What is behind all those lifetimes anyway, and where do we go from here?
Author(s):
Yi-Chun Chen;
Bryan Q. Spring;
Chittanon Buranachai;
George Malachowski;
Robert M. Clegg
Show Abstract
Fluorescence lifetime-resolved imaging microscopy (FLIM) has made tremendous strides in the last two decades.
Exciting applications are being presented weekly, an extensive diversity of instrumentation and commercial devices have
appeared and have improved dramatically, sophisticated algorithms for analysis and interpretation are now available, and
FLIM is being coupled to other imaging modalities, such as spectral dispersion and anisotropy. In other words, FLIM
has matured considerably, and is approaching a point where it can be routinely applied by researchers who are not
involved with the instrumentation and analysis side of things. The number of interested users in FLIM has almost
certainly surpassed that of the audience that previously employed single channel fluorescence lifetime measurements (in
a cuvette). The reason is, of course, the imaging capability of FLIM and its exciting possibilities for biological
applications, especially FRET. In this lecture I will attempt to give an overview of where we have come from together
with a personal judgment on where we stand, propose possible areas of growth that will be of importance for the future
of FLIM, and discuss some specific challenges that remain, especially considering the unique nature of the FLIM
measurement and the complex biological and medical systems that require innovative and novel solutions from the
FLIM community.
Multicolor fluctuation spectroscopy in cells
Author(s):
Michelle A. Digman;
Enrico Gratton
Show Abstract
Based on the Fluctuation Correlation principle we have developed a method that is capable of measuring the
stoichiometry of molecular complexes and mapping dynamic processes in living cells. The method is based on
measuring simultaneously fluctuations of the fluorescence intensity at two image channels, each detecting a different
kind of protein. This is an extension of the number and brightness analysis in which we use the use of the ratio between
the variance and the average intensity to obtain the brightness of molecules.
Fast-acquisition multispectral FLIM by parallel TCSPC
Author(s):
Wolfgang Becker;
Bertram Su;
Axel Bergmann
Show Abstract
Currently used TCSPC FLIM systems are characterised by high counting efficiency, high time resolution, and multiwavelength
capability. The systems are, however, restricted to count rates on the order of a few MHz. In the majority of
applications, such as FRET or tissue autofluorescence, the photostability of the samples limits the count rate to much
lower values. The limited counting capability of the hardware is therefore no problem. However, if FLIM is used for
samples containing highly photostable fluorophores at high concentrations the available count rates can exceed the
counting capability of a single TCSPC channel. In this paper we describe a TCSPC FLIM system that uses 8 parallel
TCSPC channels to record FLIM data at a peak count rate on the order of 50•106 s-1. By using a polychromator for
spectral dispersion and a multi-channel PMT for detection we obtain multi-spectral FLIM data at acquisition times on
the order of one second. We demonstrate the system for recording transient lifetime effects in the chloroplasts in live
plants.
The characterization of optimized fluorescent proteins for Förster resonance energy transfer microscopy
Author(s):
Richard N. Day;
Yuansheng Sun;
Cynthia F. Booker;
Sangeeta Kumari;
Ammasi Periasamy;
Mike Davidson
Show Abstract
The genetically encoded fluorescent proteins (FP), used in combination with Förster resonance energy transfer (FRET)
microscopy, provide the tools necessary for the direct visualization of protein interactions inside living cells. Currently,
the FPs most commonly used for live-cell FRET studies are the Cerulean and Venus variants of the cyan and yellow FPs.
However, there are problems associated with this donor-acceptor pair, and these might be overcome by exploiting the
characteristics of some of the newer FPs. For example, earlier we showed that the monomeric teal FP (mTFP) has
advantages over Cerulean as a FRET donor for Venus. Here, using mTFP as the common donor fluorophore, we
characterize a variety of different yellow, orange and red FPs as potential acceptors of FRET. We employed a "FRET
standard" genetic construct to minimize variability in the separation distance and positioning of the fused donor and
acceptor FPs. Using spectral FRET imaging and fluorescence lifetime measurements from living cells expressing the
fused proteins, we characterized both sensitized acceptor emission and the shortening of the donor lifetime resulting
from quenching for each of the fused FP pairs. Surprisingly, we found disagreements between the spectral FRET and
lifetime measurements for some of the different FP pairs. Our results appear to indicate that some of the orange and red
FPs can quench the mTFP donor while yielding little sensitized emission. We are characterizing the basis for this
observation.
Adding new dimensions to fluorescence microscopy
Author(s):
Christoph Biskup;
Jana Kusch;
Eckhard Schulz;
Birgit Hoffmann;
Vasilica Nache;
Frank Schwede;
Frank Lehmann;
Klaus Benndorf
Show Abstract
Global analysis algorithms perform better than unconstrained data fitting and can improve the accuracy and precision of
the experimental data. Here, we report on different strategies that can be pursued to improve the results derived from
fluorescence measurements. We point out the benefits of acquiring fluorescence data in a temporally and spectrally
resolved manner and show how these data sets can be used to evaluate FRET measurements.
Fluorescence measurements can be also combined with other methods such as the patch-clamp technique. This
combination allows to record simultaneously fluorescence signals and electrical currents of ion channels in membrane
patches. Global analysis of the data can yield valuable information about the processes underlying channel activation.
We used this approach to study the activation of homotetrameric CNGA2 channels in inside-out membrane patches. By
using fluorescent analogues of cyclic nucleotides as ligands we were able to simultaneously determine ligand binding
and channel activation.
Metabolic mapping of cell culture growth by NADH fluorescence lifetime imaging
Author(s):
Vladimir Ghukasyan;
Tatyana Buryakina;
Fu-Jen Kao
Show Abstract
Fluorescence lifetime imaging microscopy (FLIM) has been demonstrated as advantageous at discrimination between
free and protein-bound forms of the NADH coenzyme, providing not only with the lifetimes of the both states (shorter τ1
and longer τ2), but also with the relative concentrations of both (fractions α1 and α2 correspondingly). Given the role of
NADH in cellular energetics, NADH FLIM has been applied for the noninvasive characterization of metabolic changes
in a range of pathologies. However, for the discrimination of pathological states, a proper characterization of NADH
fluorescence lifetime dynamics at physiological conditions has to be conducted. We have applied FLIM NADH for the
characterization of metabolic changes during cell culture growth. Our results demonstrate that during the exponential
growth stage there's a well expressed trends of gradual decrease of the free/bound ratio, as measured from the center
from the cell colonies. At the same time the cells at the edges of a colony exhibit higher values of the ratio. Several
possible reasons for the phenomena observed are discussed.
SLIM: multispectral FLIM with wide applications in cell biology
Author(s):
A. Rück;
F. Dolp;
C. Steinmetz;
B. von Einem;
C. A. F. von Arnim
Show Abstract
The fluorescence decay of a fluorophore in many cases does not show a simple monoexponential profile. A very complex situation arises, when more than one compound must be analyzed. A considerable improvement of the measurement could be achieved when time-resolved and spectral-resolved techniques are simultaneously incorporated. SLIM (spectral fluorescence lifetime imaging) is a new technique, which combines both. Time-correlated single photon counting (TCSPC) enables high counting efficiency for biomedical applications. For spectral resolved detection a polychromator in the detection path together with a 16-channel multianode photomultiplier tube and appropriate TCSPC routing electronics are used as a highly sophisticated system. The various possibilities which SLIM offers to improve molecular imaging in living cells will be discussed as well as successfully realized applications. These include FRET (resonant energy transfer) measurements for protein interactions, related to Alzheimer's disease. Special attention will be focused on molecules involved in the processing and trafficking of the amyloid precursor protein (APP), as trafficking proteins of the GGA family and β-secretase (BACE). Taking into account also the lifetime of the acceptor could enhance reliability of the FRET result.
New multichannel photon timing instrumentation with independent synchronized channels and high count rate for FLIM and correlation analysis
Author(s):
Michael Wahl;
Gerald Kell;
Peter Kapusta;
Hans-Jürgen Rahn;
Tino Roehlicke;
Rainer Erdmann
Show Abstract
Time-Correlated Single Photon Counting (TCSPC) with multiple detector channels is invaluable in many fluorescence
sensing applications. However, existing instrumentation is often limited in its number of independent input channels.
Solutions based on multiplexing ("routing") provide input channels that are not truly independent, causing artifacts in
correlation measurements and limiting applications with high count rates. On the other hand, parallel operation of
complete TCSPC units, each with their own host computer interface, will result in multiple data streams arriving at the
host computer. This causes complications in real-time analysis of time-tagged photon data where the order and temporal
relation of events across all channels is critical. Here we present a new modular architecture allowing scalability in terms
of the number of input channels (currently 8 but potentially 64), while using one common synchronization channel,
delivering a single output data stream that contains time-tag records for all events from all inputs in correct temporal
order, despite any variations in event rates across the channels. We discuss the influence of pile-up, resulting limitations
and solutions based on parallelization. This is especially important when measuring strong cellular samples or
autofluorescent species. Selected application results will be shown.
