Fluorescence excitation-scanning hyperspectral imaging with scalable 2D-3D deep learning framework for colorectal cancer detection.

Colorectal cancer is one of the top contributors to cancer-related deaths in the United States, with over 100,000 estimated cases in 2020 and over 50,000 deaths. The most common screening technique is minimally invasive colonoscopy using either reflected white light endoscopy or narrow-band imaging. However, current imaging modalities have only moderate sensitivity and specificity for lesion detection.

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.

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.

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 While HSI was originally developed for remote sensing applications, modern uses include agriculture, historical document authentication, and medicine.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.

Algorithm for biological second messenger analysis with dynamic regions of interest.

Physiological function is regulated through cellular communication that is facilitated by multiple signaling molecules such as second messengers. Analysis of signal dynamics obtained from cell and tissue imaging is difficult because of intricate spatially and temporally distinct signals. Signal analysis tools based on static region of interest analysis may under- or overestimate signals in relation to region of interest size and location.

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

Second messenger signals, e.g., Ca and cyclic nucleotides, orchestrate a wide range of cellular events.

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.

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.

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.

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.

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. Fӧrster 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.

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 Fӧrster resonance energy transfer (FRET). FRET-based probes are comprised of a cAMP binding domain sandwiched between donor and acceptor fluorophores.

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

Ca 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.

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.

COVID-19 and Long-Term Outcomes: Lessons from Other Critical Care Illnesses and Potential Mechanisms.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a novel coronavirus that is currently causing a pandemic and has been termed coronavirus disease (COVID-19). The elderly or those with preexisting conditions like diabetes, hypertension, coronary heart disease, chronic obstructive pulmonary disease, cerebrovascular disease, or kidney dysfunction are more likely to develop severe cases when infected. Patients with COVID-19 admitted to the ICU have higher mortality than non-ICU patients.

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.

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.

Phosphodiesterase 4 mediates interleukin-8-induced heterologous desensitization of the β -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 (β -AR) led to decreased beta-agonist-induced mobilization of cyclic adenosine monophosphate (cAMP).

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

Colorectal cancer is the 3 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).

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 Fӧrster resonance energy transfer (FRET) based biosensors has furthered the ability to measure and visualize cAMP signals in cells.

Development of an endothelial cell-restricted transgenic reporter rat: a resource for physiological studies of vascular biology.

Here, we report the generation of a Cre-recombinase (iCre) transgenic rat, where iCre is driven using a vascular endothelial-cadherin (CDH5) promoter. The CDH5 promoter was cloned from rat pulmonary microvascular endothelial cells and demonstrated ~60% similarity to the murine counterpart. The cloned rat promoter was 2,508 bp, it extended 79 bp beyond the transcription start site, and it was 22,923 bp upstream of the translation start site.

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.

Spectral Illumination System Utilizing Spherical Reflection Optics.

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.

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.

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 3 leading cancer for incidence and mortality rates which is potentially linked to missed early lesions which allow for increased growth and metastatic potential.