High-speed fluorescence excitation-scanning hyperspectral imaging microscopy using thin-film tunable filters.

Hyperspectral imaging (HSI) technologies have enabled a range of experimental techniques and studies in the fluorescence microscopy field. Unfortunately, a drawback of many HSI microscope platforms is increased acquisition time required to collect images across many spectral bands, as well as signal loss due to the need to filter or disperse emitted fluorescence into many discrete bands. We have previously demonstrated that an alternative approach of scanning the fluorescence excitation spectrum can greatly improve system efficiency by decreasing light losses associated with emission filtering. Our initial system was configured using an array of thin-film tunable filters (TFTFs, VersaChrome, Semrock) mounted in a tiltable filter wheel (VF-5, Sutter) that required ~150-200 ms to switch between wavelengths. Here, we present a new configuration for high-speed switching of TFTFs to allow rapid time-lapse HSI microscopy. A TFTF array was mounted in a custom holder that was attached to a piezoelectric rotation mount (ThorLabs), allowing high-speed rotation. Switching between adjacent filters was achieved using the internal optics of a DG-4 lightsource (Sutter Instrument), including a pair of off-axis parabolic mirrors and galvanometers. Output light was coupled to a liquid lightguide and into an inverted widefield fluorescence microscope (TI-2, Nikon Instruments). Initial tests indicate that the HSI system provides a 15-20 nm bandwidth tunable excitation band and ~10-20 ms wavelength switch time, allowing for high-speed HSI imaging of dynamic cellular events. This work was supported by NIH P01HL066299, R01HL169522, NIH TL1TR003106, and NSF MRI1725937.

Hyperspectral imaging and dynamic region of interest tracking approaches to quantify localized cAMP signals.

Cyclic adenosine monophosphate (cAMP) is a ubiquitous second messenger known to orchestrate a myriad of cellular functions over a wide range of timescales. In the last 20 years, a variety of single-cell sensors have been developed to measure second messenger signals including cAMP, Ca2+, and the balance of kinase and phosphatase activities. These sensors utilize changes in fluorescence emission of an individual fluorophore or Förster resonance energy transfer (FRET) to detect changes in second messenger concentration. cAMP and kinase activity reporter probes have provided powerful tools for the study of localized signals. Studies relying on these and related probes have the potential to further revolutionize our understanding of G protein-coupled receptor signaling systems. Unfortunately, investigators have not been able to take full advantage of the potential of these probes due to the limited signal-to-noise ratio of the probes and the limited ability of standard epifluorescence and confocal microscope systems to simultaneously measure the distributions of multiple signals (e.g. cAMP, Ca2+, and changes in kinase activities) in real time. In this review, we focus on recently implemented strategies to overcome these limitations: hyperspectral imaging and adaptive thresholding approaches to track dynamic regions of interest (ROI). This combination of approaches increases signal-to-noise ratio and contrast, and allows identification of localized signals throughout cells. These in turn lead to the identification and quantification of intracellular signals with higher effective resolution. Hyperspectral imaging and dynamic ROI tracking approaches offer investigators additional tools with which to visualize and quantify multiplexed intracellular signaling systems.

Comparing Performance of Spectral Image Analysis Approaches for Detection of Cellular Signals in Time-Lapse Hyperspectral Imaging Fluorescence Excitation-Scanning Microscopy.

