New Dynamic Imaging Modality
Krisztia ´n Szigeti, Szabolcs Osva ´th.
Semmelweiss University, Budapest, Hungary.
A new imaging method (patent pending) was developed. This technology alters
existing 2D or 3D X-ray imaging methods to create a completely new type of
image along with the traditional one. The new image shows previously inacces-
sible information about movements hidden inside living bodies or objects, thus
providing functional information. The new technology provides four images at
once without increasing the necessary measurement time or radiation dose. The
most important of these images is a new ‘‘dynamic’’ image that represents local
motions inside the patient or studied object. Besides the dynamic image, the
new technology also reconstructs a ‘‘static’’ image very similar to the existing
conventional images. This static image, however, is an improved version of the
conventional image, since it is less affected by motion artifacts. The new tech-
nology reconstructs two more images which show the point-wise errors of the
static and dynamic images. The two error images are very important for
computer-based noise reduction and data analysis. The new technique gives
a better estimation of errors than present methods because it is based on the
measurement of physical parameters rather than a priori assumptions. Efficient
noise handling and good image quality are especially important in the medical
practice. A better estimation of the error of the image helps to optimise radia-
tion intensity and the measurement time necessary to get the diagnostic infor-
mation. This, in turn, facilitates avoiding unnecessary patient doses. This work
was funded by Ja ´nos Bolyai Research Scholarship of the Hungarian Academy
of Sciences grant num- ber BO/00468/09.
Temperature Measurement in Single Living Cells using a Hydrophilic
Fluorescent Nanogel Thermometer
Kohki Okabe1, Seiichi Uchiyama1, Yoshie Harada2, Takashi Funatsu1.
1University of Tokyo, Tokyo, Japan,2Kyoto University, Kyoto, Japan.
The temperature of a living cell changes with every cellular event. Thus, mea-
suring intracellular temperature will contribute to the explanation of intricate
biological processes and the development of novel diagnoses. Here we demon-
strate the first intracellular thermometry with a newly developed fluorescent
nanogel thermometer (FNT). The temperature-sensing function of FNT is un-
dertaken by the thermo-responsive polyNIPAM unit combined with a water-
sensitive fluorophore. Furthermore, two structural modifications were made
taking into consideration its functions in intracellular environments. The first
was gelation at a nanometer scale using a crosslinker, allowing the nanogel
to remove undesirable responses originating from interactions between cellular
components and the fluorophores. The second was the enrichment of ionic sul-
troscopic study, the fluorescence enhancement of FNT with increasing
temperature was independent of KCl concentration (100 to 200 mM), environ-
mental pH (4 to 10), or surrounding proteins. Then FNT was microinjected into
living COS7 cells, followed by imaging with an epi-fluorescence microscope.
The total fluorescence intensity of FNT in single COS7 cells showed the
temperature-dependent enhancement upon heating, which provides the calibra-
tion curve for intracellular thermometry using FNT. The temperature resolution
was evaluated to be 0.29-0.50?C (27-33?C). Next, intracellular temperature
variations induced by FCCP (mitochondria uncoupler) was investigated.
FCCP provoked the fluorescence enhancement of FNT, indicating intracellular
heating by 0.45?C for 30 min. This result suggests that our thermometer FNT
successfully detected the intrinsic and significant intracellular temperature
change in response to stimulation. In conclusion, novel thermometer FNT is su-
perior to other candidate thermometers in terms of biocompatibility (i.e., size,
sensitivity, and solubility) and functional independence (i.e., negligible interac-
tions with cellular components), enabling intracellular temperature measure-
ment in single living cells.
Photosensitization Mechanism in Lipid Membranes: The Role of Hydro-
Helena C. Junqueira1, Mauricio Baptista2, Rosangela Itri1.
1Physics Institute - University of Sao Paulo, Sa ˜o Paulo, Brazil,
2Chemistry Institute - University of Sao Paulo, Sa ˜o Paulo, Brazil.
Oxidative stress causes lipid peroxidation, generating membrane damages.
These lead to signaling cascades culminating on cellular death by apoptosis.
The aim of this work is to investigate how the presence of hydroperoxized
lipids in the membrane composition impacts on the lipid bilayer physical prop-
erties. Moreover, the importance of hydroperoxides generation and the role of
this species in lipid rafts formation will be also addressed. We studied the
effects of two oxidized lipids in giant unilamellar vesicles (GUVs) to verify
the lipid bilayer integrity. One oxidized lipid was commercially available
and one hydroperoxidized lipid was synthesized in our lab. The results showed
that smaller and more fragile vesicles were produced as the concentration of
oxidized lipids in their membranes increases. The membrane permeability
lost was verified by irradiating GUVs dispersed in methylene blue solutions.
