JOURACA_SP_2017

5 dimensional (5D) Hyperspectral Measurement of
Fluorescence and FRET Signals in Living Cells
Mohammed El-Sharkh
Cyclic AMP (cAMP) is a ubiquitous second
messenger that regulates diverse cellular functions.
In the past, cAMP distribution has been
thought to be non-uniform; however, cAMP
gradients have not been measured in three spatial
dimensions (x, y, and z). Because the spatiotemporal
cAMP distribution has not been measured,
it is difficult to understand how cAMP signals
simultaneously regulate dozens of cellular
functions. A standard imaging technique used to
investigate cellular functions is Förster resonance
energy transfer (FRET). FRET probes
have been used to measure cAMP levels for
more than a decade. One reason 3D cAMP distributions
have not been measured is the low
signal-to-noise ratio inherent to FRET measurements.
We have previously reported that hyperspectral
imaging improves the signal-to-noise
ratio of FRET measurements. Here we present
hyperspectral imaging-based measurements of
intracellular cAMP in five dimensions (5D):
three spatial dimensions (x, y, z), time (t), and
wavelength of emitted light (?). 5D imaging was
used to measure cAMP levels in pulmonary microvascular
endothelial cells (PMVECs) transfected
with the H188 FRET probe. The H188
probe contains a cAMP binding domain sandwiched
between donor (Turquoise) and acceptor
(Venus) fluorescent proteins. Hyperspectral images
were taken with a Nikon A1R inverted
confocal microscope and analyzed with the accompanying
Elements software and custom
MATLAB scripts. Results demonstrate realtime
measurement of cAMP in 5D.
College of Allied Health Professions
Mentors: E. Spencer, C. Montalvo
Electrical and Computer Engineering
They also illustrate related problems including photobleaching
and interfering fluorescence signals,
likely from non-uniform background fluo-rescence.
Interfering fluorescence can be over-come by including
background fluorescence spectra in the
spectral library. Subsequent spec-tral unmixing of
hyperspectral image stacks al-lows quantitative assessment
of spatially varying FRET and background
fluorescence signals. The approach was validated
in single PMVECs. cAMP production was
triggered with 100 nM PGE1, which resulted in an
increase in cAMP. A subset of cells were pretreated
with 100 nM thapsigargin, which inhibited PGE1-
triggered cAMP accumulation, consistent with
Ca2+- mediated inhibition of cAMP production.
Treat-ment with thapsigargin alone did not alter
cAMP levels. Overall, these data suggest that 5D
hyper-spectral imaging allows real-time measurement
of intracellular cAMP signals in three spatial
dimensions.
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