Hyperspectral imaging (HSI) technology has been applied in a range of fields for target detection and mixture analysis. While HSI was originally developed for remote sensing applications, modern uses include agriculture, historical document authentication, and medicine. HSI has also shown great utility in fluorescence microscopy. However, traditional fluorescence microscopy HSI systems have suffered from limited signal strength due to the need to filter or disperse the emitted light across many spectral bands. We have previously demonstrated that sampling the fluorescence excitation spectrum may provide an alternative approach with improved signal strength. Here, we report on the use of excitation-scanning HSI for dynamic cell signaling studies-in this case, the study of the second messenger Ca2+. Time-lapse excitation-scanning HSI data of Ca2+ signals in human airway smooth muscle cells (HASMCs) were acquired and analyzed using four spectral analysis algorithms: linear unmixing (LU), spectral angle mapper (SAM), constrained energy minimization (CEM), and matched filter (MF), and the performances were compared. Results indicate that LU and MF provided similar linear responses to increasing Ca2+ and could both be effectively used for excitation-scanning HSI. A theoretical sensitivity framework was used to enable the filtering of analyzed images to reject pixels with signals below a minimum detectable limit. The results indicated that subtle kinetic features might be revealed through pixel filtering. Overall, the results suggest that excitation-scanning HSI can be employed for kinetic measurements of cell signals or other dynamic cellular events and that the selection of an appropriate analysis algorithm and pixel filtering may aid in the extraction of quantitative signal traces. These approaches may be especially helpful for cases where the signal of interest is masked by strong cellular autofluorescence or other competing signals.

Combined hyperspectral imaging, monolayer stress microscopy, and S8 image analysis approaches for simultaneously interrogating cellular signals and biomechanics.

Second messenger signals, e.g., Ca2+ and cyclic nucleotides, orchestrate a wide range of cellular events. The methods by which second messenger signals determine specific physiological responses are complex. Recent studies point to the importance of temporal and spatial encoding in determining signal specificity. Studies also indicate the importance of mechanical stimuli, substrate stiffness, and mechanical responses - the "mechanosome" - in regulating physiology. Hence, approaches that probe both chemical and mechanical signals are needed. Here, we report preliminary efforts to combine hyperspectral imaging for second messenger signal measurements, monolayer stress microscopy for mechanical force measurements, and S8 analysis software for quantifying localized signals - specifically, Ca2+ dynamics and mechanical forces in human airway smooth muscle cells (HASMCs). HASMCs were prepared as confluent monolayers on 11 kPa gels with embedded fluorescent microparticles that serve as fiducial markers as well as smaller microparticles to measure deformation (strain). Imaging was performed using a custom excitation-scanning hyperspectral microscope. Hyperspectral images were unmixed to identify signals from cellular fluorescent labels (e.g., CAL 590-AM) and fluorescent microparticles. Images were analyzed to quantify localized force dynamics through monolayer stress microscopy. S8 software was used to identify, track, and quantify spatially-localized Ca2+ activity. Results indicate that localized and transient cellular signals and forces can be quantified and mapped within cell populations. Importantly, these results establish a method for simultaneous interrogation of cellular signals and mechanical forces that may play synergistic roles in regulating downstream cellular physiology in confluent monolayers. This work was supported by NIH P01HL066299, R01HL137030, R01HL058506, and NSF MRI1725937. Drs. Leavesley and Rich disclose financial interest in a university start-up company, SpectraCyte LLC, to commercialize spectral imaging technologies.

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.

Endoscopy Lifetime Systems Architecture: Scoping Out the Past to Diagnose the Future Technology.

Systems engineering captures the desires and needs of the customer to conceptualize a system from the overall goal down to the small details prior to any physical development. While many systems projects tend to be large and complicated (i.e., cloud-based infrastructure, long-term space travel shuttles, missile defense systems), systems engineering can also be applied to smaller, complex systems. Here, the system of interest is the endoscope, a standard biomedical screening device used in laparoscopic surgery, screening of upper and lower gastrointestinal tracts, and inspection of the upper airway. Often, endoscopic inspection is used to identify pre-cancerous and cancerous tissues, and hence, a requirement for endoscopic systems is the ability to provide images with high contrast between areas of normal tissue and neoplasia (early-stage abnormal tissue growth). For this manuscript, the endoscope was reviewed for all the technological advancements thus far to theorize what the next version of the system could be in order to provide improved detection capabilities. Endoscopic technology was decomposed into categories, using systems architecture and systems thinking, to visualize the improvements throughout the system's lifetime from the original to current state-of-the-art. Results from this review were used to identify trends in subsystems and components to estimate the theoretical performance maxima for different subsystems as well as areas for further development. The subsystem analysis indicated that future endoscope systems will focus on more complex imaging and higher computational requirements that will provide improved contrast in order to have higher accuracy in optical diagnoses of early, abnormal tissue growth.