Filtered FCS and species cross correlation function
Author(s):
Suren Felekyan;
Stanislav Kalinin;
Alessandro Valeri;
Claus A. M. Seidel
Show Abstract
An extended analysis method of time, polarization and color resolved fluorescence correlation spectroscopy (filtered
FCS) is introduced. It uses multiparameter fluorescence detection (MFD) [1-3] to separate pure fluorescence signal of
multiple species and scatter contributions. This method allows monitoring of simultaneous and independent diffusion of
several molecular species in one sample and makes possible accurate and quantitative analyses of fractions. The
proposed method is simple to implement experimentally, because it requires only single wavelength excitation and MFD
widely used in single molecule experiments. In comparison to recently introduced fluorescence lifetime correlation
spectroscopy (FLCS) [4-7] this method is able to distinguish species when they have very close or even the same
fluorescence lifetime using just differences in time resolved fluorescence anisotropy.
Angular domain fluorescent lifetime imaging in turbid media
Author(s):
Fartash Vasefi;
Eldon Ng;
Bozena Kaminska;
Glenn H. Chapman;
Jeffrey J. L. Carson
Show Abstract
We describe a novel florescent lifetime imaging methodology applicable to fluorophores embedded in turbid media.
The method exploits the collimation detection capabilities of an angular filter device to extract photons emitted by a
fluorophore embedded at depth within the medium. A laser source is used to excite the fluorophore within the medium.
Photons emitted by the fluorophore that are not scattered to a high degree pass through the angular filter array and are
detected by the intensified CCD camera (200 ps minimum gate width). Scattered photons are rejected by the filter and
do not pass through to the camera. We fabricated angular filter arrays using silicon bulk micromachining and found that
an array of 80 μm square aperture micro-tunnels, 1.5 cm in length accepted photons with trajectories within 0.4° of the
axes of the micro-tunnels. The small acceptance angle rejected most of the scattered light exiting the turbid medium.
Voltage sensitive dyes used for FLIM studied by two-photon-excitation
Author(s):
T. Gensch
Show Abstract
Several voltage sensitive dyes and other dyes with high membrane affinity have been investigated with respect to their
use in multiphoton fluorescence microscopy with special emphasis on fluorescence lifetime imaging. The fluorescence
decay behavior in several cell types and in the retina, the dye distribution pattern and changes upon external stimuli are
found to be very different.
Analysis of NAD(P)H fluorescence components in cardiac myocytes from human biopsies: a new tool to improve diagnostics of rejection of transplanted patients
Author(s):
Y. Cheng;
A. Mateasik;
N. Poirier;
J. Miró;
N. Dahdah;
D. Chorvat Jr.;
A. Chorvatova
Show Abstract
Tissue autofluorescence is one of the most versatile non-invasive tools for mapping the metabolic state in living tissues.
Increasing interest in the imaging and diagnosis of living cells and tissues, based on their intrinsic fluorescence rather than fluorescence labeling, is closely connected to the latest developments in high-performance spectroscopic and microscopic techniques. We investigate metabolic state of cardiac cells isolated from one additional human biopsy from transplanted pediatric patients presenting either no rejection (R0) or mild rejection (R1). Two different approaches for isolation of human cardiac myocytes are also compared. Spectrally-resolved fluorescence lifetime detection of NAD(P)H fluorescence (excitation by pulsed 375 nm picosecond laser) is tested as a promising new tool for quantitative analysis of intrinsic cellular autofluorescence signals in living cardiomyocytes. This work opens new horizons in the evaluation of cardiac transplant rejection using latest fluorescence imaging approaches.
Deep-tissue multiphoton fluorescence lifetime microscopy for intravital imaging of protein-protein interactions
Author(s):
G. O. Fruhwirth;
D. R. Matthews;
A. Brock;
M. Keppler;
B. Vojnovic;
T. Ng;
S. Ameer-Beg
Show Abstract
Fluorescent lifetime imaging microscopy (FLIM) has proven to be a valuable tool in beating the Rayleigh criterion for
light microscopy by measuring Förster resonance energy transfer (FRET) between two fluorophores. Applying
multiphoton FLIM, we previously showed in a human breast cancer cell line that recycling of a membrane receptorgreen
fluorescent protein fusion is enhanced concomitantly with the formation of a receptor:protein kinase C α complex
in the endosomal compartment. We have extended this established technique to probe direct protein-protein interactions
also in vivo. Therefore, we used various expressible fluorescent tags fused to membrane receptor molecules in order to
generate stable two-colour breast carcinoma cell lines via controlled retroviral infection. We used these cell lines for
establishing a xenograft tumour model in immune-compromised Nude mice. Using this animal model in conjunction
with scanning Ti:Sapphire laser-based two-photon excitation, we established deep-tissue multiphoton FLIM in vivo.
For the first time, this novel technique enables us to directly assess donor fluorescence lifetime changes in vivo and we
show the application of this method for intravital imaging of direct protein-protein interactions.
Optimizing CARS signal using coherent control methods
Author(s):
Vladimir S. Malinovsky
Show Abstract
We demonstrate the adiabatic passage based method to maximize CARS coherence and present numerical results
of the roof scheme implementation without making an assumption of adiabatic elimination of detuned excited
electronic states. Also we analyze influence of fast decoherence in the molecular samples on the results of proposed
scheme. It is shown that the adiabatic method allows achieving chemical sensitivity with high resolution and
can be used to obtain CARS signal with efficiently suppressed background in molecular systems with coherence
times of several hundred of femtoseconds.
CARS microscopy using linearly chirped ultrafast laser pulses
Author(s):
Israel Rocha-Mendoza;
Wolfgang Langbein;
Paola Borri
Show Abstract
We have developed a home-built CARS microscope which exploits linearly-chirped ultrafast laser pulses. By using
glass of high group-velocity dispersion, Stokes and Pump pulses of 150 fs duration Fourier-limited are equally
chirped to pulse durations in the 0.5 ps-2.8 ps range. In this way we reduce the spectral width of the instantaneous
frequency difference to the Fourier limit of the chirped pulse duration (spectral focussing). As a proof of
principle, CARS spectroscopy with high spectral resolution is demonstrated on polystyrene beads. We also
show, both theoretically and experimentally, that for chirped pulse durations shorter than or comparable to the
Raman coherence time, maximum CARS signal occurs for a Pump arriving after the Stokes pulse. Furthermore,
we demonstrate the applicability of our CARS microscope to biological sciences by performing CARS microspectroscopy
on different live cells and fixed tissue samples.
CARS and SHG microscopy for the characterization of bacterial cellulose
Author(s):
Annika Enejder;
Christian Brackmann;
Aase Bodin;
Madeleine Åkeson;
Paul Gatenholm
Show Abstract
We have developed a protocol employing dual-mode non-linear microscopy for the monitoring of the
biosynthesis of bacterial cellulose at a single-fiber level, with the fundamental aim to achieve a
product with material properties similar to those of human blood vessels. Grown in a tubular geometry
it could then be used as a natural and biocompatible source of replacement tissue in conjunction with
cardiovascular surgery. The bacteria (Acetobacter xylinum) were selectively visualized based on the
CH2 vibration of its organic macromolecular contents by the Coherent Anti-Stokes Raman Scattering
(CARS) process and, simultaneously, the non-centrosymmetrically ordered, birefringent cellulose
fibers were depicted by the Second Harmonic Generation (SHG) process. This dual-channel detection
approach allows the monitoring of cellulose-fiber formation in vivo and to determine the influence of
e.g. different growth conditions on fiber thickness and orientation, their assembling into higher-order
structures and overall network density. The bacterial and fiber distributions were monitored in a
simple microscope cultivation chamber, as well as in samples harvested during the actual fermentation
process of tubular cellulose grafts. The CARS and SHG
co-localization images reveal that highest
bacterial population densities can be observed in the surface regions of the cellulose tissue, where the
primary growth presumably takes place. The cellulose network morphology was also compared with
that of human arteries and veins, from which we conclude that the cellulose matrix is comparatively
homogeneous in contrast to the wavy band-like supra-formations of collagen in the native tissue. This
prompts for sophisticated fermentation methods by which tunnels and pores of appropriate sizes and
shapes can be introduced in the cellulose network in a controllable way. With this protocol we hope to
contribute to the fundamental knowledge required for optimal production of bioengineered cellulose
tissues, eventually being available for clinical use.
