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.
This PDF file contains the front matter associated with SPIE Proceedings Volume 8228, including the Title Page, Copyright information, Table of Contents, Introduction, and the Conference Committee listing.
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.
Utilizing quantum properties of light to break the diffraction limit has been the goal of intense research in the
recent years. This paper is a progress report on a study aimed at experimentally demonstrating a superresolution
microscopy technique enabled by photon antibunching, a non-classical emission statistics feature exhibited by
most emitters used as fluorescent markers. We find that photon antibunching gives rise to correlations that encode
high spatial frequency information on the distribution of fluorescent emitters. Detecting these correlations using
photon counting instrumentation in a standard fluorescence microscope setting allows for three-dimensional
superresolution imaging of fluorophore stained samples. The technique provides a quantum alternative to the
established superresolution tools.
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.
We combine pulsed laser, supercontinuum radiation source and fast single-photon counting peripherals to obtain a multifunctional
micro/nano-scope. This provides us with better spatial and temporal resolution to observe fast dynamics.
Performing fluorescence correlation spectroscopy for fast dynamics (<μs) with sub-diffraction limit resolution to observe
the initial state of single-lipid dynamics in supported lipid bilayers and living cells is our goal. Lipid raft serves as a
platform for recruiting signaling components of effective signal transduction. However, the dynamics of sub-200nm
rapidly aggregated lipid rafts are still not elucidated in living cells. We here report our recent progress on the
construction of this multi-functional stage-scanning fluorescence micro/nanoscope for single-lipid dynamics study.
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.
The ABEL trap allows trapping of single biomolecules in solution for extended observation without immobilization. The
essential idea combines fluorescence-based position estimation with fast electrokinetic feedback in a microfluidic
geometry to counter the Brownian motion of a single nanoscale object, hence maintaining its position in the field of view
for hundreds of milliseconds to seconds. Such prolonged observation of single proteins allows access to slow dynamics,
as probed by any available photophysical observables. We have used the ABEL trap to study conformational dynamics
of the β2-adrenergic receptor, a key G-protein coupled receptor and drug target, in the absence and presence of agonist.
A single environment-sensitive dye reports on the receptor microenvironment, providing a real-time readout of
conformational change for each trapped receptor. The focus of this paper will be a quantitative comparison of the ligandfree
and agonist-bound receptor data from our ABEL trap experiments. We observe a small but clearly detectable shift in
conformational equilibria and a lengthening of fluctuation timescales upon binding of agonist. In order to quantify the
shift in state distributions and timescales, we apply nonparametric statistical tests to place error bounds on the resulting
single-molecule distributions.
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.
We have used single molecule fluorescence in situ hybridization (smFISH) to study cell-to-cell heterogeneity of
messenger RNA (mRNA) copy numbers for human host cells subject to a variety of external stimuli. In order to study
the effect of various stimuli and genetic modifications on mRNA copy number, we have constructed an automated highthroughput
multiplexed imaging system and data analysis package capable of localizing large numbers of individual
mRNA transcripts in three dimensions. These experimental distributions of mRNA are used to refine and down-select
regulatory models. Here we present a case example of Interleukin 1 alpha mRNA production in response to immune
system stimulation. We propose a methodology for extending these methods to study the effect of small RNA on genetic
expression by combining multiplexed imaging and numerical modeling at the system-level.
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.
Thermophilic enzymes operate at high temperatures but show reduced activities at room temperature. They are in
general more stable during preparation and, accordingly, are considered to be more rigid in structure. Crystallization is
often easier compared to proteins from bacteria growing at ambient temperatures, especially for membrane proteins.
The ATP-producing enzyme FoF1-ATP synthase from thermoalkaliphilic Caldalkalibacillus thermarum strain TA2.A1 is driven by a Fo motor consisting of a ring of 13 c-subunits. We applied a single-molecule Förster resonance energy transfer (FRET) approach using duty cycle-optimized alternating laser excitation (DCO-ALEX) to monitor the expected
13-stepped rotary Fo motor at work. New FRET transition histograms were developed to identify the smaller step sizes
compared to the 10-stepped Fo motor of the Escherichia coli enzyme. Dwell time analysis revealed the temperature and
the LDAO dependence of the Fo motor activity on the single molecule level. Back-and-forth stepping of the Fo motor
occurs fast indicating a high flexibility in the membrane part of this thermophilic enzyme.
