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.

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.

Hyperspectral imaging microscopy for measurement of localized second messenger signals in single cells.

Ca2+ and cAMP are ubiquitous second messengers known to differentially regulate a variety of cellular functions over a wide range of timescales. Studies from a variety of groups support the hypothesis that these signals can be localized to discrete locations within cells, and that this subcellular localization is a critical component of signaling specificity. However, to date, it has been difficult to track second messenger signals at multiple locations within a single cell. This difficulty is largely due to the inability to measure multiplexed florescence signals in real time. To overcome this limitation, we have utilized both emission scan- and excitation scan-based hyperspectral imaging approaches to track second messenger signals as well as labeled cellular structures and/or proteins in the same cell. We have previously reported that hyperspectral imaging techniques improve the signal-to-noise ratios of both fluorescence and FRET measurements, and are thus well suited for the measurement of localized second messenger signals. Using these approaches, we have measured near plasma membrane and near nuclear membrane cAMP signals, as well as distributed signals within the cytosol, in several cell types including airway smooth muscle, pulmonary endothelial, and HEK-293 cells. We have also measured cAMP and Ca2+ signals near autofluorescent structures that appear to be golgi. Our data demonstrate that hyperspectral imaging approaches provide unique insight into the spatial and kinetic distributions of cAMP and Ca2+ signals in single cells.

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.

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.

Endoscopic hyperspectral imaging: light guide optimization for spectral light source.

Hyperspectral imaging (HSI) is a technology used in remote sensing, food processing and documentation recovery. Recently, this approach has been applied in the medical field to spectrally interrogate regions of interest within respective substrates. In spectral imaging, a two (spatial) dimensional image is collected, at many different (spectral) wavelengths, to sample spectral signatures from different regions and/or components within a sample. Here, we report on the use of hyperspectral imaging for endoscopic applications. Colorectal cancer is the 3rd leading cancer for incidences and deaths in the US. One factor of severity is the miss rate of precancerous/flat lesions (~65% accuracy). Integrating HSI into colonoscopy procedures could minimize misdiagnosis and unnecessary resections. We have previously reported a working prototype light source with 16 high-powered light emitting diodes (LEDs) capable of high speed cycling and imaging. In recent testing, we have found our current prototype is limited by transmission loss (~99%) through the multi-furcated solid light guide (lightpipe) and the desired framerate (20-30 fps) could not be achieved. Here, we report on a series of experimental and modeling studies to better optimize the lightpipe and the spectral endoscopy system as a whole. The lightpipe was experimentally evaluated using an integrating sphere and spectrometer (Ocean Optics). Modeling the lightpipe was performed using Monte Carlo optical ray tracing in TracePro (Lambda Research Corp.). Results of these optimization studies will aid in manufacturing a revised prototype with the newly designed light guide and increased sensitivity. Once the desired optical output (5-10 mW) is achieved then the HIS endoscope system will be able to be implemented without adding onto the procedure time.

Design of a modified endoscope illuminator for spectral imaging of colorectal tissues.

The gold standard for locating colonic polyps is a white light endoscope in a colonoscopy, however, polyps smaller than 5 mm can be easily missed. Modified procedures such as narrow band imaging have shown only marginal increases in detection rates. Spectral imaging is a potential solution to improve the sensitivity and specificity of colonoscopies by providing the ability to distinguish molecular fluorescence differences in tissues. The goal of this work is to implement a spectral endoscopic light source to acquire spectral image data of colorectal tissues. A beta-version endoscope light source was developed, by retrofitting a white light endoscope light source (Olympus, CLK-4) with 16 narrow band LEDs. This redesigned, beta-prototype uses high-power LEDs with a minimum output of 500 mW to provide sufficient spectral output (0.5 mW) through the endoscope. A mounting apparatus was designed to provide sufficient heat dissipation. Here, we report recent results of our tests to characterize the intensity output through the light source and endoscope to determine the flat spectral output for imaging and intensity losses through the endoscope. We also report preliminary spectral imaging data from transverse pig colon that demonstrates the ability to result in working practical spectral data. Preliminary results of this revised prototype spectral endoscope system demonstrate that there is sufficient power to allow the imaging process to continue and potentially determine spectral differences in cancerous and normal tissue from imaging ex vivo pairs. Future work will focus on building a spectral library for the colorectal region and refining the user interface the system for in vivo use.

5D imaging approaches reveal the formation of distinct intracellular cAMP spatial gradients.