Enhancing two-color absorption, self-phase modulation, and Raman microscopy signatures in tissue with femtosecond laser pulse shaping
Author(s):
Martin C. Fischer;
Ivan Piletic;
Dan Fu;
Thomas E. Matthews;
Henry Liu;
Prathyush Samineni;
Baolei Li;
Warren S. Warren
Show Abstract
Nonlinear microscopies (most commonly, two-photon fluorescence, second harmonic generation, and coherent
anti-Stokes Raman scattering (CARS)) have had notable successes in imaging a variety of endogenous and exogenous targets
in recent years. These methods generate light at a color different from any of the exciting laser pulses, which makes the
signal relatively easy to detect. Our work has focused on developing microscopy techniques using a wider range of
nonlinear signatures (two-photon absorption of nonfluorescent species, self phase modulation) which have some specific
advantages - for example, in recent papers we have shown that we can differentiate between different types of melanin
in pigmented lesions, image hemoglobin and its oxygenation, and measure neuronal activity. In general, these signatures
do not generate light at a different color and we rely on the advantages of femtosecond laser pulse shaping methods to
amplify the signals and make them visible (for example, using heterodyne detection of the induced signal with one of the
co-propagating laser pulses). Here we extend this work to stimulated Raman and CARS geometries. In the simplest
experiments, both colors arise from filtering a single fs laser pulse, then modulating afterwards; in other cases, we
demonstrate that spectral reshaping can retain high frequency resolution in Raman and CARS geometries with
femtosecond laser pulses.
Ultrafast multiphoton microscopy with high-order spectral phase distortion compensation
Author(s):
Yair Andegeko;
Dmitry Pestov;
Vadim V. Lozovoy;
Marcos Dantus
Show Abstract
High-order dispersion of ultrashort laser pulses (with ~100 nm bandwidth) is shown to account for significant reduction
of two-photon excitation fluorescence and second harmonic generation signal produced at the focal plane of a laser-scanning
two-photon microscope. The second- and third-order corrections recover 20-40% of the signal intensity
expected for a transform-limited laser pulse, while the rest depends on the proper compensation of higher-order terms. It
can be accomplished through the use of a pulse shaper by measuring and correcting all nonlinear spectral phase
distortions.
Spectral phase shaping for high resolution CARS spectroscopy around 3000 cm-1
Author(s):
A. C. W. van Rhijn;
S. Postma;
J. P. Korterik;
J. L. Herek;
H. L. Offerhaus
Show Abstract
By spectral phase shaping of both the pump and probe pulses in coherent anti-Stokes Raman scattering (CARS)
spectroscopy we demonstrate the extraction of the frequencies of vibrational lines using an unamplified oscillator.
Furthermore we demonstrate chemically selective broadband CARS microscopy on a mixed sample of 4 μm
diameter polystyrene (PS) and poly(methyl methacrylate) (PMMA) beads. The CARS signal from either the
PS or the PMMA beads is shown to be enhanced or suppressed, depending on the phase profile applied to the
broadband spectrum. Using a combination of negative and positive (sloped) π-phase steps in the pump and
probe spectrum the purely non-resonant background signal is removed.
Pulse shaping for background free broadband CARS
Author(s):
Young Jong Lee;
Marcus T. Cicerone
Show Abstract
We demonstrate that pulse shaping of a narrowband pulse can suppress the nonresonant background (NRB) contribution and retrieve resonant Raman signals efficiently in a broadband coherent anti-Stokes Raman scattering (CARS) spectrum. A pulse shaper prepares a probe pulse with two spectral components of differing phase. When the CARS fields
generated by these two out-of-phase components are optically mixed, the NRB signal is greatly reduced while a resonant CARS signal survives with minimal attenuation. We discuss three model schemes for the interfering pulse components: (1) two pulses with different bandwidths and the same center frequency (ps-fs scheme); (2) two pulses with the same bandwidth and shifted center frequencies
(ps-ps scheme); and (3) a pulse with different phases across the center frequency (fs(+/-) scheme). In all schemes, only the resonant signal from the "3-color" CARS mechanism survives. The resonant signal from "2-color" CARS mechanism vanishes along with the NRB. We discuss optimization conditions for signal intensity and shape of resonant CARS peaks.
High performance multimodal CARS microscopy using a single femtosecond source
Author(s):
Adrian F. Pegoraro;
Andrew Ridsdale;
Douglas J. Moffatt;
John P. Pezacki;
Albert Stolow
Show Abstract
We demonstrate high performance coherent anti-Stokes Raman scattering (CARS) microscopy using a single
femtosecond Ti:Sapphire laser source combined with a photonic crystal fiber (PCF). By adjusting the chirp of
the femtosecond pump and Stokes laser pulses, we achieve high quality multimodal imaging (simultaneous CARS,
two-photon fluorescence, and second harmonic generation) of live cells and tissues. The tuneable Ti:sapphire
output provides the pump beam directly, while part of this is converted to the red-shifted Stokes pulse using a
PCF having two close-lying zero dispersion wavelengths. This PCF gives good power and stability over Stokes
shifts ranging from below 2300 cm-1 to over 4000 cm-1. This tuning range can be accessed by simply controlling
the time delay between the input pulses. This allows fast, continuous computer-controlled tuning of the Stokes
shift over a broad range, without involving any adjustment of either the femtosecond laser or the PCF. The
simultaneous optimization of CARS, two-photon fluorescence and second harmonic generation is achieved by
controlling the degree of chirp and involves a trade-off between spectral resolution of the CARS process and
signal strength. This is illustrated by showing applications of the multimodal CARS imaging and optimization
technique to biomedical problems involving both live cells and tissues.
Background free CARS imaging by local phase detection
Author(s):
M. Jurna;
J. P. Korterik;
C. Otto;
L. Herek;
H. L. Offerhaus
Show Abstract
In this article we show that heterodyne CARS, based on a controlled and stable phase-preserving chain, can be
used to measure amplitude and phase information of molecular vibration modes. The technique is validated by
a comparison of the imaginary part of the heterodyne CARS spectrum to the spontaneous Raman spectrum of
polyethylene. The detection of the phase allows for rejection of the non-resonant background from the data. The
resulting improvement of the signal to noise ratio is shown by measurements on a sample containing lipid.
Optical pulse shaping for selective excitation of coherent molecular vibrations by stimulated Raman scattering
Author(s):
Joseph B. Geddes III;
Daniel L. Marks;
Stephen A. Boppart M.D.
Show Abstract
Coherent anti-Stokes Raman scattering (CARS) can be used to identify biological molecules from their vibrational
spectra in tissue. A single double-chirped broadband optical pulse can excite a broad spectrum of resonant
molecular vibrations in the fingerprint spectral region. Such a pulse also excites nonresonant CARS, particularly
from water. We describe a theoretical technique to design an optical pulse to selectively excite coherent vibrations
in a target molecular species so that the CARS signal generated is increased. The signal from other molecules
is reduced, since the incident pulse does not excite them to have coherent vibrations. As an example, we apply
the technique to design pulses to elicit increased CARS signal from a mixture of one or more of the alcohols
methanol, ethanol, and isopropanol. We also show how such pulse designs can be used to selectively excite one
member of closely related complex biological species. As measured interferometrically, the CARS signal from
three phosphodiester stretch modes of DNA can be increased to more than ten times that of the analogous signal
from RNA when the pulse design technique is used.
Coherent anti-Stokes generation from single nanostructures
Author(s):
Hyunmin Kim;
Tatyana Sheps;
David K. Taggart;
Philip G. Collins;
Reginald M. Penner;
Eric O. Potma
Show Abstract
Dual color four-wave-mixing is used to visualize individual gold nanowires and single carbon nanotubes. The
strong nonlinear signals, which are detected at the anti-Stokes frequency, originate from the electronic response
of the nanostructures. In gold nanowires, the collective electron motions produce detectable coherent anti-Stokes signals that can be used to study the orientation and relative strength of the structure's plasmon resonances. In single walled carbon nanotubes, coherent anti-Stokes contrast can be used to map the orientation of the electronic resonances in single tubes. Coherent anti-Stokes imaging of the material's electronic response allows the first
close-ups of the coherent nonlinear properties of individual structures and molecules.
A new light source for multimodal multiphoton microscopy including CARS
Author(s):
Ingo Rimke;
Volker Siffrin;
Raluca Niesner;
Tina Leuenberger;
Frauke Zipp;
Edlef Büttner;
Gero Stibenz
Show Abstract
We will present a new flexible laser source for multimodal Multiphoton excitation microscopy including CARS. It
consists of a tuneable femtosecond-Ti:Sapphire laser and an optical parametric oscillator (OPO). The new OPO-design
allows for high flexibility in pump- and output wavelengths giving rise to for instance image EGFP with the Ti:Sapphire
and tdRFP with the OPO simultaneously. This is presented on living mouse brain tissue.
