CARS and SHG microscopy of artificial bioengineered tissues
Author(s):
Annika Enejder;
Christian Brackmann;
Jan-Olof Dahlberg;
Engin Vrana;
Paul Gatenholm
Show Abstract
Major efforts are presently made to develop artificial replacement tissues with optimal architectural and
material characteristics, mimicking those of their natural correspondents. Encouraged by the readiness with
which cellulose fibers woven by the bacteria Acetobacter xylinum can be formed into organ-like macroscopic
shapes and with different microscopic textures, it emerges as an interesting material within tissue engineering.
We have developed a protocol employing simultaneous CARS and SHG microscopy for monitoring the
cellulose network characteristics and its impact on the integration of smooth muscle cells (SMCs) for
functionalized artificial tissues. CARS and SHG overlay images of the cells and the cellulose fibers reveal an
immediate interaction irrespective of scaffold morphology and that the SMCs attach to the cellulose fibers
already during the first cultivation day without cell-adhesive coatings. During the subsequent 28 days, SMCs
were found to readily proliferate and differentiate on the cellulose scaffold without the need for exogenous
growth factors. However, the efficiency with which this occurred depended on the topography of the cellulose
constructs, benefited by porous and less compact matrices. This brings forward the need for in-depth studies
on how the microstructure of tissue scaffolds influences and can be optimized for native cell integration and
proliferation, studies where the benefits of multi-modal non-linear microscopy can be fully exploited.
High performance multimodal CARS microscopy using a single femtosecond source
Author(s):
Adrian F. Pegoraro;
Aaron D. Slepkov;
Andrew Ridsdale;
Rodney K. Lyn;
Douglas J. Moffatt;
John Paul Pezacki;
Brian K. Thomas;
Libin Fu;
Liang Dong;
Martin E. Fermann;
Albert Stolow
Show Abstract
Traditional CARS microscopy using picosecond (ps) lasers has been applied to a wide variety of applications;
however, the lasers required are expensive and require an environmentally stable lab. In our work, we demonstrate
CARS microscopy using a single femtosecond (fs) laser combined with a photonic crystal fiber (PCF) and optimal
chirping to achieve similar performance to the ps case with important added advantages: fs-CARS utilizes
versatile source that allows CARS to be combined with other multiphoton techniques (e.g. SHG, TPF) for
multimodal imaging without changing laser sources. This provides an attractive entry point for many researchers
to the field. Furthermore, optimal chirping in fs-CARS also opens the door to the combination and extension
of other techniques used in ps CARS microscopy such as multiplex and FM imaging. The key advantage with
chirped fs pulses is that time delay corresponds to spectral scanning and allows for rapid modulation of the
resonant CARS signal. The combination of a fs oscillator with a PCF leads to a natural extension of the
technology towards an all-fiber source for multimodal multiphoton microscopy. An all-fiber system should be
more robust against environmental fluctuations while being more compact than free-space systems. We have
constructed and demonstrated a proof of concept all-fiber based source that can be used for simultaneous CARS,
TPF and SHG imaging. This system is capable of imaging tissue samples and live cell cultures with 4 μs/pixel
dwell time at low average powers.
Frequency modulation coherent anti-Stokes Raman scattering (FM-CARS) microscopy based on spectral focusing of chirped laser pulses
Author(s):
Bi-Chang Chen;
Jiha Sung;
Sang-Hyun Lim
Show Abstract
We demonstrate a new CARS microscopy method based on fast switching of effective vibrational excitation frequency
from chirped femtosecond laser pulses. Broadband pump and Stokes pulses excite a single vibrational mode with a high
spectral resolution when the two pulses are identically chirped and their pulse durations are approaching the dephasing
time of the excited vibrational state. This "spectral focusing" mechanism is applied to CARS microscopy with a single
broadband Ti:Sapphire laser. The vibrational excitation frequency is controlled simply by the time delay between the
pump and Stokes pulses and fast switching of the excitation frequency (~100 kHz) is achieved with a Pockels cell and
polarization optics. Lock-in detection of the difference between the two CARS signals at nearby vibrational frequencies
not only eliminates the non-resonant background but also generates a spectral line shape similar to the spontaneous
Raman scattering. We demonstrate both micro-spectroscopy and vibrational imaging with various samples.
Vibrational phase contrast CARS microscopy for quantitative analysis
Author(s):
M. Jurna;
E. T. Garbacik;
J. P. Korterik;
C. Otto;
J. L. Herek;
H. L. Offerhaus
Show Abstract
In biological samples the resonant CARS signal of less abundant constituents can be overwhelmed by the nonresonant
background, preventing detection of those molecules. We demonstrate a method to obtain the phase of
the oscillators in the focal volume that allows discrimination of those hidden molecules. The phase is measured
with respect to the local excitation fields using a cascaded
phase-preserving chain. It is measured point-bypoint
and takes into account refractive index changes in the sample, phase curvature over the field-of-view and
interferometric instabilities. The detection of the phase of the vibrational motion can be regarded as a vibrational
extension of the linear (refractive index) phase contrast microscopy introduced by Zernike around 1933.
