Hoppe, Adam D.

Straight, Samuel S.

Swanson, Joel A.

Elsevier - Publisher Connector 

Monday, March 7, 2011 183ato study gene regulation of processes in living cells. The approach can be read-
ily applied to other viruses as well.
1005-Plat
Quantitative TCSPC FRET-FLIM Applied to Donors Exhibiting Multi-
Exponential Decays: Spatio-Temporal Interaction Between Calmodulin
and the Kv7.2 Potassium Channel in Living Cells
Sergi Padilla-Parra, Gloria de las Heras, Juncal Fernandez-Orth,
Sonia Lopez-Fernandez, Alvaro Villarroel, Jose Requejo-Isidro.
Unidad de Biofı´sica, CSIC-UPV/EHU, Leioa, Spain.
The spatio-temporal study of the dynamics of molecular interactions using
FRET-FLIM is generally compromised by the large number of photons re-
quired to fit the multiple-lifetime decay of the donor population in each pixel
of an image. Long acquisitions prevent interacting dynamics to be detected
in an image, while the use of high excitation intensities results in artifactual
measurements due to bleaching. The computation of the minimal fraction of
donor molecules (mfD) undergoing FRET allows quantitative imaging of
molecular interactions with either single or multi-lifetime donors such as
CFP, where the complexity associated to fitting a fluorescent decay with
more than two components hampers quantification with traditional least-
squares fitting strategies. This novel non-fitting analysis has been recently ap-
plied to wide-field time gated FLIM systems and we now extend it to more
widely available TCSPC systems.
The use of mfD analysis has allowed us to study the spatio-temporal dynamics
of the interaction between CFP-tagged Kv7.2 channels (donor) and YFP-
labeled calmodulin (acceptor) in HEK293 living cells on a TCSPC system.
We show the existence of discrete interacting domains that can be followed
as a function of time (every 12-15s), where the fraction of interacting
KCNQ2 with calmodulin oscillates at least between 15% and 25%.
In summary, we have shown that the mfD analysis allows quantitative study of
the spatio-temporal dynamics of molecular interactions on TCSPC systems.
Using this non-fitting strategy we have quantitatively imaged the interacting
dynamics of CFP-tagged Kv7.2 channels and YFP-labeled calmodulin in living
cells.
1006-Plat
N-Way FRET Microscopy for Imaging Multiple Protein Interactions
Within a Single Living Cell
Adam D. Hoppe1, Samuel S. Straight2, Joel A. Swanson2.
1South Dakota State University, Brookings, SD, USA, 2University of
Michigan, Ann Arbor, MI, USA.
Fluorescence Resonance Energy Transfer (FRET) microscopy has emerged as
a powerful tool for probing nanoscale protein interactions while capturing the
microscale organization of these interactions. However, current FRET micros-
copy approaches are limited to the analysis two interacting fluorescently
labeled proteins at a time. This limitation precludes the use of FRET micros-
copy for simultaneous measurement of multiple biochemical activities. Here
we present a new FRET microscopy method that generalizes quantitative
FRET microscopy to any number of fluorophores interacting in any combina-
tion. This approach makes use of Parallel Factor Analysis (PARAFAC) to
define excitation/emission spectral fingerprints for FRET between any number
of interacting fluorophores from observations of reference samples on any
instrument. The resultant spectral fingerprints are then used in a simple linear
unmixing model recover the distributions of free and interacting fluorophores
as well as their apparent FRET efficiencies. Input data can consist of either
complete spectral data or filter-based methods. The approach was validated
using control constructs consisting of fluorescent protein fusions inside living
cells. This method will enable intracellular analysis of sequential biochemical
interactions that could not previously be observed.
1007-Plat
Single Cell Sensing and Manipulation by Scanning Nanopore Microscopy
R. Adam Seger, Paolo Actis, Boaz Vilozny, Nader Pourmand.
UC Santa Cruz, Santa Cruz, CA, USA.
SignalTransduction by IonNano-Gating (STING) technology is a label-free bio-
sensor based on quartz nanopipettes capable of detecting DNA and proteins.
Using conical quartz nanopipettes a Scanning Ion Conductance Microscope
(SICM) was developed and used for single cell imaging, sensing and manipula-
tion. The height of the nanopore above a surface can be accurately controlled by
measuring the ionic current through the pore as it approaches a surface. Bymain-
taining a predefined distance from a surface and raster scanning the pore, a topo-
graphical image can be acquired. Additionally, material can be ejected through
nanopipette using electrophoretic and electroosmotic properties. The cost of fab-
rication and ease of use of nanopipettes provides an ideal system for developing
cellular systems for both cell detection and control.We demonstrate the capabil-ity of this sensor to image living cells, deposit material for controlled single cell
growth on a substrate, and a platform for single cell injection and detection using
