More Like this

1. A spherical mirror-based illumination system for fluorescence excitation-scanning hyperspectral imaging

   https://doi.org/10.1117/12.2510553  

   Gunn Mayes, Samantha ; Mayes, Samuel A. ; Browning, Craig ; Parker, Marina ; Rich, Thomas C. ; Leavesley, Silas J. ; Farkas, Daniel L. ; Leary, James F. ; Tarnok, Attila ( March 2019 , Proc. SPIE 10881, Imaging, Manipulation, and Analysis of Biomolecules, Cells, and Tissues XVII, 108810N)

   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. Inmore »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.« less
2. Hyperspectral imaging fluorescence excitation scanning (HIFEX) microscopy for live cell imaging

   https://doi.org/10.1117/12.2510562  

   Leavesley, Silas J. ; Griswold, John R. ; Deal, Joshua A. ; McAlister, Kathleen ; Mayes, Sam A. ; Browning, Craig ; Parker, Marina ; Gunn Mayes, Samantha ; Rich, Thomas C. ; Brown, Thomas G. ; et al ( February 2019 , Proc. SPIE 10883, Three- Dimensional and Multidimensional Microscopy: Image Acquisition and Processing XXVI, 108831A)

   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-scanningmore »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.« less
3. Colorectal cancer detection by hyperspectral imaging using fluorescence excitation scanning

   https://doi.org/10.1117/12.2290696  

   Leavesley, Silas J. ; Deal, Joshua ; Martin, Will A. ; Lall, Malvika ; Lopez, Carmen ; Rich, Thomas C. ; Boudreaux, Carole W. ; Rider, Paul F. ; Hill, Shante ( February 2018 , Proc. SPIE 10489, Optical Biopsy XVI: Toward Real- Time Spectroscopic Imaging and Diagnosis, 104890K)

   Hyperspectral imaging technologies have shown great promise for biomedical applications. These techniques have been especially useful for detection of molecular events and characterization of cell, tissue, and biomaterial composition. Unfortunately, hyperspectral imaging technologies have been slow to translate to clinical devices – likely due to increased cost and complexity of the technology as well as long acquisition times often required to sample a spectral image. We have demonstrated that hyperspectral imaging approaches which scan the fluorescence excitation spectrum can provide increased signal strength and faster imaging, compared to traditional emission-scanning approaches. We have also demonstrated that excitation-scanning approaches may bemore »able to detect spectral differences between colonic adenomas and adenocarcinomas and normal mucosa in flash-frozen tissues. Here, we report feasibility results from using excitation-scanning hyperspectral imaging to screen pairs of fresh tumoral and nontumoral colorectal tissues. Tissues were imaged using a novel hyperspectral imaging fluorescence excitation scanning microscope, sampling a wavelength range of 360-550 nm, at 5 nm increments. Image data were corrected to achieve a NIST-traceable flat spectral response. Image data were then analyzed using a range of supervised and unsupervised classification approaches within ENVI software (Harris Geospatial Solutions). Supervised classification resulted in >99% accuracy for single-patient image data, but only 64% accuracy for multi-patient classification (n=9 to date), with the drop in accuracy due to increased false-positive detection rates. Hence, initial data indicate that this approach may be a viable detection approach, but that larger patient sample sizes need to be evaluated and the effects of inter-patient variability studied.« less
4. Excitation-scan mirror array system advancements to hyperspectral imaging applications

   https://doi.org/10.1117/12.2546485  

   Parker, Marina ; Browning, Craig M. ; Mayes, Sam A. ; Deal, Joshua ; Gunn Mayes, Samantha ; Rich, Thomas C. ; Leavesley, Silas J. ; Brown, Thomas G. ; Wilson, Tony ; Waller, Laura ( February 2020 , Proc. SPIE 11245, Three-Dimensional and Multidimensional Microscopy: Image Acquisition and Processing XXVII)

   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 modelmore »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.« less
5. Comparison of spectral FRET microscopy approaches for single-cell analysis

   https://doi.org/10.1117/12.2546308  

   Deal, Joshua ; Annamdevula, Naga S. ; Pleshinger, D J. ; Griswold, John Robert ; Odom, Aliyah ; Tayara, Alia ; Lall, Malvika ; Browning, Craig M. ; Parker, Marina ; Rich, Thomas C. ; et al ( February 2020 , Proc. SPIE 11243, Imaging, Manipulation, and Analysis of Biomolecules, Cells, and Tissues XVIII)

   Förster resonance energy transfer (FRET) is a valuable tool for measuring molecular distances and the effects of biological processes such as cyclic nucleotide messenger signaling and protein localization. Most FRET techniques require two fluorescent proteins with overlapping excitation/emission spectral pairing to maximize detection sensitivity and FRET efficiency. FRET microscopy often utilizes differing peak intensities of the selected fluorophores measured through different optical filter sets to estimate the FRET index or efficiency. Microscopy platforms used to make these measurements include wide-field, laser scanning confocal, and fluorescence lifetime imaging. Each platform has associated advantages and disadvantages, such as speed, sensitivity, specificity, out-of-focusmore »fluorescence, and Zresolution. In this study, we report comparisons among multiple microscopy and spectral filtering platforms such as standard 2-filter FRET, emission-scanning hyperspectral imaging, and excitation-scanning hyperspectral imaging. Samples of human embryonic kidney (HEK293) cells were grown on laminin-coated 28 mm round gridded glass coverslips (10816, Ibidi, Fitchburg, Wisconsin) and transfected with adenovirus encoding a cAMP-sensing FRET probe composed of a FRET donor (Turquoise) and acceptor (Venus). Additionally, 3 FRET “controls” with fixed linker lengths between Turquoise and Venus proteins were used for inter-platform validation. Grid locations were logged, recorded with light micrographs, and used to ensure that whole-cell FRET was compared on a cell-by-cell basis among the different microscopy platforms. FRET efficiencies were also calculated and compared for each method. Preliminary results indicate that hyperspectral methods increase the signal-to-noise ratio compared to a standard 2-filter approach.« less