Further, we investigated the influence of hydroperoxides in model membranes
made of binary lipid mixtures (POPC:DPPC and POPC:Cholesterol). The fluo-
rescence optical microscopy has been used to follow phase separation on mem-
brane depending on lipid composition and amount of oxidized species. The
overall results help us to better understand the role of hydroperoxide lipids in
the photosensitization mechanisms.
Keywords: hydroperoxide lipids, GUV, photosensitization, methylene blue
Work supported by FAPESP.
Three-Dimensional Multifluorophore FRET Microscopy
Brandon L. Scott, Jia Lin, Adam D. Hoppe.
South Dakota State University, Brookings, SD, USA.
Biochemical pathways are comprised of spatially organized interactions be-
tween multiple proteins. Fluorescent resonance energy transfer (FRET) micros-
copy techniques allow imaging of these interactions within living cells.
Recently, these techniques have been extended to allow imaging of more than
two fluorescently-tagged molecules, but these methods are limited to only two
dimensions. Here, we explore the extension of multifluorophore FRET imaging
to three-dimensional microscopy by image reconstruction methods. This work
takes advantage of our previously developed three-dimensional FRET stoichi-
ometry reconstruction (3D-FSR) and multifluorophore FRET microscopy
(MFM). Three-dimensional multifluorophore FRET microscopy (3D-MFM)
was utilized to observe the oligomerization of the HIV structural protein Gag,
in the three-dimensional space of both fixed and living cells.
Clear and Flexible Thin Films for Simultaneous Mechanical Loading
and Imaging of Cells
Bao-Ngoc Nguyen1, Joshua Chetta1, Sameer Shah2.
1University of Maryland, College Park, MD, USA,
2University of California - San Diego, La Jolla, CA, USA.
Cultured neurons undergo morphological changes when placed under a tensile
load. Stretch can occur during growth or joint movement, but can also have
damaging effects during surgery or limb lengthening. To study the cellular me-
chanics governing these responses, we have previously studied the effects of
applied stretch on axons of cultured rat sensory neurons. Our initial device re-
quired the inversion of a flexible membrane, on which neurons were seeded, in
close proximity to a glass cover slip to allow for high resolution microscopy
during stretch. This design proved to be generally effective, yet provided lim-
ited access to the cells due to substrate inversion. To enable solution exchange
or drug delivery to cells during experimentation, an optically clear and flexible
substrate compatible with current devices and optical microscopy techniques is
necessary. We present a method to integrate thin films made from polydime-
thylsiloxane (PDMS) into a cell stretching device. PDMS is a cheap silicone
elastomer which is optically clear and biocompatible. The polymer was spun
thickness,which was dictated to be compatible with the maximum workingdis-
tance of the microscope objective (typically <200 microns). The thin films
have shown to equally distribute tensile loading when uniaxial strain is applied
through a cell stretcher. In addition, the optically clear property coupled with
the appropriatethickness of the film enabled real time imagingof neuronalcells
and the analysis of the cytoskeletal component actin in response to tensile load-
ing. The upright film provides easy access to cells for drug delivery or other
chemical reagents. Future applications include films with microfluidic con-
structed channels for localized drug delivery to cells.
Millisecond Spatiotemporal Dynamics of FRET Biosensors by the Pair
Correlation Function and the Phasor Approach to FLIM
Elizabeth Hinde1, Michelle A. Digman1, Christopher Welch2,
Klaus M. Hahn2, Enrico Gratton1.
1University fo California, Irvine, CA, USA,2University fo North Carolina,
Chapel Hill, NC, USA.