The minimum energy difference between Ti:Sapphire and
OPO-wavelengths achievable is 2500cm-1. Thus CARS
imaging of lipids is possible. Due to synchronous pumping of the OPO the pump- and OPO pulses are intrinsically
locked in time to each other thus they can be brought to perfect overlap of pump and stokes pulses. Uncaging
multiphoton microscopy is also possible with this system due to the low minimum OPO pump wavelength of 730nm.
Coupling CARS with multiphoton fluorescence and high harmonic generation imaging modalities using a femtosecond laser source
Author(s):
Hongtao Chen;
Mikhail N. Slipchenko;
Jiabin Zhu;
Kimberly K. Buhman;
Ji-Xin Cheng D.V.M.
Show Abstract
Multimodal nonlinear optical imaging has opened new opportunities and becomes a powerful tool for imaging complex
tissue samples with inherent 3D spatial resolution.. We present a robust and easy-to-operate approach to add the coherent
anti-stokes Raman scattering (CARS) imaging modality to a widely used multiphoton microscope. The laser source
composed of a Mai Tai femtosecond laser and an optical parametric oscillator (OPO) offers one-beam, two-beam and
three-beam modalities. The Mai Tai output at 790 nm is split into two beams, with 80% of the power being used to pump
the OPO. The idler output at 2036 nm from OPO is doubled using a periodically poled lithium niobate (PPLN) crystal.
This frequency-doubled idler beam at 1018 nm is sent through a delay line and collinearly combined with the other Mai
Tai beam for CARS imaging on a laser-scanning microscope. This Mai Tai beam is also used for multiphoton
fluorescence and second harmonic generation (SHG) imaging. The signal output at 1290 nm from OPO is used for SHG
and third-harmonic generation (THG) imaging. External detectors are installed for both forward and backward detection,
whereas two internal lamda-scan detectors are employed for microspectroscopy analysis. This new system allows
vibrationally resonant CARS imaging of lipid bodies, SHG imaging of collagen fibers, and multiphoton fluorescence
analysis in fresh tissues. As a preliminary application, the effect of diacylglycerol acyltransferase 1 (DGAT1) deficiency
on liver lipid metabolism in mice was investigated.
Analytical capabilities of nonlinear Raman microspectroscopic imaging
Author(s):
R. Arora;
G. I. Petrov;
V. V. Yakovlev
Show Abstract
Nonlinear Raman scattering is an emerging spectroscopy technique for non-invasive microscopic imaging. It can
produce a fluorescence background free vibrational spectrum from a microscopic volume of a sample providing
chemically specific information about its molecular composition.
Fiber lasers for CARS microscopy
Author(s):
F. W. Wise;
K. Kieu;
Brian Saar;
Gary Holtom;
Sunney Xie
Show Abstract
We report a fiber-based high-power picosecond laser system for coherent Raman microscopy (CRM). This source
generates 3-ps pulses with 6 W average power at 1030 nm.
Frequency-doubling yields more than 2 W of green light,
which can be used to pump a commercial optical parametric oscillator to produce the pump and Stokes beams for CRM.
The design and performance of the laser are described, along with an application to CARS imaging.
Clinical multiphoton tomography and clinical two-photon microendoscopy
Author(s):
Karsten König;
Rainer Bückle;
Martin Weinigel;
Peter Elsner;
Martin Kaatz
Show Abstract
We report on applications of high-resolution clinical multiphoton tomography based on the femtosecond laser system
DermaInspectTM with its flexible mirror arm in Australia, Asia, and Europe. Applications include early detection of
melanoma, in situ tracing of pharmacological and cosmetical compounds including ZnO nanoparticles in the epidermis
and upper dermis, the determination of the skin aging index SAAID as well as the study of the effects of anti-aging
products. In addition, first clinical studies with novel rigid
high-NA two-photon 1.6 mm GRIN microendoscopes have
been conducted to study the effect of wound healing in chronic wounds (ulcus ulcera) as well as to perform intrabody
imaging with subcellular resolution in small animals.
Two-photon imaging and nanoprocessing of stem cells with sub-20 fs laser pulses
Author(s):
A. Uchugonova;
A. Isemann;
R. Bückle;
W. Watanabe;
K. König
Show Abstract
Novel ultracompact multiphoton sub-20 femtosecond near infrared MHz laser scanning microscopes and conventional
250 fs laser microscopes have been used to perform high resolution multi-photon imaging of stem cell clusters as well as
targeted intracellular nanoprocessing and knock-out of living single stem cells within a 3D microenvironment. Also
lethal exposure of large parts of cell clusters was successfully probed while maintaining single cells of interest alive.
Mean powers in the milliwatt range for 3D nanoprocessing and microwatt powers for two-photon imaging were found to
be sufficient. Ultracompact low power sub-20 fs laser systems may become interesting tools for nanobiotechnology such
as optical cleaning of stem cell clusters and optical transfection.
Three-dimensional (3D) high-speed imaging and fabrication system based on ultrafast optical pulse manipulation
Author(s):
Daekeun Kim;
Peter T. C. So
Show Abstract
Laser scanning systems for two-photon microscopy and fabrication have been proven to be excellent in depth-resolving
capability for years. However, their applications have been limited to laboratory use due to their intrinsic slow nature.
The recently introduced temporal focusing concept enables wide-field optical sectioning and thus has potential in both
high-speed 3D imaging and 3D mass-production fields. In this paper, we use the ultrafast optical pulse manipulation to
generate two-photon excitation depth-resolved wide-field illumination (TPEDRWFI). The design parameters for the
illumination were chosen based on numerical simulation of the temporal focusing. The imaging system was
implemented, and the optical sectioning performance was compared with experimental result.
Adaptive optics for multiphoton microscopy
Author(s):
Delphine Débarre;
Tony Wilson;
Martin J. Booth
Show Abstract
Specimen-induced aberrations are frequently encountered in high resolution microscopy, particularly when high
numerical aperture lenses are used to image deep into biological specimens. These aberrations distort the focal
spot causing a reduction in resolution and, often more importantly, reduced signal level and contrast. This is
particularly problematic in multiphoton microscopy, where the
non-linear nature of the signal generation process
means that the signal level is strongly affected by changes in the focal spot intensity. We have developed an
adaptive two-photon fluorescence microscope to correct for these aberrations. Unlike a conventional adaptive
optics system, the microscope does not include a wavefront sensor but uses an efficient sensorless optimisation
scheme to obtain optimum aberration correction.
Multiphoton microscopy by multiexcitonic ladder climbing in colloidal quantum dots
Author(s):
Nir Rubin Ben Haim;
Dan Oron
Show Abstract
Depth resolved multiphoton microscopy is performed by collecting the fluorescent emission of two-exciton states
in colloidal quantum dots. The biexciton is formed via two sequential resonant absorption events. Due to the
large absorption cross-section and the long lifetime of the intermediate (singly excited) state, unprecedented
low excitation energy and peak powers (down to 105W/cm2) are required to generate this nonlinear response.
Depending on the quantum dot parameters, the effective two-photon cross section can be as large as 1010 GM,
orders of magnitude higher than for nonresonant excitation. The biexciton emission can be differentiated from
that of the singly excited state by utilizing its different transient dynamics. Alternate methods for discrimination
are also discussed. This system is ideal for performing
three-dimensional microscopy using low excitation power.
Moreover, it enables to perform multiphoton imaging even with
near-infrared emitting quantum dots, which
are highly compatible with imaging deep into a scattering tissue. The depth resolution of our microscope is
shown to be equivalent to a standard two-photon microscope. The system also shows slow saturation due to the
contribution of higher (triply and above) excited states to the emitted signal.
Surface plasmon-enhanced two-photon fluorescence microscopy for live cell membrane imaging
Author(s):
R.-Y. He;
K.-C. Cho;
N.-S. Chang;
Y.-D. Su;
S.-J. Chen
Show Abstract
A surface plasmon-enhanced two-photon total-internal-reflection fluorescence (TIRF) microscope has been developed to
provide the fluorescent images of living cell membranes. The proposed microscope with the helps of surface plasmons
(SPs) not only provides brighter fluorescent images based on the mechanism of local electromagnetic field enhancement,
but also reduces photobleaching due to having shorter fluorophore lifetime. In comparison with one-photon TIRF, the
two-photon TIRF can achieve higher signal-to-noise ratio cell membrane imaging due its smaller excitation volume and
lower scattering. Combining with the SP enhancement and two-photon excitation TIRF, the microscope has
demonstrated the brighter and more contrast fluorescence membrane images of living monkey kidney COS-7 fibroblasts
transfected with an EYFP-MEM or EGFP-WOX1 construct.
