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.
KEYWORDS: Fluorescence resonance energy transfer, Data modeling, Visualization, Interference (communication), Finite element methods, Signal to noise ratio, Hyperspectral imaging, Lung, Signal detection, Sensors
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. 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.
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. 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.
Cyclic AMP (cAMP) is a second messenger that regulates a wide variety of cellular functions. There is increasing evidence suggesting that signaling specificity is due in part to cAMP compartmentalization. In the last 15 years, development of cAMP-specific Förster resonance energy transfer (FRET) probes have allowed us to visualize spatial distributions of intracellular cAMP signals. The use of FRET-based sensors is not without its limitations, as FRET probes display low signal to noise ratio (SNR). Hyperspectral imaging and analysis approaches have, in part, allowed us to overcome these limitations by improving the SNR of FRET measurements. Here we demonstrate that the combination of hyperspectral imaging approaches, linear unmixing, and adaptive thresholding allow us to visualize regions of elevated cAMP (regions of interest – ROIs) in an unbiased manner. We transfected cDNA encoding the H188 FRET-based cAMP probe into pulmonary microvascular endothelial cells. Application of isoproterenol and prostaglandin E1 (PGE1) triggered complex cAMP responses. Spatial and temporal aspects of cAMP responses were quantified using an adaptive thresholding approach and compared between agonist treatment groups. Our data indicate that both the origination sites and spatial/temporal distributions of cAMP signals are agonist dependent in PMVECs. We are currently analyzing the data in order to better quantify the distribution of cAMP signals triggered by different agonists.
F¨orster resonance energy transfer (FRET) is a tool used for studying various biological process as well as for measuring molecular distances. This process can occur when the emission spectrum of the donor fluorophore overlaps with the excitation spectrum of the acceptor, and the fluorophores are in close enough distance for the energy to pass from donor to acceptor non-radiatively. The efficiency of this energy transfer is dependent on the distance and orientation of the fluorophores, in addition to their overlapping spectra. Here we present a study to assess the impact of tissue autofluorescence on estimates of FRET efficiency and fluorophore abundance within experimental cellular images in tissue. To accomplish this we performed a theoretical sensitivity analysis on FRET rat kidney control images with varying ranges of donor and acceptor fluorophores to observe their pixel by pixel responses. In the experimental data, the donor was the Turquoise fluorescent protein and the acceptor fluorophore was the Venus fluorescent protein. Detection of the acceptor was more difficult due to its excitation spectrum closely resembling the autofluorescence spectrum from the base image while the emission spectrum of the Turquoise donor was more unique and easier to detect. In addition, variable FRET efficiencies were added to data at different fluorophore levels to compare the visible abundance of FRET to the autofluorescence in the resultant images. This analysis can benefit future work by furthering the understanding of the donor and acceptor concentrations needed for strong FRET measurements.
Structural complexity and heterogeneity may play critical roles in pathophysiology and therapeutic effect, at length scales ranging from subcellular (nm) to whole organ (cm). Here, we report on efforts to utilize a multidimensional spectral FRET imaging approach for visualization of the effects of pharmacologic treatments on tissues. We have developed a transgenic rat line expressing a cAMP FRET reporter for visualization of second messenger signaling in tissues in response to treatment and pathological conditions. When utilized with vascular and lung preparations, transgenic FRET tissues displayed the ability to elicit agonist-induced cAMP responses in single cells.
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 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.
Access to the requested content is limited to institutions that have purchased or subscribe to SPIE eBooks.
You are receiving this notice because your organization may not have SPIE eBooks access.*
*Shibboleth/Open Athens users─please
sign in
to access your institution's subscriptions.
To obtain this item, you may purchase the complete book in print or electronic format on
SPIE.org.
INSTITUTIONAL Select your institution to access the SPIE Digital Library.
PERSONAL Sign in with your SPIE account to access your personal subscriptions or to use specific features such as save to my library, sign up for alerts, save searches, etc.