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.
Single-molecule Förster resonance energy transfer (smFRET) is a powerful tool for extracting distance information
between two fluorophores (a donor and acceptor dye) on a nanometer scale. This method is commonly used to monitor
binding interactions or intra- and intermolecular conformations in biomolecules freely diffusing through a focal volume
or immobilized on a surface. The diffusing geometry has the advantage to not interfere with the molecules and to give
access to fast time scales. However, separating photon bursts from individual molecules requires low sample
concentrations. This results in long acquisition time (several minutes to an hour) to obtain sufficient statistics. It also
prevents studying dynamic phenomena happening on time scales larger than the burst duration and smaller than the
acquisition time. Parallelization of acquisition overcomes this limit by increasing the acquisition rate using the same low
concentrations required for individual molecule burst identification. In this work we present a new two-color smFRET
approach using multispot excitation and detection. The donor excitation pattern is composed of 4 spots arranged in a
linear pattern. The fluorescent emission of donor and acceptor dyes is then collected and refocused on two separate areas
of a custom 8-pixel SPAD array. We report smFRET measurements performed on various DNA samples synthesized
with various distances between the donor and acceptor fluorophores. We demonstrate that our approach provides
identical FRET efficiency values to a conventional single-spot acquisition approach, but with a reduced acquisition time.
Our work thus opens the way to high-throughput smFRET analysis on freely diffusing molecules.
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.
In this study, we have developed a four-channel Stokes vector formalism based second harmonic generation (SHG)
microscopy to map and analyze SHG signal. A four-channel Stokesmeter setup is calibrated and integrated into a laser
scanning microscope to measure and characterize the SH's corresponding Stokes parameters. We are demonstrating the use
of SH and its Stokes parameters to visualize the birefringence and crystalline orientation of KDP and collagen. We believe
the developed method can reveal unprecedented information for biomedical and biomaterial studies.
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.
Reducing reagent needs and costs while increasing throughput constitute important needs for assays in pharmaceutical
drug discovery. We are developing an ultrasensitive, fluorescence-based detection system in highly parallel microfluidic
channels with kHz readout rates in each channel. Prototype microfluidic devices with an array of >150 microchannels
have been fabricated by direct femtosecond laser machining of fused silica substrates. A device is placed in a custombuilt,
wide-field microscope where a line-generating red diode laser provides uniform epi-illumination just a few
microns high across a 500 micron field of view. Single-molecule levels in the probe volumes can be attained by flowing
suitably dilute aqueous solutions (~pM) of fluorescently labeled biomolecules through the microchannels. Fluorescence
is detected with an electron-multiplying CCD camera allowing readout rates up to 7 kHz for each microchannel. Rapid
initial assessment of detected fluorescence signals is performed through digital filtering derived from simulations based
on experimental parameters. Fluorescence correlation spectroscopy can then provide more detailed analysis of the
sample within each microchannel. Optimized microfluidic devices could be mass-produced in low-cost polymers using
imprint lithography.
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.
Over the past few years there has been a growing interest in monolithic arrays of single photon avalanche diodes (SPAD)
for spatially resolved detection of faint ultrafast optical signals. SPADs implemented in planar technologies offer the
typical advantages of microelectronic devices (small size, ruggedness, low voltage, low power, etc.). Furthermore, they
have inherently higher photon detection efficiency than PMTs and are able to provide, beside sensitivities down to
single-photons, very high acquisition speeds. In order to make SPAD array more and more competitive in time-resolved
application it is necessary to face problems like electrical crosstalk between adjacent pixel, moreover all the singlephoton
timing electronics with picosecond resolution has to be developed.
