise fluorescence spectroscopic data, harnessing the autofluorescence of tissue, adding a hyperspectral contrast to colorectal cancer detection while maintaining spatial resolution at video-rate speeds. We have previously designed a multi-furcated LED-based spectral light source to prove this concept. Our results demonstrated that the technique is feasible, but the initial prototype has a high light transmission loss (~98%) minimizing spatial resolution and slowing video acquisition. Here, we present updated results in developing an optical ray-tracing model of light source geometries to maximize irradiance throughput for excitation-scanning hyperspectral imaging. Results show combining solid light guide branches have a compounding light loss effect, however, there is potential to minimize light loss through the use of optical claddings. This simulation data will provide the necessary metrics to verify and validate future physical optical components within the hyperspectral endoscopic system for detecting colorectal cancer.

Spectral Illumination System Utilizing Spherical Reflection Optics.

Fluorescence imaging microscopy has traditionally been used because of the high specificity that is achievable through fluorescence labeling techniques and optical filtering. When combined with spectral imaging technologies, fluorescence microscopy can allow for quantitative identification of multiple fluorescent labels. We are working to develop a new approach for spectral imaging that samples the fluorescence excitation spectrum and may provide increased signal strength. The enhanced signal strength may be used to provide increased spectral sensitivity and spectral, spatial, and temporal sampling capabilities. A proof of concept excitation scanning system has shown over 10-fold increase in signal to noise ratio compared to emission scanning hyperspectral imaging. Traditional hyperspectral imaging fluorescence microscopy methods often require minutes of acquisition time. We are developing a new configuration that utilizes solid state LEDs to combine multiple illumination wavelengths in a 2-mirror assembly to overcome the temporal limitations of traditional hyperspectral imaging. We have previously reported on the theoretical performance of some of the aspects of this system by using optical ray trace modeling. Here, we present results from prototyping and benchtop testing of the system, including assembly, optical characterization, and data collection. This work required the assembly and characterization of a novel excitation scanning hyperspectral microscopy system, containing 12 LEDs ranging from 365-425 nm, 12 lenses, a spherical mirror, and a flat mirror. This unique approach may reduce the long image acquisition times seen in traditional hyperspectral imaging while maintaining high specificity and sensitivity for multilabel identification and autofluorescence imaging in real time.

Excitation-scan Mirror Array System Advancements to Hyperspectral Imaging Applications.

Hyperspectral imaging (HSI) technology has been applied in a range of fields for target detection and mixture analysis. While its original applications were in remote sensing, modern uses include agriculture, historical document authentications and medicine. HSI has shown great utility in fluorescence microscopy; however, acquisition speeds have been slow due to light losses associated with spectral filtering. We are currently developing a rapid hyperspectral imaging platform for 5-dimensional imaging (RHIP-5D), a confocal imaging system that will allow users to obtain simultaneous measurements of many fluorescent labels. We have previously reported on optical modeling performance of the system. This previous model investigated geometrical capability of designing a multifaceted mirror imaging system as an initial approach to sample light at many wavelengths. The design utilized light-emitting diodes (LEDs) and a multifaceted mirror array to combine light sources into a liquid light guide (LLG). The computational model was constructed using Monte Carlo optical ray software (TracePro, Lambda Research Corp.). Recent results presented here show transmission has increased up to 9% through parametric optimization of each component. Future work will involve system validation using a prototype engineered based on our optimized model. System requirements will be evaluated to determine if potential design changes are needed to improve the system. We will report on spectral resolution to demonstrate feasibility of the RHIP-5D as a promising solution for overcoming current HSI acquisition speed and sensitivity limitations.

A Spherical Mirror-based Illumination System for Fluorescence Excitation-Scanning Hyperspectral Imaging.

