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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Applications and assessment of an excitation-scanning hyperspectral imaging system
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.
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- Award ID(s):
- 1725937
- NSF-PAR ID:
- 10064179
- Date Published:
- Journal Name:
- Proc. SPIE 10497, Imaging, Manipulation, and Analysis of Biomolecules, Cells, and Tissues XVI
- Volume:
- 10497
- Page Range / eLocation ID:
- 1049706
- 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/10.1117/12.2290120