In this paper we present a new instrument suitable for single-photon imaging applications and made up of 32 timeresolved
parallel channels. The 32x1 pixel array that includes SPAD detectors represents the system core, and an
embedded data elaboration unit performs on-board data processing for single-photon counting applications. Photontiming
information is exported through a custom parallel cable that can be connected to an external multichannel TCSPC
system.
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.
A general framework to include fluctuations in the single molecule fluorescence intensity (FI) signal due to random changes in molecule dipole orientation was introduced at Optics Express (21, 2007). By assuming continuous changes in dipole orientation described by Brownian rotational diffusion, this research derives the probability density function (PDF) equation of FI fluctuations. Solution of the proposed equation for several limiting cases and different correlation times yields the short time behavior of FI fluctuations. Monte Carlo simulations results, in accordance with those found in theory will be presented during our talk.
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.
Fluorescent proteins are invaluable molecules in fluorescence microscopy and spectroscopy. The size and brightness of
fluorescent proteins often dictates the application they may be used for. While a monomeric protein may be the least
perturbative structure for labeling a protein in a cell, often oligomers (dimers and tetramers) of fluorescent proteins can
be more stable. However, from a quantitative microscopy standpoint, it is important to realize the photophysical
properties of monomers do not necessarily multiply by their number when they form oligomers. In this work we studied
oligomerization states of the Azami Green (AG) protein with fluorescence correlation spectroscopy (FCS) and photon
antibunching or photon pair correlation spectroscopy (PPCS). FCS was used to measure the hydrodynamic size of the
oligomers, whereas antibunching was used to count the number of fluorescent emitters in the oligomers. The results
exhibited that the dimers of AG were single emitters and the tetramers were dual-emitters, indicative of dipole-dipole
interactions and energy transfer between the monomeric units. We also used these methods to estimate the number of
fluorescent proteins displayed on T7 phage molecules.
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.
Typical problems in molecular biology, like oligomerization of proteins, appear on non-resolvable length scales.
Therefore a method which allows counting numbers of fluorescent emitters beyond this barrier can help to
unveil these questions. One approach engaging this task makes use of the photon antibunching (PAB) effect.
Most fluorophores are single photon emitters. Therefore upon a narrow excitation pulse they might only run
through one excitation cycle and emit one photon at a time. This behavior is known as PAB. By analyzing
coincident events of photon detections (Coincidence Analysis, CCA ) over many excitation cycles the number of
fluorophores residing in the confocal volume can be estimated. Simulations have shown that up to 40 fluorophores
can be distinguished with a reasonable error. In follow-up experiments five fluorophores could be distinguished
by CCA. In this work the method is applied to a whole sample set and statistical variance and robustness are
determined. CCA is critical to several parameters like photo stability, background noise, label efficiency and
photopysical properties of the dye, like brightness and blinking. Therefore a reasonable scheme for analysis is introduced and setup parameters are optimized. To proof the superiority of CCA, it has been applied to estimate the number of dyes for a well-defined probe and the results have been compared with bleach step analysis (BS analysis), a method based on the ability to observe single bleach-steps.
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.
The epidermal growth factor receptor (EGFR), which is over-expressed in tumors cells of epithelial origin is one of the
determinants of tumor responses to ionizing radiation. Recently, it has been shown that higher EGFR expression levels
lead to higher tumor resistance to radiation therapy through the activation of cell proliferation and survival pathways. In
this study, a raster-scan imaging technique known as Number and Brightness (N&B) analysis has been employed to
demonstrate the nuclear translocation of EGFR in living cells under a variety of experimental conditions. About 80% of
wild type (WT) EGFR translocated to the nucleus after γ-irradiation while the L858R and ▵E746-E750 mutant EGFR
did not. Subsequently, the effects of γ-irradiation together with an EGFR-blocking antibody (cetuximab) were monitored
simultaneously in the same cell lines expressing EGFR and its mutants. In the combined radiation and cetuximab
treatment, about 26 % of WT were translocated to the nucleus, while the L858R and ▵E746-E750 mutant EGFR did not.