Microscopy is better in color: development of a streamlined spectral light path for real-time multiplex fluorescence microscopy.

Spectroscopic image data has provided molecular discrimination for numerous fields including: remote sensing, food safety and biomedical imaging. Despite the various technologies for acquiring spectral data, there remains a trade-off when acquiring data. Typically, spectral imaging either requires long acquisition times to collect an image stack with high spectral specificity or acquisition times are shortened at the expense of fewer spectral bands or reduced spatial sampling. Hence, new spectral imaging microscope platforms are needed to help mitigate these limitations. Fluorescence excitation-scanning spectral imaging is one such new technology, which allows more of the emitted signal to be detected than comparable emission-scanning spectral imaging systems. Here, we have developed a new optical geometry that provides spectral illumination for use in excitation-scanning spectral imaging microscope systems. This was accomplished using a wavelength-specific LED array to acquire spectral image data. Feasibility of the LED-based spectral illuminator was evaluated through simulation and benchtop testing and assessment of imaging performance when integrated with a widefield fluorescence microscope. Ray tracing simulations (TracePro) were used to determine optimal optical component selection and geometry. Spectral imaging feasibility was evaluated using a series of 6-label fluorescent slides. The LED-based system response was compared to a previously tested thin-film tunable filter (TFTF)-based system. Spectral unmixing successfully discriminated all fluorescent components in spectral image data acquired from both the LED and TFTF systems. Therefore, the LED-based spectral illuminator provided spectral image data sets with comparable information content so as to allow identification of each fluorescent component. These results provide proof-of-principle demonstration of the ability to combine output from many discrete wavelength LED sources using a double-mirror (Cassegrain style) optical configuration that can be further modified to allow for high speed, video-rate spectral image acquisition. Real-time spectral fluorescence microscopy would allow monitoring of rapid cell signaling processes (i.e., Ca2+ and other second messenger signaling) and has potential to be translated to clinical imaging platforms.

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.

Measurement of agonist-induced Ca2+ signals in human airway smooth muscle cells using excitation scan-based hyperspectral imaging and image analysis approaches.

Ca2+ and cAMP are ubiquitous second messengers known to differentially regulate a variety of cellular functions over a wide range of timescales. Studies from a variety of groups support the hypothesis that these signals can be localized to discrete locations within cells, and that this subcellular localization is a critical component of signaling specificity. However, to date, it has been difficult to track second messenger signals at multiple locations. To overcome this limitation, we utilized excitation scan-based hyperspectral imaging approaches to track second messenger signals as well as labeled cellular structures and/or proteins in the same cell. We have previously reported that hyperspectral imaging techniques improve the signal-to-noise ratios of both fluorescence measurements, and are thus well suited for the measurement of localized Ca2+ signals. We investigated the spatial spread and intensities of agonist-induced Ca2+ signals in primary human airway smooth muscle cells (HASMCs) using the Ca2+ indicator Cal520. We measured responses triggered by three agonists, carbachol, histamine, and chloroquine. We utilized custom software coded in MATLAB and Python to assess agonist induced changes in Ca2+ levels. Software algorithms removed the background and applied correction coefficients to spectral data prior to linear unmixing, spatial and temporal filtering, adaptive thresholding, and automated region of interest (ROI) detection. All three agonists triggered transient Ca2+ responses that were spatially and temporally complex. We are currently analyzing differences in both ROI area and intensity distributions triggered by these agonists. This work was supported by NIH awards P01HL066299, K25HL136869, and R01HL137030 and NSF award MRI1725937.

Novel Hyperspectral imaging approaches allow 3D measurement of cAMP signals in localized subcellular domains of human airway smooth muscle cells.

