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Colorectal cancer is the 3^rdleading cancer for incidence and mortality rates. Positive treatment outcomes have been associated with early detection; however, early stage lesions have limited contrast to surrounding mucosa. A potential technology to enhance early stagise detection is hyperspectral imaging (HSI). While HSI technologies have been previously utilized to detect colorectal cancerex vivoor post-operation, they have been difficult to employ in real-time endoscopy scenarios. Here, we describe an LED-based multifurcated light guide and spectral light source that can provide illumination for spectral imaging at frame rates necessary for video-rate endoscopy. We also present an updated light source optical ray-tracing model that resulted in further optimization and provided a ∼10X light transmission increase compared to the initial prototype. Future work will iterate simulation and benchtop testing of the hyperspectral endoscopic system to achieve the goal of video-rate spectral endoscopy.

 

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 While its original applications were in remote sensing, modern uses include agriculture, historical document authentications and medicine.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. 

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. 

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, excitationscanning 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. 

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. 

The majority of microscopic and endoscopic technologies utilize white light illumination. For a number of applications, hyper-spectral imaging can be shown to have significant improvements over standard white-light imaging techniques. This is true for both microscopy and in vivo imaging. However, hyperspectral imaging methods have suffered from slow application times. Often, minutes are required to gather a full imaging stack. Here we will describe and evaluate a novel excitation-scanning hyperspectral imaging system and discuss some applications. We have developed and are optimizing a novel approach called excitation-scanning hyperspectral imaging that provides an order of magnitude increased signal strength. This excitation scanning technique has enabled us to produce a microscopy system capable of high speed hyperspectral imaging with the potential for live video acquisition. The excitation-scanning hyperspectral imaging technology we developed may impact a range of applications. The current design uses digital strobing to illuminate at 16 wavelengths with millisecond image acquisition time. Analog intensity control enables a fully customizable excitation profile. A significant advantage of excitation-scanning hyperspectral imaging is can identify multiple targets simultaneously in real time. Finally, we are exploring utilizing this technology for a variety of applications ranging from measuring cAMP distribution in three dimensions within a cell to electrophysiology.