A CARS solution with high temporal resolution
Author(s):
Vanessa Lurquin;
William C. Hay;
Stefanie Landwehr;
Vishnu Krishnamachari
Show Abstract
Confocal and multiphoton microscopy are powerful fluorescence techniques for morphological and dynamics studies of
labeled elements. For non-fluorescent components, CARS (Coherent Anti-Stokes Raman Scattering) microscopy can be
used for imaging various elements of cells such as lipids, proteins, DNA, etc. This technique is based on the intrinsic
vibrational properties of the molecules. Leica Microsystems has combined CARS technology with its TCS SP5 II
confocal microscope to provide several advantages for CARS imaging. The Leica TCS SP5 II combines two
technologies in one system: a conventional scanner for maximum resolution and a resonant scanner for high time
resolution. For CARS microscopy, two picosecond near-infrared lasers are tightly overlapped spatially and temporally
and sent directly into the confocal system. The conventional scanner can be used for morphological studies and the
resonant scanner for following dynamic processes of unstained living cells. The fast scanner has several advantages over
other solutions. First, the sectioning is truly confocal and does not suffer from spatial leakage. Second, the high speed
(29 images/sec @ 512×512 pixels) provides fast data acquisition at video rates, allowing studies at the sub-cellular level.
In summary, CARS microscopy combined with the tandem scanner makes the Leica TCS SP5 II a powerful tool for
multi-modal and three-dimensional imaging of chemical and biological samples. We will present our solution and show
results from recent studies with the Leica instrument to illustrate the high flexibility of our system.
Nonlinear Raman microscopy: improving detection through nonlinear optical interaction
Author(s):
G. I. Petrov;
V. V. Yakovlev
Show Abstract
Raman spectroscopy based on spontaneous scattering cannot compete with nonlinear Raman spectroscopy, when the
sample volume is large enough to take advantage of the coherent nature of nonlinear optical interactions. However, when
the interaction volume is small, the number of optical photons generated through spontaneous Raman scattering can be
larger than the number of photons generated by coherent Raman process. In this work, the signal-to-noise ratio for
Raman spectroscopy is carefully evaluated, and, by optically amplifying the weak Raman signal, Raman spectra, free of
background noise are achieved.
Comparing coherent and spontaneous Raman scattering signals for biological imaging applications
Author(s):
Brandon R. Bachler;
Meng Cui;
Sarah R. Nichols;
Jennifer P. Ogilvie
Show Abstract
We present a systematic comparison between coherent and spontaneous Raman scattering under conditions
relevant for biological imaging. Using spectral domain imaging, we find that the signal levels for each method
are comparable at the low excitation power and low concentrations appropriate for biological samples. For
samples of polystyrene beads with a molecular concentration of 10 M, we determine the critical power at which
the two methods give equal signal levels to be ~1.3 mW. The advantages offered by coherent Raman methods
are mitigated by the low excitation power, low sample concentrations, and short interaction lengths involved
with biological imaging. We present calculations to support our measurements.
Polarization-resolved coherent anti-Stokes Raman scattering microscopy
Author(s):
Fabiana Munhoz;
Sophie Brustlein;
Sophie Brasselet;
Hervé Rigneault
Show Abstract
We have implemented a polarization-resolved coherent anti-Stokes Raman scattering (CARS) microscopy, based
on the continuous variation of the incident linear polarization at the pump and Stokes wavelengths, together
with a polarized analysis of the anti-Stokes signal. In isotropic media, such as solutions, this technique can
be a powerful way to probe microscopic-scale information, such as the vibrational symmetry properties of the
molecular bonds. In ordered media, additional macroscopic-scale structural information can be obtained, such
as the orientation of the unit-cell of a crystal in 3D.
High-speed CARS spectral imaging using acousto optic tunable filter
Author(s):
Mamoru Hashimoto;
Takeo Minamikawa;
Tsutomu Araki
Show Abstract
We developed a high speed CARS (coherent anti-Stokes Raman scattering) spectral-imaging system using an
acousto optic tunable filter and multi-focus excitation system. We compared two methods of CARS emission
filtering and CARS excitation filtering. In both case, two laser pulses with narrow band (picosecond laser) and
broad band (femotosecond laser)were used for the light source. For CARS emission filtering, the generated CARS
was filtered by an AOTF, and for excitation filtering the broad band femtosecond laser pulse were filtered by an
AOTF before excitation. The experimental results indicated that the CARS emission filtering was suitable for
CARS microscopy.
Better FLIM and FCS data by GaAsP hybrid detectors
Author(s):
Wolfgang Becker;
Bertram Su;
Axel Bergmann
Show Abstract
The principle of the hybrid PMT is known for about 15 years: Photoelectrons emitted by a photocathode are accelerated
by a strong electrical field, and directly injected into an avalanche diode chip. Until recently, the gain of hybrid PMTs
was too low for picosecond-resolution photon counting. Now devices are available that reach a total gain of a few
100,000, enough to detect single photons at ps resolution. Compared with conventional PMTs, multi-channel PMTs, and
SPADs (single-photon avalanche photodiodes) hybrid PMTs have a number of advantages: With a modern GaAsP
cathode the detection quantum efficiency reaches the efficiency of a SPAD. However, the active area is on the order of
5 mm2, compared to 2.5 10-3 mm2 for a SPAD. A hybrid PMT can therefore be used for non-descanned detection in a
multiphoton microscope. The TCSPC response is clean, without the bumps typical for PMTs, and without the diffusion
tail typical for SPADs. Most important, the hybrid PMT is free of afterpulsing. So far, afterpulsing has been present in
all photon counting detectors. It causes a signal-dependent background in FLIM measurements, and a typical
afterpulsing peak in FCS. With a hybrid PMT, FLIM measurements reach a much higher dynamic range. Clean FCS
data are obtained from a single detector. Compared to cross-correlation of the signals of two detectors an increase in
FCS efficiency by a factor of four is obtained. We demonstrate the performance of the new detector for a number of
applications.
pH and chloride recordings in living cells using two-photon fluorescence lifetime imaging microscopy
Author(s):
Mattes Lahn;
Carsten Hille;
Felix Koberling;
Peter Kapusta;
Carsten Dosche
Show Abstract
Today fluorescence lifetime imaging microscopy (FLIM) has become an extremely powerful technique in life sciences.
