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.
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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.
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- Award ID(s):
- 1725937
- NSF-PAR ID:
- 10106734
- Date Published:
- Journal Name:
- Proc. SPIE 10881, Imaging, Manipulation, and Analysis of Biomolecules, Cells, and Tissues XVII, 108810N
- Volume:
- 10881
- Page Range / eLocation ID:
- 22
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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- Free Publicly Accessible Full Text
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Accepted Manuscript1.0
- Conference Paper:
- https://doi.org/10.1117/12.2510553