Imaging dental sections with polarization-resolved SHG and time-resolved autofluorescence
Author(s):
Jun Huang Chen;
Po-Yen Lin;
Stephen C. Y. Hsu;
Fu-Jen Kao
Show Abstract
In this study, we are using two-photon (2-p) excited autofluorescence and second harmonic (SH) as imaging modalities
to investigate dental sections that contains the enamel and the dentin. The use of near-infrared wavelengths for multiphoton
excitation greatly facilitates the observation of these sections due to the hard tissue's larger index of refraction
and highly scattering nature. Clear imaging can be achieved without feature altering preparation procedures of the
samples. Specifically, we perform polarization resolving on SH and lifetime analysis on autofluorescence. Polarization
resolved SH reflects the preferred orientation of collagen while very different autofluorescence lifetimes are observed
from the dentin and the enamel. The origin of 2-p autofluorescence and SH signals are attributed to hydroxyapatite
crystals and collagen fibrils, respectively. Hydroxyapatite is found to be present throughout the sections while collagen
fibrils exist only in the dentin and dentinoenamel junctions.
Second harmonic generating (SHG) nanoprobes: a new tool for biomedical imaging
Author(s):
Periklis Pantazis;
Ye Pu;
Demetri Psaltis;
Scott Fraser
Show Abstract
Fluorescence microscopy has profoundly changed how cell and molecular biology is studied in almost
every aspect. However, the need of characterizing biological targets is largely unmet due to deficiencies
associated with the use of fluorescent agents. Dye bleaching, dye signal saturation, blinking, and tissue
autofluorescence can severely limit the signal-to-noise ratio (SNR). Given the photophysical properties are
fundamentally different to the fluorescent agents currently used in biomedical research, second harmonic
generating (SHG) nanoprobes can be suitable for biomedical imaging and can eliminate most of the
drawbacks encountered in classical fluorescence systems.
Infrared-based least-invasive third and second harmonic generation imaging of ocular tissues
Author(s):
Szu-Yu Chen;
Han-Chieh Yu;
I-Jong Wang;
Chi-kuang Sun
Show Abstract
Cornea functions as an outermost lens and plays an important role in vision. For cornea diagnosis and treatment, a
microscopic imaging system with cellular resolution and high eye safety is strongly desired. Recently, the cell
morphology of corneal epithelium and endothelium can be revealed by confocal or two-photon fluorescence microscopy,
while the collagen fibers in the corneal stroma can be shown by second harmonic generation (SHG) microscopy.
However, in most of the developed imaging tools, visible to near-infrared light sources were used. To increase the eye
safety, a light source with longer wavelength would be needed. In this presentation, a study using an infrared laser based
nonlinear microscopy to investigate the ocular tissues of a mouse eye will be demonstrated. Since most of autofluorescence
was suppressed under infrared excitation, third harmonic generation (THG) microscopy was used to reveal
the cellular morphology and ~700μm penetrability could be achieved. Combining SHG with THG, in an intact mouse
eye, not only the cornea but also the upper half of the lens could be observed with cellular resolution. Our study
indicated that infrared-based SHG and THG microscopy could provide a useful in vivo investigating tool for
ophthalmology.
Multimodal multiphoton microscopy
Author(s):
François Légaré;
Christian P. Pfeffer M.D.;
Feruz Ganikhanov
Show Abstract
Multiphoton microscopy is a powerful technique for high spatial resolution thick tissue imaging. In its simple version, it
uses a high repetition rate femtosecond oscillator laser source focussed and scanned across biological sample that contains fluorophores. However, not every biological structure is inherently fluorescent or can be stained without causing biochemical changes. To circumvent these limitations, other non-invasive nonlinear optical imaging approaches are currently being developed and investigated with regard to different applications. These techniques are: (1) second harmonic generation (SHG), (2) third harmonic generation (THG), and (3) coherent anti-Stokes Raman scattering
(CARS) microscopy. The main advantage of the above mentioned techniques is that they derive their imaging contrast
from optical nonlinearities that do not involve fluorescence process. As a particular application example we investigated
collagen arrays. We show that combining SHG-THG-CARS onto a single imaging platform provides complementary information about the sub-micron architecture of the tissue. SHG microscopy reveals the fibrillar architecture of collagen arrays and confirm a rather high degree of heterogeneity of χ(2) within the focal volume, THG highlights the boundaries between the collagen sheets, and CH2 spectroscopic contrast with CARS.
Third and second harmonic generation imaging of human articular cartilage
Author(s):
Ming-Rung Tsai;
Chih-Hwa Chen;
Chi-Kuang Sun
Show Abstract
Cartilage matrix is damaged in diseased states such as osteoarthritis, while adult articular cartilage does not have the
capacity to repair structural damage. A least invasive mean to diagnose these diseased states of human articular cartilage
with a high spatial resolution is thus highly desired. In this paper, we present our harmonic generation microscopic
studies on the human articular cartilage samples. Without any staining, third and second harmonic generation can
provide strong contrast in chondrocytes and collagen matrix, respectively. Our study indicates the high capability of
harmonic generation microscopy for future articular cartilage disease diagnosis.
Structural and molecular conformation of myosin in intact muscle fibers by second harmonic generation
Author(s):
V. Nucciotti;
C. Stringari;
L. Sacconi;
F. Vanzi;
M. Linari;
G. Piazzesi;
V. Lombardi;
F. S. Pavone
Show Abstract
Recently, the use of Second Harmonic Generation (SHG) for imaging biological samples has been explored
with regard to intrinsic SHG in highly ordered biological samples. As shown by fractional extraction of
proteins, myosin is the source of SHG signal in skeletal muscle. SHG is highly dependent on symmetries
and provides selective information on the structural order and orientation of the emitting proteins and the
dynamics of myosin molecules responsible for the mechano-chemical transduction during contraction. We
characterise the polarization-dependence of SHG intensity in three different physiological states: resting,
rigor and isometric tetanic contraction in a sarcomere length range between 2.0 μm and 4.0 μm. The
orientation of motor domains of the myosin molecules is dependent on their physiological states and
modulate the SHG signal. We can discriminate the orientation of the emitting dipoles in four different
molecular conformations of myosin heads in intact fibers during isometric contraction, in resting and rigor.
We estimate the contribution of the myosin motor domain to the total second order bulk susceptibility from
its molecular structure and its functional conformation. We demonstrate that SHG is sensitive to the
fraction of ordered myosin heads by disrupting the order of myosin heads in rigor with an ATP analog. We
estimate the fraction of myosin motors generating the isometric force in the active muscle fiber from the
dependence of the SHG modulation on the degree of overlap between actin and myosin filaments during an
isometric contraction.
Polarization-resolved second-harmonic-generation imaging of photoaged dermal collagen fiber
Author(s):
Takeshi Yasui;
Yu Takahashi;
Tsutomu Araki
Show Abstract
Polarization-resolved second-harmonic-generation (SHG) microscopy is useful for assessment of collagen fiber
orientation in tissues. In this paper, we investigated the relation between wrinkle direction and collagen orientation in
ultraviolet-B-exposed (UVB-exposed) skin using polarization-resolved SHG microscopy. A polarization anisotropic
image of the SHG light indicated that wrinkle direction in UVB-exposed skin is predominantly parallel to the orientation
of dermal collagen fibers whereas no-UVB-exposed skin was dominated by collagen orientation parallel to the meridian
line of body. The method proposed has the potential to become a powerful non-invasive tool for assessment of cutaneous
photoaging.