These results are consistent with findings attained by standard molecular techniques and support the hypothesis that a
cytosolic pool of EGFR exists that cannot be accessed by cetuximab and can therefore contribute to treatment resistance.
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.
This paper describes recent efforts to characterize the diffusion of single molecule SERS analytes adsorbed to the surface of aggregated silver nanoparticles. Using a two-dimensional Gaussian function to fit the point spread function of a single diffraction-limited SERS emission image, the centroid of the emission can be located with precision better than 5nm. Tracking the centroid over time allows a diffusion trajectory to be constructed. By calculating the mean-r-squared displacement as a function of time lag, the diffusion properties of the SERS centroid can be tracked. For the single molecule SERS centroids presented herein, the diffusion analysis shows signatures of subdiffusion, indicating barriers to Brownian diffusion on the nanoparticle surface.
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.
Superresolution localization microscopy (e.g. PALM, STORM) builds images with sub-wavelength resolution
by analyzing a series of frames containing sparse, non-overlapping single-fluorophore images. A different set
of fluorophores is activated in each frame, and the key parameter controlled by the user is the fraction of
fluorophores activated. Using variational techniques, we show that the optimal activation probability, which
is the result of a tradeoff between speed and accuracy, depends sensitively on two factors: Whether activation
and fluorescent emission are controlled by separate wavelengths or by the same wavelength, and (in the single
wavelength case) the detailed kinetics of the bleaching process. Here our approach is extended to the case of
multi-fluorophore superresolution techniques, where we show that situations that would be ill-suited to singlefluorophore
localization are well-suited to multi-fluorophore localization.
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.
In the presented study we characterized the suitability of 15 conventional fluorescence dyes for GSDIM microscopy. For
all dyes involved in the screening labeled secondary antibodies for immunohistochemistry are commercially available.
The dye performance was tested after staining to fixed mammalian cells. Chemical environments were chosen to be
compatible with the applicative and spectroscopic demands. Investigated watery environments are suitable for TIRF
based applications. To the best of our knowledge, we present for the first time systematic screening for configurations of
dyes embedded in solid polymer. The polymer mounting matches well to the refractive index of oil immersion optics.
This is crucial for applications at high penetration depth into the sample and suitable for long-term sample storage.
We rated the final super-resolution image quality additional to quantitative characterization of important spectroscopic
parameters. Therefore, this dye screening is optimized for various biological imaging applications. Control of the single molecule blinking rate by 405nm light exposure is quantified, as well. It is shown that this important effect is applicable to numerous fluorescent dyes. Thus, the controlled application of low intensities of 405nm light allows to maximize recording speed. As this option is already included in commercial GSDIM microscopes the results of our study allow optimized super-resolution imaging down to ~20nm with multiple dyes and multi-color staining.
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.
Lately quite a plethora of concepts have been successfully developed, which take resolution beyond the classical limits
of a light microscope. Among these structured illumination microscopy (SIM) and photo activated localization
microscopy (PALM) hold the promise to provide biologists with unprecedented insights into sub-cellular
organizations. A combination of these methods seems particularly attractive as it allows adapting to the required
resolution and enables to map single molecules or molecule ensembles in the context of highly resolved structures.
SIM achieves two fold resolution enhancements in both lateral and axial directions, so structures can be highly
resolved in 3D. Adapting the structuring to the wavelength opens up the avenue for multi-color staining. Hence the
distribution of one protein and its associated structure can be viewed in the context of others. Since all common
fluorescent dyes can be used sample preparation is straightforward. Besides the classical approach to obtain highly
resolved structures with up to 10 times the classical resolution, the power of PALM lies additionally in its ability to
count and observe single molecules. As such clustering of molecules can be studied as well as many molecules tracked
simultaneously to study their diffusion. New strategies open up the possibility to obtain resolution enhancement in the
axial direction as well. These applications start already to have an impact on our view how a cell is organized and how
different proteins contribute to its make-up.
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.