Many hardware approaches have been developed for implementing hyperspectral imaging on fluorescence microscope systems; each with tradeoffs in spectral sensitivity and spectral, spatial, and temporal sampling. For example, tunable filter-based systems typically have limited wavelength switching speeds and sensitivities that preclude high-speed spectral imaging. Here, we present a novel approach combining multiple illumination wavelengths using solid state LEDs in a 2-mirror configuration similar to a Cassegrain reflector assembly. This approach provides spectral discrimination by scanning a range of fluorescence excitation wavelengths, which we have previously shown can improve spectral image acquisition time compared to traditional fluorescence emission-scanning hyperspectral imaging. In this work, the geometry of the LED and other optical components was optimized. A model of the spectral illuminator was designed using TracePro ray tracing software (Lambda Research Corp.) that included an emitter, lens, Spherical mirror, flat mirror, and liquid light guide input. A parametric sensitivity study was performed to optimize the optical throughput varying the LED viewing angle, properties of the Spherical reflectors, the lens configuration, focal length, and position. The following factors significantly affected the optical throughput: LED viewing angle, lens position, and lens focal length. Several types of configurations were evaluated, and an optimized lens and LED position were determined. Initial optimization results indicate that a 10% optical transmission can be achieved for either a 16 or 32 wavelength system. Future work will include continuing to optimize the ray trace model, prototyping, and experimental testing of the optimized configuration.

Hyperspectral imaging fluorescence excitation scanning (HIFEX) microscopy for live cell imaging.

In the past two decades, spectral imaging technologies have expanded the capacity of fluorescence microscopy for accurate detection of multiple labels, separation of labels from cellular and tissue autofluorescence, and analysis of autofluorescence signatures. These technologies have been implemented using a range of optical techniques, such as tunable filters, diffraction gratings, prisms, interferometry, and custom Bayer filters. Each of these techniques has associated strengths and weaknesses with regard to spectral resolution, spatial resolution, temporal resolution, and signal-to-noise characteristics. We have previously shown that spectral scanning of the fluorescence excitation spectrum can provide greatly increased signal strength compared to traditional emission-scanning approaches. Here, we present results from utilizing a Hyperspectral Imaging Fluorescence Excitation Scanning (HIFEX) microscope system for live cell imaging. Live cell signaling studies were performed using HEK 293 and rat pulmonary microvascular endothelial cells (PMVECs), transfected with either a cAMP FRET reporter or a Ca2+ reporter. Cells were further labeled to visualize subcellular structures (nuclei, membrane, mitochondria, etc.). Spectral images were acquired using a custom inverted microscope (TE2000, Nikon Instruments) equipped with a 300W Xe arc lamp and tunable excitation filter (VF-5, Sutter Instrument Co., equipped with VersaChrome filters, Semrock), and run through MicroManager. Timelapse spectral images were acquired from 350-550 nm, in 5 nm increments. Spectral image data were linearly unmixed using custom MATLAB scripts. Results indicate that the HIFEX microscope system can acquire live cell image data at acquisition speeds of 8 ms/wavelength band with minimal photobleaching, sufficient for studying moderate speed cAMP and Ca2+ events.

Sensitivity analysis of a multibranched light guide for real time hyperspectral imaging systems.

Hyperspectral imaging (HSI) is a spectroscopic technique which captures images at a high contrast over a wide range of wavelengths to show pixel specific composition. Traditional uses of HSI include: satellite imagery, food distribution quality control and digital archaeological reconstruction. Our lab has focused on developing applications of HSI fluorescence imaging systems to study molecule-specific detection for rapid cell signaling events or real-time endoscopic screening. Previously, we have developed a prototype spectral light source, using our modified imaging technique, excitation-scanning hyperspectral imaging (HIFEX), coupled to a commercial colonoscope for feasibility testing. The 16 wavelength LED array was combined, using a multi-branched solid light guide, to couple to the scope's optical input. The prototype acquired a spectral scan at near video-rate speeds (∼8 fps). The prototype could operate at very rapid wavelength switch speeds, limited to the on/off rates of the LEDs (∼10 µs), but imaging speed was limited due to optical transmission losses (∼98%) through the solid light guide. Here we present a continuation of our previous work in performing an in-depth analysis of the solid light guide to optimize the optical intensity throughput. The parameters evaluated include: LED intensity input, geometry (branch curvature and combination) and light propagation using outer claddings. Simulations were conducted using a Monte Carlo ray tracing software (TracePro). Results show that transmission within the branched light guide may be optimized through LED focusing lenses, bend radii and smooth tangential branch merges. Future work will test a new fabricated light guide from the optimized model framework.