Imaging of collagen matrix remodeling in three-dimensional space using second harmonic generation and two photon excitation fluorescence
Author(s):
Thomas Abraham;
Jon Carthy;
Bruce McManus
Show Abstract
Second harmonic generation (SHG), a nonlinear optical phenomenon, exhibits several in-common characteristics of twophoton
excited fluorescence (TPEF) microscopy. These characteristics include identical equipment requirements from
experiment to experiment and the intrinsic capability of generating 3-dimensional (D) high resolution images. Structural
protein arrays that are highly ordered, such as collagen, produce strong SHG signals without the need for any exogenous
label (stain). SHG and TPEF can be used together to provide information on structural rearrangements in 3D space of the
collagen matrix associated with various physiological processes. In this study, we used SHG and TPEF to detect cellmediated
structural reorganization of the extracellular collagen matrix in 3D space triggered by dimensional changes of
embedded fibroblasts. These fibroblasts were cultured in native type I collagen gels and were stimulated to contract for a
period of 24 hours. The gels were stained for cell nuclei with Hoechst and for actin with phalloidin conjugated to Alexa
Fluor 488. We used non-de-scanned detectors and spectral scanning mode both in the reflection geometry for generating
the 3D images and for SHG spectra, respectively. We used a tunable infrared laser with 100-fs pulses at a repetition rate
of 80-MHz tuned to 800-nm for Hoechst and Alexa 488 excitations. We employed a broad range of excitation
wavelengths (800 to 880-nm) with a scan interval of 10 nm to detect the SHG signal. We found that spectrally clean
SHG signal peaked at 414-nm with excitation wavelength of 830-nm. The SHG spectrum has a full width half maximum
(FWHM) bandwidth of 6.60-nm, which is consistent with its scaling relation to FWHM bandwidth 100-fs excitation
pulses. When stimulated to contract, we found the fibroblasts to be highly elongated as well as interconnected in 2D
space, and the collagen matrix, in the form of a visibly clear fibril structure, accumulated around the cells. In the absence
of contraction, on the other hand, the cells were predominantly round in shape and no sign of collagen accumulation
around the cell was evident despite the presence of SHG signal as well as the fibrillar collagen morphology in the
collagen matrix. We here conclude that SHG in conjunction with TPEF can serve as a noninvasive method to provide
spatially resolved 3D structural reorganization of collagen matrices triggered by various physiological processes.
Stimulated Raman scattering microscopy for biomedical imaging
Author(s):
Wei Min;
Christian W. Freudiger;
Sijia Lu;
Chengwei He;
Jing X. Kang;
X. Sunney Xie
Show Abstract
Label-free chemical contrast is highly desirable in biomedical imaging. Spontaneous
Raman microscopy provides specific vibrational signatures of chemical bonds, but is often
hindered by low sensitivity. Here we report a 3D multi-photon vibrational imaging
technique based on stimulated Raman scattering (SRS). The sensitivity of SRS is
significantly greater than that of spontaneous Raman scattering, and is further enhanced
by high-frequency (MHz) phase-sensitive detection. SRS microscopy has a major advantage
over previous coherent Raman techniques in that it offers
background-free and easily
interpretable chemical contrast. We show a variety of biomedical applications, such as
differentiating distributions of omega-3 fatty acids and saturated lipids in living cells,
imaging of brain and skin tissues based on intrinsic lipid contrast.
Fluorescence lifetime based contrast imaging using variable period excitation pulse trains
Author(s):
M. D. Holton;
O. F. Silvestre;
R. J. Errington;
P. J. Smith;
P. Rees;
H. D. Summers
Show Abstract
The development of an experimental setup capable of contrasting fluorescent materials by their recombinative lifetimes
in an imaging mode is discussed. Such materials might include molecular dyes and QDs. The system is comprised of a
standard upright microscope fitted with an imaging CCD, and a white light laser that illuminates a circular region within
the field of view with variable period excitation pulse trains. Different fluorescent species within this region absorb the
laser light and fluoresce with a recombination lifetime dependent on material composition and local environment.
Species with differing fluorescent lifetimes can be distinguished in an imaging mode by their contrasting intensity
response to the pulse train at the range of different pulse frequencies. The technique is discussed and applied to samples
containing both CdTe (705 nm) and CdSe (611 nm) QDs, showing contrast between long (70-100 ns) and (relatively)
short (25-35 ns) lifetime within an image.
Improving image formation from the illumination side: linear and non-linear excitation cases
Author(s):
Emiliano Ronzitti;
Francesca Cella;
Alberto Diaspro
Show Abstract
Diffraction imposes for each optical system a resolution limit which could be described by using the vectorial theory of
Richards and Wolf. This theory defines the intensity distribution of a point like source imaged by a lens assuming ideal
imaging conditions. Unfortunately, these conditions can not be completely achieved in practical situations as a recorded
microscope image is always affected by noise which makes the resolution limit worse.
In this work we propose and analyze optical set-up schemes towards an image quality improvement in terms of Signal
to Noise Ratio (SNR) in linear and non-linear fluorescence microscopy. In order to reach this purpose we insert, on the
illumination arm of the microscope, a proper amplitude ring filter inducing laterally interfering beams. The effect
induced by the filter results in a shape engineering of the 3D-PSF and in a redistribution of the spatial frequencies of the
OTF. In particular, the high frequencies information are collected at improved SNR.
In order to implement such schemes we use a computational simulation mainly based on a vectorial approach analyzing
the results in both space and frequency domain to characterize the optical system response.
Analysis reveals that, although the theoretical resolution of the system is unchanged, when we impose a certain noise
level the practical imaging quality could be improved in the ring filtering scheme. The results suggest that further
improvement can be reached by the usage of the proposed annular filers in combination with image restoration.
A comparison between linear and non-linear excitation cases is presented.
Non-linear effects and role of scattering in multiphoton imaging of thick biological samples
Author(s):
Francesca Cella;
Zeno Lavagnino;
Alberto Diaspro
Show Abstract
Non linear optical scanning microscopy has became a useful tool for living tissue imaging. Biological tissues
are highly scattering media and this leads to an exponential attenuation of the excitation intensity as the light
travels into the sample. While performing imaging of biological scattering tissues in non linear excitation regime,
the localization of the maximum 2PE intensity was found to shift closer to the surface1 and the 2PE imaging
depth limit appears strongly limited by near surface fluorescence.2 In this work we computed the illumination
and the photobleaching distribution3 in order to characterize the effects induced by scattering. The simulations
have been performed for different scattering coefficients and different focus depth. An experimental test has
been carried out by imaging, with 0.9 numerical aperture objective, thick scattering fluorescent immobile sample
(polyelectrolyte gel). Results confirm that under these conditions no photobleaching effects due to scattering
occur close to the surface.
Mechanical properties of tissue determined by multiphoton microscopy
Author(s):
Magnus B. Lilledahl;
Bjørn Skallerud;
Catharina Davies
Show Abstract
Mechanical models are important in many areas of medicine and physiological research. These are usually based
on continuum mechanics using macroscopic mechanical parameters. However, knowledge of the microscopic
structure of tissue, that is, the organization of structural proteins, gives useful information for improving such
models for a given tissue. In this paper we image the structural changes in these proteins under strain by using
multiphoton microscopy. This gives insight into the response to the tissue at microscopic level and can be used
to modify existing mechanical models of tissue. Specifically we imaged the straightening of collagen fibers in
bovine chordae tendinae and related this to the macroscopic strain applied to the tissue.
Fluorescence lifetime dynamics of eGFP in protein aggregates with expanded polyQ
Author(s):
Vladimir Ghukasyan;
Chih-Chun Hsu;
Chia-Rung Liu;
Fu-Jen Kao;
Tzu-Hao Cheng
Show Abstract
Expanding a polyglutamine (polyQ) stretch at the N-terminus of huntingtin protein is the main cause of the
neurodegenerative disorder Huntington's disease (HD). Expansion of polyQ above 39 residues has an inherent propensity
to form amyloid-like fibrils and aggregation of the mutant protein is found to be a critical component for abnormal
pathology of HD. Using yeast Saccharomyces cerevisiae as a model system, we have observed a decrease in
fluorescence lifetime of the enhanced green fluorescence protein (eGFP) fused to 97 successive glutamine residues
(97Q). Compared to the sample expressing evenly distributed eGFP, the 97Q-eGFP fusion proteins show the formation
of grain-like particles and the reduction of eGFP lifetime by ~250 ps as measured by time-correlated single-photon
counting technique (TCSPC). More importantly, this phenomenon does not appear in Hsp104-deficient cells. The gene
product of HSP104 is required for the formation of polyQ aggregates in yeast cells; therefore, the cellular 97Q-eGFP
become soluble and evenly distributive in the absence of Hsp104. Under this condition, the lifetime value of 97Q-eGFP
is close to the one exhibited by eGFP alone. The independence of the effect of the environmental parameters, such as pH
and refraction index is demonstrated. These data indicate that the fluorescence lifetime dynamics of eGFP is linked to the
process of polyQ protein aggregation per se.