Studies of the cAMP signaling pathway have led to the hypothesis that localized cAMP signals regulate distinct cellular responses. Much of this work focused on measurement of localized cAMP signals using cAMP sensors based upon FÓ§rster resonance energy transfer (FRET). FRET-based probes are comprised of a cAMP binding domain sandwiched between donor and acceptor fluorophores. Binding of cAMP triggers a conformational change which alters FRET efficiency. In order to study localized cAMP signals, investigators have targeted FRET probes to distinct subcellular domains. This approach allows detection of cAMP signals at distinct subcellular locations. However, these approaches do not measure localized cAMP signals per se, rather they measure cAMP signals at specific locations and typically averaged throughout the cell. To address these concerns, our group implemented hyperspectral imaging approaches for measuring highly multiplexed signals in cells and tissues. We have combined these approaches with custom analysis software implemented in MATLAB and Python. Images were filtered both spatially and temporally, prior to adaptive thresholding (OTSU) to detect cAMP signals. These approaches were used to interrogate the distributions of isoproterenol and prostaglandin-triggered cAMP signals in human airway smooth muscle cells (HASMCs). Results demonstrate that cAMP signals are spatially and temporally complex. We observed that isoproterenol- and prostaglandin-induced cAMP signals are triggered at the plasma membrane and in the cytosolic space. We are currently implementing analysis approaches to better quantify and visualize the complex distributions of cAMP signals. This work was supported by NIH P01HL066299, R01HL058506, and S10RR027535.

Improving Visualization of cAMP Gradients Using Algorithmic Modelling.

A ubiquitous second messenger molecule, cAMP is responsible for orchestrating many different cellular functions through a variety of pathways. FÓ§rster resonance energy transfer (FRET) probes have been used to visualize cAMP spatial gradients in pulmonary microvascular endothelial cells (PMVECs). However, FRET probes have inherently low signal-to-noise ratios; multiple sources of noise can obscure accurate visualization of cAMP gradients using a hyperspectral imaging system. FRET probes have also been used to measure cAMP gradients in 3D; however, it can be difficult to differentiate between true FRET signals and noise. To further understand the effects of noise on experimental data, a model was developed to simulate cAMP gradients under experimental conditions. The model uses a theoretical cAMP heatmap generated using finite element analysis. This heatmap was converted to simulate the FRET probe signal that would be detected experimentally with a hyperspectral imaging system. The signal was mapped onto an image of unlabeled PMVECs. The result was a time lapse model of cAMP gradients obscured by autofluorescence, as visualized with FRET probes. Additionally, the model allowed the simulated expression level of FRET signal to be varied. This allowed accurate attribution of signal to FRET and autofluorescence. Comparing experimental data to the model results at different levels of FRET efficiency has allowed improved understanding of FRET signal specificity and how autofluorescence interferes with FRET signal detection. In conclusion, this model can more accurately determine cAMP gradients in PMVECs. This work was supported by NIH award P01HL066299, R01HL58506 and NSF award 1725937.

Extracellular vesicle-induced cyclic AMP signaling.

Second messenger signaling is required for cellular processes. We previously reported that extracellular vesicles (EVs) from stimulated cultured endothelial cells contain the biochemical second messenger, cAMP. In the current study, we sought to determine whether cAMP-enriched EVs induce second messenger signaling pathways in naïve recipient cells. Our results indicate that cAMP-enriched EVs increase cAMP content sufficient to stimulate PKA activity. The implications of our work are that EVs represent a novel intercellular mechanism for second messenger, specifically cAMP, signaling.

Automated Image Analysis of FRET Signals for Subcellular cAMP Quantification.

A variety of FRET probes have been developed to examine cAMP localization and dynamics in single cells. These probes offer a readily accessible approach to measure localized cAMP signals. However, given the low signal-to-noise ratio of most FRET probes and the dynamic nature of the intracellular environment, there have been marked limitations in the ability to use FRET probes to study localized signaling events within the same cell. Here, we outline a methodology to dissect kinetics of cAMP-mediated FRET signals in single cells using automated image analysis approaches. We additionally extend these approaches to the analysis of subcellular regions. These approaches offer a unique opportunity to assess localized cAMP kinetics in an unbiased, quantitative fashion.