Wemultiplexthe phasorapproachto biosensorFRET detectionbyfluorescence
lifetime imaging microscopy (FLIM) with pair correlation function (pCF) anal-
ysis along a line scan acquisition, to measure the spatiotemporal dynamics of
Rac1 and RhoA activation at the front and back of the cell upon growth factor
Sunday, February 26, 2012
stimulation. We recently demonstrated the phasor approach to biosensor FRET
detection by FLIM as a method that is robust towards biosensor design (single
and dual chain) as well as the fluorescence artifacts inherent to the cellular en-
vironment. Using a frame mode acquisition we were able to map the spatial lo-
calization and quantify the fractional contribution of the free and bound state of
a dual chain biosensor or the low and high FRET species of a single chain bio-
sensor in each pixel of an image. To increase temporal resolution we find that
line acquisition of FLIM data increases the total pixel integration and allows us
to probe millisecond to second dynamics of RhoA and Rac1 activity across the
cell. Given that this timescale is comparable to the diffusive rate at which Rac1
and RhoA traverse the cell upon activation we concomitantly perform pair cor-
relation function (pCF) analysis along the line scan and investigate the molec-
ular flow patternof RhoAand Rac1 upongrowthfactorstimulation.Wefind for
RhoA and Rac1 there are distinct gradients of activation from back to front
(FLIM data) and a molecular flow pattern (pCF) that explains the observed po-
larized GTPase activity.
Numerical Methods for Improving the Reliability of Number and Bright-
ness (N&B) Analysis
Antonio Trullo1,2, Valeria Rosaria Caiolfa1,2, Moreno Zamai1,2.
1San Raffaele Scientific Institute, Milan, Italy,2Centro Nacional de
Investigaciones Cardiovasculares, Madrid, Spain.
N&B is a technique based on moment-analysis for the measurement of the av-
erage number of molecules and brightness in each pixel in fluorescence corre-
lation microscopy images. The average brightness of the particle is obtained
from the ratio of the variance to the average intensity at each pixel (Digman
et al., 2008).
N&B is useful for determining stoichiometry and oligomerization of protein
complexes in live cells. However, the signal is generally affected by some
effects lead to an overestimation of variance, and consequently of brightness.
In this work we present a protocol for correcting N&B analysis for these sour-
ces of extra-variance.
We sort out the errors due to translational motion by realigning the images of
the time series (~250 frames) using a simple routine based on correlation argu-
ments. This correction is useful because N&B is a pixel-based technique. After
realignment, a given region of the cell can be associated to each pixel, avoiding
signal fluctuations due to the displacement of the cell.
Moreover, we use a particular high-pass filter, the ‘boxcar’ filter, to correct for
tion of the response of the fluorophore to laser excitation. Since photobleaching
is negligible within a short time period (i.e., a small data segment), the boxcar
the signal. The final result is the average over the total collected frames of the
brightness values computed for each segment (Hellriegel et al, 2011).
We demonstrate the efficiency of these corrections using simulations of mem-
brane motion and photobleaching. We also provide examples of correction ap-
plied to data acquired on EGFP constructs expressed in live cells.
A Novel FRET Biosensor For Measuring Glycolytic Activity: A Study of
Matthew J. Merrins, Leslie S. Satin.
University of Michigan, Ann Arbor, MI, USA.
Pulses of insulin from pancreatic beta-cells help maintain blood glucose in
a narrow range, although the source of these pulses is unclear. We propose
that a positive feedback circuit exists within the glycolytic pathway employing
the allosteric enzyme phosphofructokinase-1 (PFK1), which endows beta-cells
with the ability to generate oscillations in metabolism via autocatalytic activa-
tion by its product Fructose-1,6-bisphosphate (Fru1,6-BP). To test this hypoth-
esis, we have engineered a family of inter- and intramolecular Cerulean/Citrine
FRET biosensors based on the glycolytic enzyme pyruvate kinase M2 (PK),
which is allosterically activated and reported to multimerize upon binding
Fru1,6-BP. When introducedinto Min6 beta-cells,intramolecular PK biosensor
apparent FRET efficiency increased dose-dependently in response to glucose
(0, 2.5, 11, and 25 mM). This change was rapid (within seconds) and reversible.
When Min6 cells were stimulated with 25 mM glucose/TEA, oscillations in PK
biosensor activity were evident as ratiometric FRET changes, and exhibited
a similar period to slow oscillations in intracellular calcium or NAD(P)H (3-
4 min). Our results suggest that glycolysis in beta-cells is oscillatory and that
PFK1 is indeed an attractive candidate for the oscillatory generator. More
broadly, this family of PK biosensor constructs could be useful for exploring
the magnitude and kinetics of glycolytic activity in living cells. Supported by
F32DK085960 (M.J.M.) and R01DK46409 (L.S.).
Approach Enables New Directions for FLIM, FRET and FCS
Samantha Fore1, Felix Koberling2, Marcelle Koenig2, Peter Kapusta2,
Bendedikt Kraemer2, Benjamin Ewers2, Rainer Erdmann2,
Steffen Ruettinger2, Julie L. Fiore3, David Nesbitt3.