Lipids distribution imaging of lipid vesicles by multi-focus excitation CARS microscope
Author(s):
Takeo Minamikawa;
Tsutomu Araki;
Mamoru Hashimoto
Show Abstract
We demonstrated high-speed imaging of the distribution of DPPC (dipalmitoylphosphatidylcholine), d62-DPPC (deuterated DPPC), and DOPC (dioleoylphosphatidylcholine) lipids in a lipid vesicle with a multi-focus excitation CARS (coherent anti-Stokes Raman scattering) microscope using a microlens array scanner. By the multi-focus excitation, the dwell time is increased in proportion to the number of focal spots compared with a single beam scanning, and high-speed and high-quality CARS imaging is possible without increasing the peak power of each spot. We demonstrated the selectively visualization of DPPC and d62-DPPC lipid vesicles, in which the vesicles contain a type of lipid, by observing at 2840 cm-1 and 2090 cm-1. We also visualized the DOPC and DPPC lipids distribution in a lipid mixture vesicle observed at 1440 cm-1 and 1655 cm-1. The image acquisition time of 10 s/image at each Raman shift was realized. The signal ratio of 1440 cm-1 and 1655 cm-1 was locally intense on the lipid vesicle. It must be because the gel phase domain of DPPC lipids was exists in the DOPC lipids which were liquid-crystalline phase at room temperature.
Optical detection of concentrations for mixed acid: HF and HNO3
Author(s):
Gumin Kang;
Kyoungsik Kim
Show Abstract
Mixed acid, which consist of HF and HNO3, is used as a good etchant for silicon dioxide in the wet etching and pickling
process of stainless steel. The optical detection of concentration for such mixed acids is crucial to optimize and cut costs
in the manufacturing process. Optical detection in the IR regime has been utilized to measure the concentration of the
mixed acid for HF and HNO3, because that has several strong absorption peaks, which is contributed by vibrational mode
of each acid molecular in this spectrum. In this research, we observed the concentrations of mixed acid to consist of HF
and HNO3, as we measured the absorption intensity of OH- stretch and NO3
- stretch band by optical spectroscopy. The
concentration range of HF over 1.5-3 wt% and that of HNO3 over 2-10 wt% were studied in room temperature.
Coherent control in multiphoton fluorescence imaging
Author(s):
Arijit Kumar De;
Debabrata Goswami
Show Abstract
In multiphoton fluorescence laser-scanning microscopy ultrafast laser pulses, i.e. light pulses having pulse-width ≤
1picosecond (1 ps = 10-12 s), are commonly used to circumvent the low multiphoton absorption cross-sections of
common fluorophores. Starting with a discussion on how amplitude modulation of ultrashort pulse-train enhances the
two-photon fluorescence providing deep insight into laser-induced photo-thermal damage, the effect of controlling time
lag between phase-locked laser pulses on imaging is described. In addition, the prospects of laser pulse-shaping in signal
enhancement (by temporal pulse-compression at the sample) and selective excitation of fluorophores (by manipulating
the phase and/or amplitude of different frequency components within the pulse) are discussed with promising future
applications lying ahead.
High-resolution wavefront correction in multiphoton microscopy
Author(s):
G. Hall;
M. Ren;
W. B. Amos;
K. W. Eliceiri;
J. G. White
Show Abstract
A system for making wavefront corrections for use in multiphoton microscopy has been constructed. Corrections are
made using a high-resolution nematic liquid crystal device which has a phase stroke of 2π. The device has a design
wavelength of 1064 nm. A simple way for setting the device up for lower wavelengths (here 800 nm) is presented. It
was found that the device has an undesired zero-order diffraction component of 30%. A scheme for filtering this portion
out is presented and it was demonstrated that this can eliminate the component completely. The device was used to
optically simulate a thin lens with a specified focal length, which was found to match within error bounds. Finally the
modulator was used to compensate for a mechanical defocus that was applied intentionally.
Comparison of two-photon imaging depths with 775 nm excitation and 1300 nm excitation
Author(s):
Demirhan Kobat;
Angela Wong;
Chris B. Schaffer;
Chris Xu
Show Abstract
We quantitatively compared the maximal two-photon fluorescence microscopy imaging depth achieved with 775 nm
excitation to that achieved with 1300 nm excitation through ex-vivo TPM of blood vessels in the mouse brain. We
achieved high contrast imaging of labeled blood vessels at approximately twice the depth at 1300 nm excitation as at 775
nm excitation. We also measured the two-photon excitation
cross-sections of several commercially available
fluorophores at 1220-1320 nm. We found that some of these fluorophores reveal comparable if not better cross-section
values than those of the widely used dyes excited by shorter wavelength light.
Localization of protein-protein interactions among three fluorescent proteins in a single living cell: three-color FRET microscopy
Author(s):
Yuansheng Sun;
Cynthia F. Booker;
Richard N. Day;
Ammasi Periasamy
Show Abstract
Förster resonance energy transfer (FRET) methodology has been used for over 30 years to localize protein-protein
interactions in living specimens. The cloning and modification of various visible fluorescent proteins (FPs) has generated
a variety of new probes that can be used as FRET pairs to investigate the protein associations in living cells. However,
the spectral cross-talk between FRET donor and acceptor channels has been a major limitation to FRET microscopy.
Many investigators have developed different ways to eliminate the bleedthrough signals in the FRET channel for one
donor and one acceptor. We developed a novel FRET microscopy method for studying interactions among three
chromophores: three-color FRET microscopy. We generated a genetic construct that directly links the three
FPs - monomeric teal FP (mTFP), Venus and tandem dimer Tomato (tdTomato), and demonstrated the occurrence of mutually
dependent energy transfers among the three FPs. When expressed in cells and excited with the 458 nm laser line, the
mTFP-Venus-tdTomato fusion proteins yielded parallel (mTFP to Venus and mTFP to tdTomato) and sequential (mTFP
to Venus and then to tdTomato) energy transfer signals. To quantify the FRET signals in the three-FP system in a single
living cell, we developed an algorithm to remove all the spectral cross-talk components and also to separate different
FRET signals at a same emission channel using the laser scanning spectral imaging and linear unmixing techniques on
the Zeiss510 META system. Our results were confirmed with fluorescence lifetime measurements and using acceptor
photobleaching FRET microscopy.
FRET imaging of multiple focal planes to analyze the organization and conformation of transferrin-receptor in polarized cells
Author(s):
Horst Wallrabe;
Ammasi Periasamy;
Margarida Barroso
Show Abstract
We developed a FRET-based assay to analyze multiple focal planes from z-staks collected using confocal imaging of polarized epithelial MDCK cells. To establish the imaging assay for multiple focal planes, we have used fixed cells, where changes in the organization and conformation of transferrin-receptor complexes between endosomes are expected to be minimized. Therefore, we indeed only see minor changes in E% throughout the endocytic pathway of polarized cells. In the future, we will apply this FRET-based assay to live polarized epithelial cells to investigate the factors involved in the regulation of the organization and conformation of membrane-bound receptors during endocytic trafficking.
Angiotensin II-induced angiotensin II type I receptor lysosomal degradation studied by fluorescence lifetime imaging microscopy
Author(s):
Hewang Li;
Peiying Yu;
Robin A. Felder;
Ammasi Periasamy;
Pedro A. Jose M.D.
Show Abstract
Upon activation, the angiotensin (Ang) II type 1 receptor (AT1Rs) rapidly undergoes endocytosis. After a series of
intracellular processes, the internalized AT1Rs recycle back to the plasma membrane or are trafficked to proteasomes or
lysosomes for degradation. We recently reported that AT1Rs degrades in proteasomes upon stimulation of the D5
dopamine receptor (D5R) in human renal proximal tubule and HEK-293 cells. This is in contrast to the degradation of
AT1R in lysosomes upon binding Ang II. However, the dynamic regulation of the AT1Rs in lysosomes is not well
understood. Here we investigated the AT1Rs lysosomal degradation using FRET-FLIM in HEK 293 cells heterologously
expressing the human AT1R tagged with EGFP as the donor fluorophore. Compared to its basal state, the lifetime of
AT1Rs decreased after a 5-minute treatment with Ang II treatment and colocalized with Rab5 but not Rab7 and LAMP1.
With longer Ang II treatment (30 min), the AT1Rs lifetime decreased and co-localized with Rab5, as well as Rab7 and
LAMP1. The FLIM data are corroborated with morphological and biochemical co-immunoprecipitation studies. These
data demonstrate that Ang II induces the internalization of AT1Rs into early sorting endosomes prior to trafficking to late
endosomes and subsequent degradation in lysosomes.
Fluorescent nanodiamonds for FRET-based monitoring of a single biological nanomotor FoF1-ATP synthase
Author(s):
M. Börsch;
R. Reuter;
G. Balasubramanian;
R. Erdmann;
F. Jelezko;
J. Wrachtrup
Show Abstract
Color centers in diamond nanocrystals are a new class of fluorescence markers that attract significant interest due to
matchless brightness, photostability and biochemical inertness. Fluorescing diamond nanocrystals containing defects
can be used as markers replacing conventional organic dye molecules, quantum dots or autofluorescent proteins. They
can be applied for tracking and ultrahigh-resolution localization of the single markers. In addition the spin properties of
diamond defects can be utilized for novel magneto-optical imaging (MOI) with nanometer resolution. We develop this
technique to unravel the details of the rotary motions and the elastic energy storage mechanism of a single biological
nanomotor FoF1-ATP synthase. FoF1-ATP synthase is the enzyme that provides the 'chemical energy currency' adenosine
triphosphate, ATP, for living cells. The formation of ATP is accomplished by a stepwise internal rotation of subunits
within the enzyme. Previously subunit rotation has been monitored by single-molecule fluorescence resonance energy
transfer (FRET) and was limited by the photostability of the fluorophores. Fluorescent nanodiamonds advance these
FRET measurements to long time scales.