Ion Channel-Based Reporters for cAMP Detection.

In the last 20 years tremendous progress has been made in the development of single cell cAMP sensors. Sensors are based upon cAMP binding proteins that have been modified to transduce cAMP concentrations into electrical or fluorescent readouts that can be readily detected using patch clamp amplifiers, photomultiplier tubes, or cameras. Here, we describe two complementary approaches for the detection and measurement of cAMP signals near the plasma membrane of cells using cyclic nucleotide (CNG) channel-based probes. These probes take advantage of the ability of CNG channels to transduce small changes in cAMP concentration into ionic flux through channel pores that can be readily detected by measuring Ca2+ and/or Mn2+ influx or by measuring ionic currents.

Suppression of Colon Tumorigenesis in Mutant Apc Mice by a Novel PDE10 Inhibitor that Reduces Oncogenic ß-Catenin.

Previous studies have reported that phosphodiesterase 10A (PDE10) is overexpressed in colon epithelium during early stages of colon tumorigenesis and essential for colon cancer cell growth. Here we describe a novel non-COX inhibitory derivative of the anti-inflammatory drug, sulindac, with selective PDE10 inhibitory activity, ADT 061. ADT 061 potently inhibited the growth of colon cancer cells expressing high levels of PDE10, but not normal colonocytes that do not express PDE10. The concentration range by which ADT 061 inhibited colon cancer cell growth was identical to concentrations that inhibit recombinant PDE10. ADT 061 inhibited PDE10 by a competitive mechanism and did not affect the activity of other PDE isozymes at concentrations that inhibit colon cancer cell growth. Treatment of colon cancer cells with ADT 061 activated cGMP/PKG signaling, induced phosphorylation of oncogenic ß-catenin, inhibited Wnt-induced nuclear translocation of ß-catenin, and suppressed TCF/LEF transcription at concentrations that inhibit cancer cell growth. Oral administration of ADT 061 resulted in high concentrations in the colon mucosa and significantly suppressed the formation of colon adenomas in the Apc+/min-FCCC mouse model of colorectal cancer without discernable toxicity. These results support the development of ADT 061 for the treatment or prevention of adenomas in individuals at risk of developing colorectal cancer. PREVENTION RELEVANCE: PDE10 is overexpressed in colon tumors whereby inhibition activates cGMP/PKG signaling and suppresses Wnt/ß-catenin transcription to selectively induce apoptosis of colon cancer cells. ADT 061 is a novel PDE10 inhibitor that shows promising cancer chemopreventive activity and tolerance in a mouse model of colon cancer.

Phosphodiesterase 4 mediates interleukin-8-induced heterologous desensitization of the ß2 -adrenergic receptor.

Acute respiratory distress syndrome (ARDS) is a life-threatening illness characterized by decreased alveolar-capillary barrier function, pulmonary edema consisting of proteinaceous fluid, and inhibition of net alveolar fluid transport responsible for resolution of pulmonary edema. There is currently no pharmacotherapy that has proven useful to prevent or treat ARDS, and two trials using beta-agonist therapy to treat ARDS demonstrated no effect. Prior studies indicated that IL-8-induced heterologous desensitization of the beta2-adrenergic receptor (ß2 -AR) led to decreased beta-agonist-induced mobilization of cyclic adenosine monophosphate (cAMP). Interestingly, phosphodiesterase (PDE) 4 inhibitors have been used in human airway diseases characterized by low intracellular cAMP levels and increases in specific cAMP hydrolyzing activity. Therefore, we hypothesized that PDE4 would mediate IL-8-induced heterologous internalization of the ß2 -AR and that PDE4 inhibition would restore beta-agonist-induced functions. We determined that CINC-1 (a functional IL-8 analog in rats) induces internalization of ß2 -AR from the cell surface, and arrestin-2, PDE4, and ß2 -AR form a complex during this process. Furthermore, we determined that cAMP associated with the plasma membrane was adversely affected by ß2 -AR heterologous desensitization. Additionally, we determined that rolipram, a PDE4 inhibitor, reversed CINC-1-induced derangements of cAMP and also caused ß2 -AR to successfully recycle back to the cell surface. Finally, we demonstrated that rolipram could reverse CINC-1-mediated inhibition of beta-agonist-induced alveolar fluid clearance in a murine model of trauma-shock. These results indicate that PDE4 plays a role in CINC-1-induced heterologous internalization of the ß2 -AR; PDE4 inhibition reverses these effects and may be a useful adjunct in particular ARDS patients.