1PicoQuant Photonics North America Inc., Westfield, MA, USA,
2PicoQuant GmbH, Berlin, Germany,3JILA, NIST and University of
Colorado, Boulder, CO, USA.
Fluorescence dynamics of single moleculescan be followed on timescales from
sub-nanoseconds to seconds and even beyond with a universal approach of
time-resolved measurements. The underlying technique (Time-Tagged Time-
Resolved (TTTR) Recording) allows one to simultaneously record timing
and fluorescence intensity information, bothspectrallyand spatially,on a single
photonbasis.Weapply photonsorting andweightingschemes determinedfrom
the nanosecond photon arrival times to extend and improve single-molecule
fluorescence methodologies which up to now commonly utilize only
intensity-based analysis, namely FCS and FRET.
In Fluorescence Lifetime Correlation Spectroscopy (FLCS) photon weighting
provides superior suppression of common parasitic contributions, e.g., Raman
scattering and detector after-pulsing. Beyond this improvement of traditional
FCS, FLCS also offers the possibility to accurately determine diffusion proper-
ties of different species only requiring that the species differ in their fluores-
cence lifetimes . In 2-focus-FCS (2fFCS), the nanosecond timing
information is used to identify the spatial origin of the photons by combining
Pulsed Interleaved Excitation (PIE) with time-gated detection . Thereby,
2fFCS dramatically improves the accuracy of measuring absolute diffusion
coefficients. In addition to this, PIE can be used to identify artifacts and sub-
populations in single-pair FRET measurements. Nanosecond time-resolved de-
tection offers a complementary approach to donor/acceptor intensity based
methods for calculating FRET efficiencies via quenching of the donor lifetime.
 Benda A., Hof. M., Wahl M., Patting M., Erdmann R., Kapusta P., Rev. Sci.
Instr., Vol.76, 033106 (2005)
 Dertinger Th., Pacheco V., von der Hocht I., Hartmann R., Gregor I., Ender-
lein J, ChemPhysChem, Vol.8, p.433 (2007)
Confocal Fluorescence Microscopy:A Generalized
Ultra-Deep Imaging with Cellular Resolution: Enhanced Two-Photon
Fluorescence Microscopy with the Use of a Wide Area Photodetector
Viera Crosignani, Alexander Dvornikov, Enrico Gratton.
Univesity of California, Irvine, Irvine, CA, USA.
We have previously shown that the use of a wide photocathode area PMT as
a detector in a two-photon fluorescence microscope allowed us to image in tur-
bid samples up to the depth of about 2.5 mm with cellular resolution. This de-
tection scheme enables a very efficient collection of fluorescence photons
directly from the wide (1’’ diameter) area of the sample, which considerably
increases the detection system sensitivity in comparison to a traditional two-
photon microscope, where fluorescence is collected by the same objective
lens used for excitation. Because the imaging depth depends on the ability of
the system to sense weak fluorescent signals, this new detection method signif-
icantly enhances the imaging depth. We have recently built a new experimental
system that works in the upright configuration, which is best suited for exper-
iments on live animals. The system employs a high power Ti:Sa Mai Tai laser
with a group velocity dispersion compensator (DeepSee) for two-photon fluo-
rescence excitation that allows us to extend the imaging depth to 3mm in sam-
ples simulating brain tissue optical properties. Imaging experiments in vivo and
in vitro have also been conducted on live animals (mice) and tissues (skin, co-
lon, small intestine).
With the aid of the new high speed response PMT that we are currently incor-
porating in the system, we will be able to perform fluorescence lifetime imag-
ing microscopy (FLIM) on whole animals and tissue samples at a few mm
depth. This double feature will particularly aid in vivo neuron imaging.
This work was supported by National Institutes of Health grants: P41-
Multi-Confocal Fluorescence Correlation Spectroscopy: A Technique for
Parallel Multi-Spot Measurements in Living Cells and its Application to
the Study of Cellular Response to Heat Shock
Meike Kloster-Landsberg1, Gae ¨tan Herbomel2, Yves Usson3, Ire `ne Wang1,
Claire Vourc’h2, Catherine Souchier2, Antoine Delon1.
1Universite ´ Joseph Fourier / CNRS, LIPhy UMR5588, Saint Martin d’He `res,
France,2Universite ´ Joseph Fourier / INSERM, IAB CRI U823 team 10,
Grenoble, France,3Universite ´ Joseph Fourier / CNRS, TIMC (IN3S),
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