Light microscopy imaging is being transformed by the application of computational methods that permit the detection of
spatial features below the optical diffraction limit. Successful localization microscopy (STORM, dSTORM, PALM,
PhILM, etc.) relies on the precise position detection of fluorescence emitted by single molecules using highly sensitive
cameras with rapid acquisition speeds. Electron multiplying CCD (EM-CCD) cameras are the current standard detector
for these applications. Here, we challenge the notion that EM-CCD cameras are the best choice for precision localization
microscopy and demonstrate, through simulated and experimental data, that certain CMOS detector technology achieves
better localization precision of single molecule fluorophores. It is well-established that localization precision is limited
by system noise. Our findings show that the two overlooked noise sources relevant for precision localization microscopy
are the shot noise of the background light in the sample and the excess noise from electron multiplication in EM-CCD
cameras. At low light conditions (< 200 photons/fluorophore) with no optical background, EM-CCD cameras are the
preferred detector. However, in practical applications, optical background noise is significant, creating conditions where
CMOS performs better than EM-CCD. Furthermore, the excess noise of EM-CCD is equivalent to reducing the
information content of each photon detected which, in localization microscopy, reduces the precision of the localization.
Thus, new CMOS technology with 100fps, <1.3 e- read noise and high QE is the best detector choice for super resolution precision localization microscopy.
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.
The diffraction limit of the conventional light microscope establishes a barrier that limits resolution and prevents
observation of fine structural details within biological specimens. A number of commercially available systems
now enable researchers to beat diffraction and achieve up to ten-fold improvements in resolution. These super
resolution systems generally rely on one of two strategies. They either add optical elements in order to overcome
the diffraction limit or they implement computational power and fitting algorithms to circumvent it. We have now
entered the next phase in the development of super resolution systems, where probes, hardware, and software are
gaining the refinements necessary to facilitate their application to a range of biological problems. Here, we
highlight the recent developments in these areas for two types of super resolution imaging systems, structured
illumination microscopy (SIM) and stochastic optical reconstruction microscopy (STORM). These improvements
are promoting the fast acquisition and processing speeds needed for live cell imaging beyond the diffraction limit.
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.
STimulated Emission Depletion (STED) microscopy enables imaging of biological samples combining significantly
improved optical resolution with all benefits of confocal microscopy. Especially, by combining multi-channel image
acquisition with high spatial resolution opens up a new understanding of co-localization experiments on nanoscales.
Such a microscope provides new insights in various fields of biology, such as cell and membrane biology, neurobiology
and physiology. We present new developments and a variety of biological examples for STED microscopy, showing
structural details on scales well below 70nm and give an overview of possible field of applications, mainly focused on live cell imaging.
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.
Quantitative fluorescent imaging requires optimization of the complete optical system, from the sample to the detector.
Such considerations are especially true for precision localization microscopy such as PALM and (d)STORM where the
precision of the result is limited by the noise in both the optical and detection systems. Here, we present a Camera
Simulation Engine (CSE) that allows comparison of imaging results from CCD, CMOS and EM-CCD cameras under
various sample conditions and can accurately validate the quality of precision localization algorithms and camera
performance. To achieve these results, the CSE incorporates the following parameters: 1) Sample conditions including
optical intensity, wavelength, optical signal shot noise, and optical background shot noise; 2) Camera specifications
including QE, pixel size, dark current, read noise, EM-CCD excess noise; 3) Camera operating conditions such as
exposure, binning and gain. A key feature of the CSE is that, from a single image (either real or simulated "ideal") we
generate a stack of statistically realistic images. We have used the CSE to validate experimental data showing that
certain current scientific CMOS technology outperforms EM-CCD in most super-resolution scenarios. Our results
support using the CSE to efficiently and methodically select cameras for quantitative imaging applications. Furthermore,
the CSE can be used to robustly compare and evaluate new algorithms for data analysis and image reconstruction. These
uses of the CSE are particularly relevant to super-resolution precision localization microscopy and provide a faster,
simpler and more cost effective means of system optimization, especially camera selection.