A setup for combined multiphoton laser scanning microscopic and multi-electrode patch clamp experiments on brain slices
Author(s):
P. Johannes Helm;
Trond Reppen;
Paul Heggelund
Show Abstract
Multi Photon Laser Scanning Microscopy (MPLSM) appears today as one of the most powerful experimental tools in
cellular neurophysiology, notably in studies of the functional dynamics of signal processing in single neurons.
Simultaneous recording of fluorescence signals at high spatial and temporal resolution and electric signals by means of
multi electrode patch clamp techniques have provided new paths for the systematic investigation of neuronal
mechanisms. In particular, this approach has opened for direct studies of dendritic signal processing in neurons.
We report about a setup optimized for simultaneous electrophysiological multi electrode patch clamp and multi photon
laser scanning fluorescence microscopic experiments on brain slices.
The microscopic system is based on a modified commercially available confocal scanning laser microscope (CLSM).
From a technical and operational point of view, two developments are important:
Firstly, in order to reduce the workload for the experimentalist, who in general is forced to concentrate on controlling the
electrophysiological parameters during the recordings, a system of shutters has been installed together with dedicated
electronic modules protecting the photo detectors against destructive light levels caused by erroneous opening or closing
of microscopic light paths by the experimentalist.
Secondly, the standard detection unit has been improved by installing the photomultiplier tubes (PMT) in a Peltier cooled
thermal box shielding the detector from both room temperature and distortions caused by external electromagnetic fields.
The electrophysiological system is based on an industrial standard multi patch clamp unit ergonomically arranged around
the microscope stage.
The electrophysiological and scanning processes can be time coordinated by standard trigger electronics.
Transient state microscopy: a new tool for biomolecular imaging
Author(s):
Tor Sandén;
Gustav Persson;
Jerker Widengren
Show Abstract
Photoinduced transient dark states are exhibited by practically all common fluorophores. However, their information
content has to date only been sparsely exploited due to methodological constraints. Here, a new concept is presented and
verified that can monitor and image these states via their photodynamic fingerprints. It unites the environmental
sensitivity of these states with the sensitivity of
fluorescence-based detection. For demonstration, triplet state images of
liposomes in different environments were generated, showing how local environmental differences can be resolved, not
clearly distinguishable via other fluorescence parameters. The concept can provide several new, useful and independent
fluorescence-based parameters in biomolecular imaging.
In vivo and in vitro investigations of retinal fluorophores in age-related macular degeneration by fluorescence lifetime imaging
Author(s):
M. Hammer;
S. Quick;
M. Klemm;
S. Schenke;
N. Mata;
A. Eitner;
D. Schweitzer
Show Abstract
Ocular fundus autofluorescence imaging has been introduced into clinical diagnostics recently for the observation of the
age pigment lipofuscin, a precursor of age-related macular degeneration (AMD). However, a deeper understanding of
the generation of single compounds contributing to the lipofuscin as well as of the role of other fluorophores such as
FAD, glycated proteins, and collagen needs their discrimination by fluorescence lifetime imaging (FLIM).
FLIM at the ocular fundus is performed using a scanning laser ophthalmoscope equipped with a picosecond laser source
(448nm or 468nm respectively, 100ps, 80 MHz repetition rate) and dual wavelength (490-560nm and 560-7600nm)
time-correlated single photon counting. A three-exponential fit of the fluorescence decay revealed associations of
decay times to anatomical structures. Disease-related features are identified from alterations in decay times and-amplitudes.
The in-vivo investigations in patients were paralleled by experiments in an organ culture of the porcine ocular fundus.
Photo-oxidative stress was induced by exposure to blue light (467nm, 0.41 mW/mm2). Subsequent analysis
(fluorescence microscopy, HPLC, LC-MS) indicated the accumulation of the pyridinium bis-retinoid A2E and its
oxidation products as well as oxidized phospholipids. These compounds contribute to the tissue auto-fluorescence and
may play a key role in the pathogenesis of AMD. Thus, FLIM observation at the ocular fundus in vivo enhances our
knowledge on the etiology of AMD and may become a diagnostic tool.
Dispersion control considerations for multiphoton and non-linear laser microscopy applications
Author(s):
C. Brideau;
P. Stys
Show Abstract
As multiphoton microscopy increases in popularity users with diverse backgrounds are exploring new applications for
the technique. With the most recent 'turnkey' systems now on the market a typical user no longer has to be a laser
physicist or engineer to employ a multiphoton system in their research. However, some basic understanding of the
mechanisms of non-linear excitation will allow a user to optimize his multiphoton system for improved performance or
extend it for new applications.
Multiphoton microscopy as a diagnostic tool for pathological analysis of sentinel lymph nodes
Author(s):
J. Lemiere;
J. Douady;
F. Estève;
D. Salameire;
S. Lantuejoul;
P. Lorimier;
C. Ricard;
B. van der Sanden;
J.-C. Vial
Show Abstract
Multiphoton microscopy has shown a powerful potential for biomedical in vivo and ex vivo analysis of tissue sections
and explants. Studies were carried out on several animal organs such as brain, arteries, lungs, and kidneys. One of the
current challenges is to transfer to the clinic the knowledge and the methods previously developed in the labs at the
preclinical level.
For tumour staging, physicians often remove the lymph nodes that are localized at the proximity of the lesion. In case of
breast cancer or melanoma, sentinel lymph node protocol is performed: pathologists randomly realize an extensive
sampling of formol fixed nodes. However, the duration of this protocol is important and its reliability is not always
satisfactory.
The aim of our study was to determine if multiphoton microscopy would enable the fast imaging of lymph nodes on
important depths, with or without exogenous staining. Experiments were first conducted on pig lymph nodes in order to
test various dyes and to determine an appropriate protocol. The same experiments were then performed on thin slices of
human lymph nodes bearing metastatic melanoma cells. We obtained relevant images with both endofluorescence plus
second-harmonic generation and xanthene dyes. They show a good contrast between tumour and healthy cells.
Furthermore, images of pig lymph nodes were recorded up to 120μm below the surface. This new method could then
enable a faster diagnosis with higher efficiency for the patient. Experiments on thicker human lymph nodes are currently
underway in order to validate these preliminary results.
Ultra-compact (palm-top size) low-cost maintenance-free (>3000 h) diode-pumped femtosecond (160 fs) solid state laser source for multiphoton microscopy
Author(s):
S. Yamazoe;
M. Katou;
T. Kasamatsu
Show Abstract
We propose and demonstrate a novel practical femtosecond laser source, which is to our knowledge, the smallest size
and potentially low cost. The innovation is the simple linear cavity design utilizing soliton mode-locking induced by
precise group velocity dispersion control. Average output power of 680 mW and pulse width of 162 fs were obtained at
around 1045 nm from a diode-pumped Yb3+:KY(WO4)2 laser. The pulse repetition rate was 2.8 GHz, leading to a pulse
peak power of 1.5 kW, which is sufficient for biomedical imaging. The laser module including the laser diode pump
system has a footprint of 8×4 cm2. Under automatic current control condition, stable operation of 3000-hour was
observed with an average power fluctuation of less than ±10 %. Furthermore, under automatic power control condition,
stable operation of 2000-hour was observed with an average power fluctuation of less than ±1 %. Using this laser
module, we successfully obtained clear two-photon fluorescence images of muntjac skin fibroblast cells stained with a
combination of fluorescent stains (Alexa Fluor 488 phalloidin and Alexa Flour 555 goat anti-mouse lgG).
SHIM and TPEM: getting more information from non linear excitation
Author(s):
Paolo Bianchini;
Alberto Diaspro
Show Abstract
Several endogenous protein structures give rise to second harmonic generation (SHG) - second order nonabsorbative
energy doubling of an excitation laser line. The orientation of collagen fibers within tissues such as tendons or ligaments
is of primary importance. In this study, we propose a method to map the orientation of collagen fibers of a tendon. The
method uses only few images acquired at specific polarizations of the input laser beam by rotating the sample on the
stage. This procedure is implemented both in backward and forward scattering pathways. The improved details should
clarify the backscattered signal nature.