Excitation-scanning hyperspectral video endoscopy: enhancing the light at the end of the tunnel.

Colorectal cancer is the 3rd leading 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 cancer ex vivo or 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.

Measurement of 3-Dimensional cAMP Distributions in Living Cells using 4-Dimensional (x, y, z, and λ) Hyperspectral FRET Imaging and Analysis.

Cyclic AMP is a second messenger that is involved in a wide range of cellular and physiological activities. Several studies suggest that cAMP signals are compartmentalized, and that compartmentalization contributes to signaling specificity within the cAMP signaling pathway. The development of FÓ§rster resonance energy transfer (FRET) based biosensors has furthered the ability to measure and visualize cAMP signals in cells. However, these measurements are often confined to two spatial dimensions, which may result in misinterpretation of data. To date, there have been only very limited measurements of cAMP signals in three spatial dimensions (x, y, and z), due to the technical limitations in using FRET sensors that inherently exhibit low signal to noise ratio (SNR). In addition, traditional filter-based imaging approaches are often ineffective for accurate measurement of cAMP signals in localized subcellular regions due to a range of factors, including spectral crosstalk, limited signal strength, and autofluorescence. To overcome these limitations and allow FRET-based biosensors to be used with multiple fluorophores, we have developed hyperspectral FRET imaging and analysis approaches that provide spectral specificity for calculating FRET efficiencies and the ability to spectrally separate FRET signals from confounding autofluorescence and/or signals from additional fluorescent labels. Here, we present the methodology for implementing hyperspectral FRET imaging as well as the need to construct an appropriate spectral library that is neither undersampled nor oversampled to perform spectral unmixing. While we present this methodology for measurement of three-dimensional cAMP distributions in pulmonary microvascular endothelial cells (PMVECs), this methodology could be used to study spatial distributions of cAMP in a range of cell types.

Optical simulations for determining efficacy of new light source designs for excitation-scanning high-speed hyperspectral imaging systems.

Positive outcomes for colorectal cancer treatment have been linked to early detection. The difficulty in detecting early lesions is the limited contrast with surrounding mucosa and minimal definitive markers to distinguish between hyperplastic and carcinoma lesions. Colorectal cancer is the 3rd leading cancer for incidence and mortality rates which is potentially linked to missed early lesions which allow for increased growth and metastatic potential. One potential technology for early-stage lesion detection is hyperspectral imaging. Traditionally, hyperspectral imaging uses reflectance spectroscopic data to provide a component analysis, per pixel, of an image in fields such as remote sensing, agriculture, food processing and archaeology. This work aims to acquire higher signal-to-noise fluorescence spectroscopic data, harnessing the autofluorescence of tissue, adding a hyperspectral contrast to colorectal cancer detection while maintaining spatial resolution at video-rate speeds. We have previously designed a multi-furcated LED-based spectral light source to prove this concept. Our results demonstrated that the technique is feasible, but the initial prototype has a high light transmission loss (~98%) minimizing spatial resolution and slowing video acquisition. Here, we present updated results in developing an optical ray-tracing model of light source geometries to maximize irradiance throughput for excitation-scanning hyperspectral imaging. Results show combining solid light guide branches have a compounding light loss effect, however, there is potential to minimize light loss through the use of optical claddings. This simulation data will provide the necessary metrics to verify and validate future physical optical components within the hyperspectral endoscopic system for detecting colorectal cancer.

Comparison of spectral FRET microscopy approaches for single-cell analysis.

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-focus fluorescence, and Z-resolution. 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.