The independency of the fluorescence decay time on fluorescence dye concentration and emission intensity circumvents
many artefacts arising from intensity based measurements. To minimize cell damage and improve scan depth, a
combination with two-photon (2P) excitation is quite promising.
Here, we describe the implementation of a 2P-FLIM setup for biological applications. For that we used a commercial
fluorescence lifetime microscope system. 2P-excitation at 780nm was achieved by a non-tuneable, but inexpensive and
easily manageable mode-locked fs-fiber laser. Time-resolved fluorescence image acquisition was performed by
objective-scanning with the reversed time-correlated single photon counting (TCSPC) technique.
We analyzed the suitability of the pH-sensitive dye BCECF and the chloride-sensitive dye MQAE for recordings in an
insect tissue. Both parameters are quite important, since they affect a plethora of physiological processes in living
tissues. We performed a straight forward in situ calibration method to link the fluorescence decay time with the
respective ion concentration and carried out spatially resolved measurements under resting conditions. BCECF still
offered only a limited dynamic range regarding fluorescence decay time changes under physiologically pH values.
However, MQAE proofed to be well suited to record chloride concentrations in the physiologically relevant range.
Subsequently, several chloride transport pathways underlying the intracellular chloride homeostasis were investigated
pharmacologically.
In conclusion, 2P-FLIM is well suited for ion detection in living tissues due to precise and reproducible decay time
measurements in combination with reduced cell and dye damages.
Regulatory assembly of the vacuolar proton pump VoV1-ATPase in yeast cells by FLIM-FRET
Author(s):
Stefan Ernst;
Claire Batisse;
Nawid Zarrabi;
Bettina Böttcher;
Michael Börsch
Show Abstract
We investigate the reversible disassembly of VOV1-ATPase in life yeast cells by time resolved confocal FRET
imaging. VOV1-ATPase in the vacuolar membrane pumps protons from the cytosol into the vacuole. VOV1-ATPase is
a rotary biological nanomotor driven by ATP hydrolysis. The emerging proton gradient is used for secondary
transport processes as well as for pH and Ca2+ homoeostasis in the cell. The activity of the VOV1-ATPase is regulated
through assembly / disassembly processes. During starvation the two parts of VOV1-ATPase start to disassemble.
This process is reversed after addition of glucose. The exact mechanisms are unknown. To follow the disassembly /
reassembly in vivo we tagged two subunits C and E with different fluorescent proteins. Cellular distributions of C
and E were monitored using a duty cycle-optimized alternating laser excitation scheme (DCO-ALEX) for time
resolved confocal FRET-FLIM measurements.
Spectrally resolved fluorescence lifetime imaging: new developments and applications
Author(s):
A. Rueck;
F. Dolp;
B. v. Einem;
C. A. F. v. Arnim;
D. Strat
Show Abstract
The fluorescence lifetime of different molecular species is calculated from the measured fluorescence intensity decrease
following short pulsed laser excitation, by a multi-channel fitting procedure. In a FRET (Förster Resonant Energy
Transfer) experiment the time dependent behaviour of the donor profile is assumed in a first view mono-exponential and
the acceptor decay profile is solved analytically. A global minimization fitting algorithm has increased information
content than a single channel fitting routine. In a normal FRET-FLIM experiment, the efficiency of FRET is calculated
only by considering the kinetics of the donor. However, as will be shown, a considerable improvement could be achieved
when time-resolved and spectral-resolved techniques are simultaneously incorporated.
Comparison of FRET microscopy imaging techniques for studying protein-protein interactions in living cells using FRET standards
Author(s):
Yuansheng Sun;
Soo-Ah Seo;
Sydney Provence;
Ammasi Periasamy
Show Abstract
Förster resonance energy transfer (FRET) microscopy is a powerful tool to localize protein-protein
interactions in living specimens. Various FRET microscopy imaging techniques have been established
and are generally categorized into intensity-based and lifetime-based methods. Based on the detection of
the acceptor sensitized emission, we have developed the FRET imaging methodologies that can be
applied in combination of wide-field, conventional confocal or spectral microscopy. All these FRET
microscopy methods have the capability to interpret the change in proximity between the donor and the
acceptor through measuring the apparent energy transfer efficiency (E). However, to answer what subtle
change of E can be detected and to link correctly FRET data to biological information, the imaging
techniques have to be well calibrated. In this regard, we compare various FRET microscopy methods and
assess their utilities using several genetic ("FRET standard") constructs where Cerulean and Venus
fluorescent proteins are tethered by different amino acid linkers.
In vivo stoichiometry monitoring of G protein coupled receptor oligomers using spectrally resolved two-photon microscopy
Author(s):
M. R. Stoneman;
D. R. Singh;
V. Raicu
Show Abstract
Resonance Energy Transfer (RET) between a donor molecule in an electronically excited state and an acceptor molecule
in close proximity has been frequently utilized for studies of protein-protein interactions in living cells. Typically, the
cell under study is scanned a number of times in order to accumulate enough spectral information to accurately
determine the RET efficiency for each region of interest within the cell. However, the composition of these regions may
change during the course of the acquisition period, limiting the spatial determination of the RET efficiency to an average
over entire cells. By means of a novel spectrally resolved two-photon microscope, we were able to obtain a full set of
spectrally resolved images after only one complete excitation scan of the sample of interest. From this pixel-level
spectral data, a map of RET efficiencies throughout the cell is calculated. By applying a simple theory of RET in
oligomeric complexes to the experimentally obtained distribution of RET efficiencies throughout the cell, a single
spectrally resolved scan reveals stoichiometric and structural information about the oligomer complex under study. This
presentation will describe our experimental setup and data analysis procedure, as well as an application of the method to
the determination of RET efficiencies throughout yeast cells (S. cerevisiae) expressing a G-protein-coupled receptor,
Sterile 2 α factor protein (Ste2p), in the presence and absence of α-factor - a yeast mating pheromone.