an integrated microfluidic chip. The combination of STING technology with
traditional SICM technology can be leveraged to acquire both topographical
data and functional data, such as local ion concentration. This form of comple-
mentarymicroscopy is henceforth termed functional Scanning Ion Conductance
Microscopy (fSICM). Preliminary fSICM results will be presented. This work
illustrates the applicability of the STING sensor to a variety of applications,
from single cell detection to definition of cellular circuits.
1008-Plat
In Vivo Identification of Changes in Metabolic State as Stem Cells
Differentiate, by Phasor Analysis of Fluorescence Lifetime Imaging
Chiara Stringari, Amanda Cinquin, Olivier Cinquin, Peter Donovan,
Enrico Gratton.
University California Irvine, Irvine, CA, USA.
In stem cell research there is a high demand of techniques to investigate self-
renewal and differentiation mechanisms and to develop stem-cell-based thera-
pies for regenerative medicine. Here we develop a label-free method to identify
and classify stem cells and differentiating cells according to their metabolic
state. We use the phasor approach to fluorescence lifetime imaging and intrinsic
biochemical fluorescence biomarkers such as NADH, flavins, retinoids and por-
phyrin. The organ studied is the C. elegans germ line, expressing a histone-GFP
fusion protein that allows us to identify the differentiation state of the germ
cells. We calculate the average phasor value of the intrinsic fluorescence of
germ cells and we plot the cell phasor fingerprints in a scatter diagram. Cell
phasor fingerprints cluster according to their differentiation state. Different
metabolic fingerprint of cells reflect changes in binding sites of NADHwith dif-
ferent coenzymes during differentiation. The phasor approach to lifetime imag-
ing provides a label-free, fit-free and sensitive method to identify different
metabolic state of cells during differentiation, to sense small changes in the re-
dox state of cells and may identify symmetric and asymmetric divisions and
predict cell fate. This method is also a promising non-invasive optical tool
for monitoring metabolic pathways during differentiation or disease progres-
sion, and for cell sorting.
Work supported in part by NIH-P41 P41-RRO3155 ,P50-GM076516, NIH
RO1 HD49488, NIH PO1 HD47675, and CIRM RC1-00110 to PD.
1009-Plat
Multiplexed Time Lapse Fluorescence Lifetime Readouts in an Optically
Sectioning Time-Gated Imaging Microscope
Anca Margineanu1, Romain Laine1, Sunil Kumar1, Clifford Talbot1,
Sean Warren1, Christopher Kimberley2, James McGinty1, Gordon Kennedy1,
Alessandro Sardini1, Christopher Dunsby1, Mark A.A. Neil1, Matilda Katan2,
Paul M.W. French1.
1Imperial College London, London, United Kingdom, 2Institute for Cancer
Research, London, United Kingdom.
The aim of our study is to follow the temporal and spatial interactions of pro-
teins within different signalling networks in live cells. To map protein-protein
interactions, we apply Fo¨rster resonant energy transfer (FRET) readout using
fluorescence lifetime imaging (FLIM) and we present an optically sectioned
FLIM microscope capable of time-lapse readouts of multiplexed FRET pairs.
We note that FLIM FRET provides robust measurements, permits the use of
dark acceptors and helps minimise the impact of spectral cross-talk.
Wide-field FLIM is implemented using a gated optical intensifier synchronized
with the excitation pulses from a fibre-laser pumped supercontinuum source or
a frequency doubled Ti:Sapphire laser and enables FLIM FRET in live cells
with acquisition times below ~10 seconds. Optical sectioning is implemented
using a Nipkow disk unit and improves quantitation compared to wide-field im-
aging, e.g. permitting separation of signals from the plasma membrane and the
cytosol.
To demonstrate the capabilities of this instrument, we have mapped the intra-
cellular changes of calcium levels using interleaved time lapse FLIM acquisi-
tions of live cells labelled with two calcium probes following stimulation with
ionomycin and calcium solutions. HEK293 cells were labelled with the genet-
ically expressed biosensor, Troponin TN-XL, for which changes in calcium
levels are read out by FRET between the CFP and YFP fluorophores, and
with a calcium sensing dye, GFP-Certified FluoForte. The emission profile
of the latter is spectrally separate from CFP and YFP, making it useful for mul-
tiplexing with the many available CFP/YFP FRET biosensors.
We aim to apply this instrument to follow the spatial and temporal changes of
simultaneous activation of signalling pathways during fibroblast migration in
a concentration gradient of a chemo-attractant (PDGF), particularly using spe-
cifically designed FRET biosensors for small GTPases (Rac), phosphoinositide
pathway (IP3) and calcium.


N-Way FRET Microscopy for Imaging Multiple Protein Interactions Within a Single Living Cell 

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N-Way FRET Microscopy for Imaging Multiple Protein Interactions Within a Single Living Cell

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