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.
There was a previous research that proposed the structured illumination confocal scanning microscope (SICSM) so as
to improve the lateral resolution of the confocal microscope. However, the image acquisition speed of the SICSM was
very slow and also an alignment error due to the mechanical rotation of a grating and a slit can easily occur. As a
theoretical study, in this paper we propose a new SI method, the cross SI method, which improves lateral resolution and
image acquisition speed. Performances of the conventional SI and the proposed SI methods are compared by analysis of
the modulation transfer function. The proposed SI method shows similar lateral resolution and can shorten the image
acquisition time compared to the conventional SI method. The cross structured illumination confocal microscope
(CSICM) is combined with the cross SI pattern optics and the line scanning confocal microscope. We have introduced a
2-D diffractive grating in order to create the cross SI pattern. The effects of the cross SI pattern, intensity and visibility,
on the system performance are analyzed. The CSICM has double the lateral resolution of the conventional microscope,
an optical sectioning ability and a fast image acquisition speed.
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.
Recently, a class of single-molecule based localization techniques such as the Photo-activated Localization Mi-
croscopy (PALM) or the Stochastic Optical Reconstruction Microscopy (STORM) has ingeniously brought light-
microscopy beyond the diraction limit. However, as the single-molecule images contain point source objects
(which have no clear edges, alignment and usually superimposed to the background), traditional restoration
techniques used for industrial vision images do not give satisfactory result on the PALM/STORM dataset. In
this work, we apply the multi-scale product of sub-band images resulting from the wavelet transformation, a
technique originally used for astronomical image restoration, for the noise ltering and single-molecule detection
in the Super-resolution images. This is an extension of the work by J.C Olivo-Marin1 on spot detection in bio-
logical images. Experimental results on real and synthetic datasets with ground-truth show that our approach
achieves very good detection rates as compared to the QuickPALM or the rapidSTORM software.
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.
DNA double-strand breaks (DSBs) are one of the most lethal types of DNA damage that occurs in eukaryotic cells.
There are two distinct pathways of repairing DSBs, homologous recombination (HR) and non-homologous end joining
(NHEJ). In the NHEJ repairing pathway, DSB recognition and repair initiation is directed by the interaction of DNAbinding
subunit Ku70/80 heterodimer with the DNA-PK protein catalytic subunit (DNA-PKcs). Mutations in these
proteins result in repair stalling and eventual DNA misrepair that may lead to genomic instability. Studying the binding
kinetics of these repair proteins is therefore important for understanding the conditions under which DSB repair stalls.
Currently open questions are, what is the minimum DNA length that this complex needs to get a foothold onto a DSB
and how tightly does DNA-PKcs bind onto the DNA-Ku70/80 complex. Fluorescence Correlation Spectroscopy (FCS)
and Fluorescence Cross-Correlation Spectroscopy (FCCS) techniques have the potential to give information about the
binding kinetics of DNA-protein and protein-protein interactions at the single-molecule level. In this work, FCS/FCCS
measurements were performed to explore the minimum DNA base-pair (bp) length that Ku70/80 needed as a foothold to
bind effectively onto the tips of different lengths of double-stranded DNA (dsDNA) fragments that mimic DSBs. 25 bp,
33 bp and 50 bp of dsDNA were used for these experiments and binding was studied as a function of salt concentration
in solution. It was found that the 25 bp binding was weak even at physiological salt concentrations while the dissociation
constant (Kd) remained constant for 33 and 50 bp dsDNA strand lengths. These studies indicated that the minimum
binding length for the Ku70/8 is in the vicinity of 25 bp. The specificity of binding of Ku70/80 was proven by
competitive binding FCCS experiments between Cy5-labeled DNA, GFP-Ku70/80 and titrations of unlabeled Ku70/80.
Finally, using FCCS it was possible to estimate the apparent Kd for DNA-PKcs binding to the DNA-Ku70/80 complex
and the induced dissociation of DNA-PKcs from that complex by phosphorylation was observed in real time.
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.