New strategies to measure intracellular sodium concentrations
Author(s):
Sascha Dietrich;
Sarmiza E. Stanca;
Charles G. Cranfield;
Birgit Hoffmann;
Klaus Benndorf;
Christoph Biskup
Show Abstract
Fluorescent ion indicators are widely used to measure ion concentrations in living cells. However, despite considerable
efforts in synthesizing new compounds, no ratiometric sodium indicator is available that can be excited at visible
wavelengths. Ratiometric indicators have an advantage in that measured fluorescence intensities can be corrected for
fluctuations of the indicator concentration and the illumination intensity, which is not possible when non-ratiometric
indicators are used. One way to circumvent this problem is to measure fluorescence lifetimes, which are independent of
these factors. Another way to overcome the disadvantages of a
non-ratiometric indicator dye is to embed it, together with
a reference dye, into nanoparticles. By relating the indicator fluorescence to the fluorescence of the reference dye,
inhomogeneities in the nanosensor concentration or the illumination intensity can be cancelled out reliably. In this study
we compare the benefits and drawbacks of these approaches.
Current developments in clinical multiphoton tomography
Author(s):
Karsten König;
Martin Weinigel;
Hans Georg Breunig;
Axel Gregory;
Peter Fischer;
Marcel Kellner-Höfer;
Rainer Bückle
Show Abstract
Two-photon microscopy has been introduced in 1990 [1]. 13 years later, CE-marked clinical multiphoton systems for 3D
imaging of human skin with subcellular resolution have been launched by the JenLab company with the tomograph
DermaInspectTM. In 2010, the second generation of clinical multiphoton tomographs was introduced. The novel mobile
multiphoton tomograph MPTflexTM, equipped with a flexible articulated optical arm, provides an increased flexibility and
accessibility especially for clinical and cosmetical examinations. The multiphoton excitation of fluorescent biomolecules
like NAD(P)H, flavins, porphyrins, elastin, and melanin as well as the second harmonic generation of collagen is induced
by picojoule femtosecond laser pulses from an tunable turn-key near infrared laser system. The ability for rapid highquality
image acquisition, the user-friendly operation of the system, and the compact and flexible design qualifies this
system to be used for melanoma detection, diagnostics of dermatological disorders, cosmetic research, and skin aging
measurements as well as in situ drug monitoring and animal research. So far, more than 1,000 patients and volunteers
have been investigated with the multiphoton tomographs in Europe, Asia, and Australia.
Long-term marker-free multiphoton imaging, targeted transfection, optical cleaning of stem cell clusters, and optical transport of microRNA with extreme ultrashort laser pulses
Author(s):
Aisada Uchugonova;
Zeno Földes-Papp;
Gerhard M. Kostner;
Karsten König
Show Abstract
The novel utrashort femtosecond laser scanning microscope FemtOgene (JenLab GmbH, Germany) with 12
femtoseconds at the focal plane have been employed in marker-free imaging and optical manipulation of stem cells as
well as for the non-contact introduction of microRNA in cancer cells. Human adult pancreatic stem cells, salivary gland
stem cells, and human dental pulp stem cells have been investigated by femtosecond laser multiphoton microscopy.
Autofluorescence based on NAD(P)H and flavoproteins and second harmonic generation due to collagen have been
imaged with submicron spatial resolution, 270 ps temporal resolution, and 10 nm spectral resolution. Major emission
peaks at 460 nm and 530 nm with typical mean fluorescence lifetimes of 1.8 ns and 2.0 ns, respectively, were measured
in a variety of stem cells using spectral imaging and time-correlated single photon counting. During differentiation, the
ratios of bound to free NAD(P)H and NAD(P)H/flavoproteins changed. In addition, the biosynthesis of lipids and
collagen was detected over a long period of time of up to 5 weeks. Nanoprocessing was performed with 12 femtosecond
laser pulses and low picojoule pulse energies to realize targeted transfection and optical cleaning of human adult stem
cell populations. Multiphoton sub-20fs microscopes may become novel non-invasive tools for marker-free optical stem
cell characterization, for on-line monitoring of differentiation within a three-dimensional microenvironment, and for
optical manipulation.
Arbitrary-scan imaging for two-photon microscopy
Author(s):
Edward Botcherby;
Christopher Smith;
Martin Booth;
Rimas Juskaitis;
Tony Wilson
Show Abstract
In this paper, we present details of a scanning two-photon fluorescence microscope we have built with a nearisotropic
scan rate. This means that the focal spot can be scanned at high speed along any direction in the
specimen, without introducing systematic aberrations. We present experimental point spread function measurements
for this system using an Olympus 0.8 NA 40X water dipping objective lens that demonstrates an axial
range of operation greater than 200 μm. We give details of a novel actuator device used to displace the focusing
element and demonstrate axial scan responses up to 3.5 kHz. Finally, we present a bioscience application of this
system to image dendritic processes that follow non-linear paths in three-dimensional space. The focal spot was
scanned along one such process at 400 Hz with an axial range of more than 90 μm.
