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.

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.

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.