Cyclic AMP (cAMP) is a ubiquitous second messenger known to differentially regulate many cellular functions. Several lines of evidence suggest that the distribution of cAMP within cells is not uniform. However, to date, no studies have measured the kinetics of 3D cAMP distributions within cells. This is largely due to the low signal-to-noise ratio of FRET-based probes. We previously reported that hyperspectral imaging improves the signal-to-noise ratio of FRET measurements. Here we utilized hyperspectral imaging approaches to measure FRET signals in five dimensions (5D) - three spatial (x, y, z), wavelength (λ), and time (t) - allowing us to visualize cAMP gradients in pulmonary endothelial cells. cAMP levels were measured using a FRET-based sensor (H188) comprised of a cAMP binding domain sandwiched between FRET donor and acceptor - Turquoise and Venus fluorescent proteins. We observed cAMP gradients in response to 0.1 or 1 µM isoproterenol, 0.1 or 1 µM PGE1, or 50 µM forskolin. Forskolin- and isoproterenol-induced cAMP gradients formed from the apical (high cAMP) to basolateral (low cAMP) face of cells. In contrast, PGE1-induced cAMP gradients originated from both the basolateral and apical faces of cells. Data suggest that 2D (x,y) studies of cAMP compartmentalization may lead to erroneous conclusions about the existence of cAMP gradients, and that 3D (x,y,z) studies are required to assess mechanisms of signaling specificity. Results demonstrate that 5D imaging technologies are powerful tools for measuring biochemical processes in discrete subcellular domains. This work was supported by NIH P01HL066299, R01HL058506, S10RR027535, AHA 16PRE27130004 and the Abraham Mitchell Cancer Research Fund.

Three dimensional measurement of cAMP gradients using hyperspectral confocal microscopy.

Cyclic AMP (cAMP) is a ubiquitous second messenger known to differentially regulate many cellular functions over a wide range of timescales. Several lines of evidence have suggested that the distribution of cAMP within cells is not uniform, and that cAMP compartmentalization is largely responsible for signaling specificity within the cAMP signaling pathway. However, to date, no studies have experimentally measured three dimensional (3D) cAMP distributions within cells. Here we use both 2D and 3D hyperspectral microscopy to visualize cAMP gradients in endothelial cells from the pulmonary microvasculature (PMVECs). cAMP levels were measured using a FRET-based cAMP sensor comprised of a cAMP binding domain from EPAC sandwiched between FRET donors and acceptors - Turquoise and Venus fluorescent proteins. Data were acquired using either a Nikon A1R spectral confocal microscope or custom spectral microscopy system. Analysis of hyperspectral image stacks from a single confocal slice or from summed images of all slices (2D analysis) indicated little or no cAMP gradients were formed within PMVECs under basal conditions or following agonist treatment. However, analysis of hyperspectral image stacks from 3D cellular geometries (z stacks) demonstrate marked cAMP gradients from the apical to basolateral membrane of PMVECs. These results strongly suggest that 2D imaging studies of cAMP compartmentalization - whether epifluorescence or confocal microscopy - may lead to erroneous conclusions about the existence of cAMP gradients, and that 3D studies are required to assess mechanisms of signaling specificity.

LED-based endoscopic light source for spectral imaging.

Colorectal cancer is the United States 3rd leading cancer in death rates.1 The current screening for colorectal cancer is an endoscopic procedure using white light endoscopy (WLE). There are multiple new methods testing to replace WLE, for example narrow band imaging and autofluorescence imaging.2 However, these methods do not meet the need for a higher specificity or sensitivity. The goal for this project is to modify the presently used endoscope light source to house 16 narrow wavelength LEDs for spectral imaging in real time while increasing sensitivity and specificity. The process to do such was to take an Olympus CLK-4 light source, replace the light and electronics with 16 LEDs and new circuitry. This allows control of the power and intensity of the LEDs. This required a larger enclosure to house a bracket system for the solid light guide (lightpipe), three new circuit boards, a power source and National Instruments hardware/software for computer control. The results were a successfully designed retrofit with all the new features. The LED testing resulted in the ability to control each wavelength's intensity. The measured intensity over the voltage range will provide the information needed to couple the camera for imaging. Overall the project was successful; the modifications to the light source added the controllable LEDs. This brings the research one step closer to the main goal of spectral imaging for early detection of colorectal cancer. Future goals will be to connect the camera and test the imaging process.