Multiphoton microscopy as a diagnostic imaging modality for lung cancer
Author(s):
Ina Pavlova;
Kelly R. Hume;
Stephanie A. Yazinski;
Rachel M. Peters;
Robert S. Weiss;
Watt W. Webb
Show Abstract
Lung cancer is the leading killer among all cancers for both men and women in the US, and is associated with one of the
lowest 5-year survival rates. Current diagnostic techniques, such as histopathological assessment of tissue obtained by
computed tomography guided biopsies, have limited accuracy, especially for small lesions. Early diagnosis of lung
cancer can be improved by introducing a real-time, optical guidance method based on the in vivo application of
multiphoton microscopy (MPM). In particular, we hypothesize that MPM imaging of living lung tissue based on twophoton
excited intrinsic fluorescence and second harmonic generation can provide sufficient morphologic and
spectroscopic information to distinguish between normal and diseased lung tissue. Here, we used an experimental
approach based on MPM with multichannel fluorescence detection for initial discovery that MPM spectral imaging
could differentiate between normal and neoplastic lung in ex vivo samples from a murine model of lung cancer. Current
results indicate that MPM imaging can directly distinguish normal and neoplastic lung tissues based on their distinct
morphologies and fluorescence emission properties in non-processed lung tissue. Moreover, we found initial indication
that MPM imaging differentiates between normal alveolar tissue, inflammatory foci, and lung neoplasms. Our long-term
goal is to apply results from ex vivo lung specimens to aid in the development of multiphoton endoscopy for in vivo
imaging of lung abnormalities in various animal models, and ultimately for the diagnosis of human lung cancer.
Controllable infrared continuum source for multiphoton imaging
Author(s):
C. de Mauro;
D. Alfieri;
M. Arrigoni;
D. Armstrong;
F. S. Pavone
Show Abstract
We report on multiphoton imaging of biological samples performed with continuum infrared source generated
in photonic crystal fibers (PCFs). We studied the spectra generated in PCFs with dispersion profiles designed
to maximize the power density in the 700-1000 nm region, where the two-photon absorption cross sections of
the most common dyes lie. Pumping in normal dispersion region, with <140 femtosecond pulses delivered by a
tunable Ti:Sa laser (Chameleon Ultra II by Coherent Inc.), results in a limitation of nonlinear broadening up to a
mean power density above 2 mW/nm. Axial and lateral resolution obtained with a scanning multiphoton system
has been measureed to be near the theoretical limit. The possibility of simultaneous two-photon excitation of
different dyes in the same sample and high image resolution are demonstrated at tens of microns in depth.
Signal-to-noise ratio and general performances are found to be comparable with those of a single wavelength
system, used for comparison.
Two-photon imaging with 80 MHz and 1-GHz repetition rate Ti:sapphire oscillators
Author(s):
Hauke Studier;
Hans Georg Breunig;
Karsten König
Show Abstract
We report on multiphoton optical imaging with a laser scanning microscope (TauMapTM, Jenlab GmbH) in combination
with two different excitation fs-lasers: a 80 MHz Ti:sapphire oscillator generating spectrally tunable 100 fs pulses and a
1 GHz Ti:sapphire oscillator producing ultra broadband 6 fs pulses. While the ultra-broadband pulses enable
simultaneous excitation of several different types of fluorophores due their large spectral width, the 100 fs pulses are
spectrally more selective and require tuning the center wavelength to cover the same excitation range. The wavelength
selectivity was confirmed in measurements with microspheres with absorption maxima in the green and blue spectral
region. Furthermore, the potential of both lasers for imaging of human skin is evaluated.
Adaptive multiphoton and harmonic generation microscopy for developmental biology
Author(s):
Anisha Thayil;
Alexander Jesacher;
Tomoko Watanabe;
Tony Wilson;
Shankar Srinivas;
Martin J. Booth
Show Abstract
Specimen-induced aberrations often affect microscopes, particularly when high numerical aperture lenses are used to
image deep into biological specimens. These aberrations cause a reduction in resolution and focal intensity. 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 applied the techniques of
adaptive optics to correct aberrations in two-photon fluorescence and harmonic generation microscopes, restoring image
quality. In particular we have used these microscopes for studies in developmental biology and for the imaging of
mammalian embryos.
Digital holography for second harmonic microscopy
Author(s):
E. Shaffer;
C. Depeursinge
Show Abstract
Quantitative phase images make digital holographic microscopy (DHM) an excellent instrument for metrological, but
also for biological applications, where it can reveal deformations and morphological details at ultrahigh resolution in the
order of a few nanometers only, while also precisely determining the refractive index across a sample (e.g. cell or
neuron). On the other hand, non-linear light-matter interactions have also proved very useful in microscopy. For
instance, second harmonic generation (SHG) allows marker-free identification of cell structures, tubulin or membranes
and, because of its coherent nature, SHG is very sensitive to the local sample structure and to the direction of the laser
polarization. In addition, since SHG does not result from light absorption and subsequent re-emission, in opposition to
fluorescence, photo-bleaching of the studied material can be avoided by a judicious selection of the laser wavelength.
These characteristics make SHG very interesting for biomedical imaging. We have designed and built a microscope that
combines the fast and precise DHM imaging with tagging capabilities of non-linear light-matter interactions. Here, we
present the technique and look into its possible applications to biological and life sciences. Among promising
applications is the 3D tracking of colloidal gold nanoparticles.
Second-order susceptibility imaging with polarization-resolved second harmonic generation microscopy
Author(s):
Wei-Liang Chen;
Tsung-Hsian Li;
Ping-Jung Su;
Chen-Kuan Chou;
Peter Tramyeon Fwu;
Sung-Jan Lin;
Daekeun Kim;
Peter T. C. So;
Chen-Yuan Dong
Show Abstract
Second harmonic generation (SHG) microscopy has become an important tool for minimally invasive biomedical
imaging. However, differentiation of different second harmonic generating species is mainly provided by morphological
features. Using excitation polarization-resolved SHG microscopy we determined the ratios of the second-order
susceptibility tensor elements at single pixel resolution. Mapping the resultant ratios for each pixel onto an image
provides additional contrast for the differentiation of different sources of SHG. We demonstrate this technique by
imaging collagen-muscle junction of chicken wing.
