5D Imaging

Prior work by Drs. Leavesley and Rich indicated that FRET-based cAMP and cGMP measurements were potentially unreliable due to low signal-to-noise ratios of genetically-encoded FRET probes, potential artifacts of cellular environment (changes in ROS, pH, etc.), and the limited sensitivity and specificity of current FRET imaging technologies.  To overcome these limitations, we have worked to develop novel imaging approaches and hardware to allow 5-dimensional (5D) imaging: imaging in the x, y, and z dimensions, as well as the λ (wavelength) and t (time) dimension.

4D Imaging

4D imaging refers to the acquisition of x,y image data over 2 additional dimensions: z (axial) spatial dimension, λ (wavelength) dimension, t (time) dimension, or others. 

4D Imaging = 2D Spatial + Hyperspectral + Timelapse: Timelapse hyperspectral microscopy allows monitoring of whole-cell and subcellular FRET efficiencies.  This study utilized a cAMP FRET reporter to study whole cell and subcellular cAMP kinetics in pulmonary microvascular endothelial cells.

Pulmonary microvascular endothelial cells expressing a Turqoise-Epac-Venus FRET probe for cAMP. Intracellular cAMP was assessed in 3-dimensions using spectral confocal microscopy. Regions of high FRET (corresponding to low cAMP) can be visualized as white, while low FRET appear as light blue.

Blue = Nuclear Stain (DRAQ5)
Red = Membrane (Wheat Germ Agglutinin)
Light Blue = Turqoise (Donor)
Yellow = Venus (Acceptor)
White = Overlap of Venus and Turqoise (regions of high FRET / low cAMP)

5D Imaging

5D imaging refers to the acquisition of x,y,z image data over 2 additional dimensions:  λ (wavelength) dimension and t (time) dimension.

Pulmonary microvascular endothelial cells treated with 100 nM Isoproteranol.  This study utilized a cAMP FRET reporter to study whole cell and 3D localized subcellular cAMP kinetics in pulmonary microvascular endothelial cells. Of particular interest, cAMP spatial gradients appear first in the axial (apical-to-basal) direction and then move outward radially.  This indicates that viewing only a single confocal slice may not capture the entire spatial dependence of cAMP signaling in pulmonary microvascular endothelial cells.

3D spatial, hyperspectral image of a single time-point illustrating how the image data can be re-sliced in the XY, XZ, and YZ planes. Regions of high FRET, corresponding to low cAMP, are shown as white. Pulmonary microvascular endothelial cells expressing a Turqoise-Epac-Venus FRET probe for cAMP. Intracellular cAMP was assessed in 3-dimensions using spectral confocal microscopy. Regions of high FRET (corresponding to low cAMP) can be visualized as white, while low FRET appear as light blue.

Orange = Plasma Membrane (Wheat Germ Agglutinin)
Red = Nuclei (DRAQ5)
Light Blue = Turqoise (FRET Donor)
Yellow = Venus (FRET Acceptor)
White = Overlap of Venus and Turqoise (regions of high FRET / low cAMP)