In vivo optical virtual biopsy of human oral cavity with harmonic generation microscopy
Author(s):
M.-R. Tsai;
S.-Y. Chen;
D.-B. Shieh;
P.-J. Lou;
C.-K. Sun
Show Abstract
Oral cancer ranked number four in both cancer incident and mortality in Taiwanese male population. Early disease
diagnosis and staging is essential for its clinical success. However, most patients were diagnosed in their late disease
stage as ideal prescreening procedures are yet to be developed especially when dealing with a large surface of
precancerous lesions. Therefore, how to detect and confirm the diagnosis of these early stage lesions are of significant
clinical value. Harmonic generation process naturally occurred in biological molecules and requires no energy deposition
to the target molecule. Thus harmonic generation microscopy (HGM) could potentially serve as a noninvasive tool for
screening of human oral mucosal diseases. The in vivo optical biopsy of human oral cavity with HGM could be achieved
with high spatial resolution to resolve dynamic physiological process in the oral mucosal tissue with equal or superior
quality but devoid of complicated physical biopsy procedures. The second harmonic generation (SHG) provide
significant image contrast for biomolecules with repetitive structures such as the collagen fibers in the lamina propria
and the mitotic spindles in dividing cells. The cell morphology in the epithelial layer, blood vessels and blood cells flow
through the capillaries can be revealed by third harmonic generation (THG) signals. Tissue transparent technology was
used to increase the optical penetration of the tissue. In conclusion, this report demonstrates the first in vivo optical
virtual biopsy of human oral mucosa using HGM and revealed a promising future for its clinical application for
noninvasive in vivo diseases diagnosis.
Nonlinear optical response of the collagen triple helix and second harmonic microscopy of collagen liquid crystals
Author(s):
A. Deniset-Besseau;
P. De Sa Peixoto;
J. Duboisset;
C. Loison;
F. Hache;
E. Benichou;
P.-F. Brevet;
G. Mosser;
M.-C. Schanne-Klein
Show Abstract
Collagen is characterized by triple helical domains and plays a central role in the formation of fibrillar and
microfibrillar networks, basement membranes, as well as other structures of the connective tissue. Remarkably, fibrillar
collagen exhibits efficient Second Harmonic Generation (SHG) and SHG microscopy proved to be a sensitive tool to
score fibrotic pathologies.
However, the nonlinear optical response of fibrillar collagen is not fully characterized yet and quantitative data are
required to further process SHG images. We therefore performed Hyper-Rayleigh Scattering (HRS) experiments and
measured a second order hyperpolarisability of 1.25 10-27 esu for rat-tail type I collagen. This value is surprisingly large
considering that collagen presents no strong harmonophore in its amino-acid sequence. In order to get insight into the
physical origin of this nonlinear process, we performed HRS measurements after denaturation of the collagen triple
helix and for a collagen-like short model peptide [(Pro-Pro-Gly)10]3. It showed that the collagen large nonlinear response
originates in the tight alignment of a large number of weakly efficient harmonophores, presumably the peptide bonds,
resulting in a coherent amplification of the nonlinear signal along the triple helix. To illustrate this mechanism, we
successfully recorded SHG images in collagen liquid solutions by achieving liquid crystalline ordering of the collagen
triple helices.
Second harmonic generation in human ovarian neoplasias
Author(s):
L. Lamonier;
F. Bottcher-Luiz;
L. Pietro;
L. A. L. A. Andrade;
A. A. de Thomaz;
C. L. Machado;
C. L. Cesar
Show Abstract
Metastasis is the main cause of death in cancer patients; it requires a complex process of tumor cell dissemination, extra
cellular matrix (ECM) remodeling, cell invasion and tumor-host interactions. Collagen is the major component of ECM;
its fiber polymerization or degradation evolves in parallel with the evolution of the cancerous lesions. This study aimed
to identify the collagen content, spatial distribution and fiber organization in biopsies of benign and malignant human
ovarian tissues. Biopsies were prepared in slides without dyes and were exposed to 800nm Ti:Sapphire laser (Spectra
Physics, 100 fs pulse duration, 800mW average power, 80MHz repetition rate). The obtained images were recorded at
triplets, corresponding to clear field, multiphoton and second harmonic generation (SHG) mycroscopy. Data showed
considerable anisotropy in malignant tissues, with regions of dense collagen arranged as individual fibers or in
combination with immature segmental filaments. Radial fiber alignment or regions with minimal signal were observed in
the high clinical grade tumors, suggesting degradation of original fibers or altered polymerization state of them. These
findings allow us to assume that the collagen signature will be a reliable and a promising marker for diagnosis and
prognosis in human ovarian cancers.
High-throughput three-dimensional (3D) lithographic microfabrication in biomedical applications
Author(s):
Daekeun Kim;
Peter T. C. So
Show Abstract
Two-photon excitation microfabrication has been shown to be useful in the field of photonics and biomedicine. It
generates 3D microstructures and provides sub-diffraction fabrication resolution. Nevertheless, laser direct writing, the
most popular two-photon fabrication technique, has slow fabrication speed, and its applications are limited to
prototyping. In this proceeding, we propose high-throughput 3D lithographic microfabrication system based on depthresolved
wide-field illumination and build several 3D microstructures with
SU-8. Through these fabrications, 3D
lithographic microfabrication has scalable function and high-throughput capability. It also has the potential for
fabricating 3D microstructure in biomedical applications, such as intertwining channels in 3D microfluidic devices for
biomedical analysis and 3D cell patterning in the tissue scaffolds.
Assessment of fibrotic liver disease with multimodal nonlinear optical microscopy
Author(s):
Fake Lu;
Wei Zheng;
Dean C. S. Tai;
Jian Lin;
Hanry Yu;
Zhiwei Huang
Show Abstract
Liver fibrosis is the excessive accumulation of extracellular matrix proteins such as collagens, which may result in
cirrhosis, liver failure, and portal hypertension. In this study, we apply a multimodal nonlinear optical microscopy
platform developed to investigate the fibrotic liver diseases in rat models established by performing bile duct ligation
(BDL) surgery. The three nonlinear microscopy imaging modalities are implemented on the same sectioned tissues of
diseased model sequentially: i.e., second harmonic generation (SHG) imaging quantifies the contents of the collagens,
the two-photon excitation fluorescence (TPEF) imaging reveals the morphology of hepatic cells, while coherent
anti-Stokes Raman scattering (CARS) imaging maps the distributions of fats or lipids quantitatively across the tissue.
Our imaging results show that during the development of liver fibrosis (collagens) in BDL model, fatty liver disease also
occurs. The aggregated concentrations of collagen and fat constituents in liver fibrosis model show a certain
correlationship between each other.
Multimodal nonlinear microscopy at 1.5 µm
Author(s):
Chun Zhan;
Chulmin Joo;
Qing Li;
Mikhail Y. Berezin;
Walter J. Akers;
Yunpeng Ye;
Samuel Achilefu;
Siavash Yazdanfar
Show Abstract
We describe multimodal nonlinear microscopy using a compact, turnkey femtosecond fiber laser at 1.5 μm. The system
allows for multiplexed detection of near infrared and visible contrast agents through two-and three-photon excitation
fluorescence microscopy as well as structural imaging viaauto-confocal microscopy (ACM). This platform expands the
available emission spectrum for multiphoton microscopy, enables simultaneous structural and functional imaging, and
offers advantages in penetration depth, contrast, and simplicity as compared to conventional MPM near 800 nm
excitation.
Multicolor excitation two-photon microscopy: in vivo imaging of cells and tissues
Author(s):
Dong Li;
Wei Zheng;
Jianan Y. Qu
Show Abstract
Two-photon microscopy based on endogenous fluorescence provides non-invasive imaging of living biological system.
Reduced nicotinamide adenine dinucleotide (NADH), flavin adenine dinucleotide (FAD), keratin, collagen and elastin
are the endogenous fluorophores widely used as the contrast agents for imaging metabolism and morphology of living
cells and tissue. The fluorescence of tryptophan, a kind of essential amino acid, conveys the information on cellular
protein content, structure and microenvironment. However, it can't be effectively excited by the commonly used
Ti:sapphire femtosecond laser. Because each endogenous fluorophore provides limited information, it is desirable to
simultaneously excite fluorescence from as many fluorophores as possible to obtain accurate biochemical and
morphological information on biomedical samples. In this study, we demonstrate that the supercontinuum generation
from a photonic crystal fiber (PCF) excited by an ultrafast source can be used to excite multiple endogenous nonlinear
optical signals simultaneously. By employing the spectral lifetime detection capability, this technology provides a unique
approach to sense the fine structure, protein distribution and cellular metabolism of cells and tissues in vivo. In particular,
with application of acetic acid, a safe contrast agent used for detection cervical cancer for many years, the tryptophan
signals reveal cellular morphology and even cell-cell junctions clearly. Moreover, it was found that the pH value
dependent lifetime of tryptophan fluorescence could provide the qualitative information on the gradient of pH value in
epithelial tissue. Finally, we will demonstrate the potential of our multi-color TPEF microscopy to investigate the early
development of cancer in epithelial tissue.
The analysis of fluorophore orientation by multiphoton fluorescence microscopy
Author(s):
Jamie M. Leeder;
David L. Andrews
Show Abstract
The accessibility of tunable, ultrafast laser sources has spurred the development and wide application of specialized
microscopy techniques based on chromophore fluorescence following
two- and three-photon absorption. The attendant
advantages of such methods, which have led to a host of important applications including three-dimensional biological
imaging, include some features that have as yet received relatively little attention. In the investigation of cellular or subcellular
processes, it is possible to discern not only on the location, concentration, and lifetime of molecular species, but
also the orientations of key fluorophores. Detailed information can be secured on the degree of orientational order in
specific cellular domains, or the lifetimes associated with the rotational motions of individual fluorophores; both are
accessible from polarization-resolved measurements. This paper reports the equations that are required for any such
investigation, determined by robust quantum electrodynamical derivation. The general analysis, addressing a system of
chromophores oriented in three dimensions, determines the fluorescence signal produced by the nonlinear polarizations
that are induced by multiphoton absorption, allowing for any rotational relaxation. The results indicate that multiphoton
imaging can be further developed as a diagnostic tool, either to selectively discriminate micro-domains in vivo, or to
monitor dynamical changes in intracellular fluorophore orientation.
Two-photon microscopy with dynamic focusing objective using a liquid lens
Author(s):
Kye-Sung Lee;
Philip Vanderwall;
Jannick P. Rolland
Show Abstract
A variable focus microscope without moving parts for axial focusing within the sample in two-photon microscopy can
provide a faster, more robust, and cost-effective solution to 3D imaging at high resolution. A custom dynamic focusing
objective was recently developed that incorporates a liquid lens within the optical design of a custom microscope. In this
paper, we investigate the use of a liquid lens embedded in an aberration-corrected custom microscope for z-dimension
scanning in 3D resolved two-photon microscopy. Results show that we can achieve imaging speed of 50 frames per sec
for an arbitrary plane in 3D space, a leap forward in scan speed for two-photon microscopy.
Pulse shaping for reducing photodamage in multiphoton microscopy
Author(s):
Dmitry Pestov;
Yair Andegeko;
Vadim V. Lozovoy;
Marcos Dantus
Show Abstract
We formulate a simple strategy for mitigation of laser-induced damage through pulse shaping and demonstrate
experimentally the effect of laser pulse duration on the degree of optically induced damage for two-photon microscopy
imaging. We use a broadband Ti:Sapphire laser source, aided with a shaper, and adjust both the laser pulse duration and
energy to maintain constant two-photon excitation efficiency. The damage is assessed by the dynamics of two-photon
excited autofluorescence intensity and sample morphology during prolonged laser exposure. We observe that for a 5-μm
layer of skin tissue the damage rate is independent of the pulse shape, which suggests that the primary damage
(bleaching) mechanism stems from the two-photon excitation itself. For optically thick dried blood samples, taken as
another example, the data suggests that the damage is driven by
one-photon absorption. In the later case, it is favorable
to use shorter laser pulses to mitigate photodamage while maintaining adequate intensity of two-photon excited
autofluorescence.
Real-time molecular imaging of organelles in living cell by multifocus excitation CARS microscope
Author(s):
Takeo Minamikawa;
Tsutomu Araki;
Mamoru Hashimoto
Show Abstract
We demonstrated real-time imaging of organelles in a living HeLa cell using a multi-focus excitation CARS
(coherent anti-Stokes Raman scattering) microscope. Chemical selective CARS imaging of lipids and proteins
was demonstrated by observing CH2 and CH3 vibrations. Real-time imaging of lipid rich organelles such as the
plasma membrane, mitochondria, and lipid rich vesicles was achieved by observing CH2 stretching vibrations
of lipids. The image acquisition rate of 5 frames per second was achieved without any staining. We also
demonstrated real-time CARS imaging of laser-induced disruption and reaction of organelles in a living HeLa
cell. A near-infrared pulsed laser beam tightly focused on an organelle in a living cell produces ablation at the
focal point, causing local disruption of the organelle. We visualized the spatial and temporal distributions of a
lipid rich organelles in the cytoplasm of a living HeLa cell in
laser-induced dissection. We also demonstrated
real-time CARS imaging of disruption of a plasma membrane and its repair.
In situ observation of collagen thermal denaturation by second harmonic generation microscopy
Author(s):
C.-S. Liao;
Z.-Y. Zhuo;
J.-Y. Yu;
P.-H. G. Chao;
S.-W. Chu
Show Abstract
Collagen denaturation is of fundamental importance for clinical treatment. Conventionally, the denaturation process
is quantified by the shrinkage of collagen fibers, but the underlying molecular origin has not been fully understood.
Since second harmonic generation (SHG) is related to the molecular packing of the triple helix in collagen fibers,
this nonlinear signal provides an insight of molecular dynamics during thermal denaturation. With the aid of SHG
microscopy, we found a new step in collagen thermal denaturation process, de-crimp. During the de-crimp step, the
characteristic crimp pattern of collagen fascicles disappeared due to the breakage of interconnecting bonds between
collagen fibrils, while SHG intensity remained unchanged, suggesting the intactness of the triple helical molecules.
At higher temperature, shrinkage is observed with strongly reduced SHG intensity, indicating denaturation at the
molecular level.
Spatio-temporal control in multiphoton fluorescence laser-scanning microscopy
Author(s):
Arijit Kumar De;
Debjit Roy;
Debabrata Goswami
Show Abstract
The broad spectral window of an ultra-short laser pulse and the broad overlapping multiphoton absorption spectra of
common fluorophores restrict selective excitation of one fluorophore in presence of others during multiphoton
fluorescence microscopy. Also spatial resolution, limited by the fundamental diffraction limit, is governed by the beam
profile. Here we show our recent work on selective fluorescence suppression using a femtosecond pulse-pair excitation
which is equivalent to amplitude shaping using a pulse shaper. In addition, prospects of laser beam shaping in imaging
are also briefly discussed.
Discrimination of collagen in normal and pathological dermis through polarization second harmonic generation
Author(s):
Ping-Jung Su;
Wei-Liang Chen;
Jin-Bon Hong;
Tsung-Hsien Li;
Ruei-Jr Wu;
Chen-Kuan Chou;
Sung-Jan Lin;
Chen-Yuan Dong
Show Abstract
We used polarization-resolved, second harmonic generation (P-SHG) microscopy at single pixel resolution for
medical diagnosis of pathological skin dermis, and found that P-SHG can be used to distinguish normal and
dermal pathological conditions of keloid, morphea, and dermal elastolysis. We find that the histograms of the
d33/d31 ratio for the pathological skins to contain two peak values and to be wider than that of the normal case,
suggesting that the pathological dermal collagen fibers tend to be more structurally heterogeneous. Our work
demonstrates that pixel-resolved, second-order susceptibility microscopy is effective for detecting heterogeneity
in spatial distribution of collagen fibers.
Multispectral autofluorescence lifetime imaging of RPE cells using two-photon excitation
Author(s):
Lingling Zhao;
Danni Chen;
Jing Qi;
Junle Qu
Show Abstract
In this paper, we present our investigation on multispectral autofluorescence lifetime imaging of RPE cells using two-photon
excitation. Morphological characters of RPE cells are obtained with high spatial resolution. Different
autofluorescence lifetime parameters have been compared at different emission bands. Spatial distribution of dominant
endogenous fluorophores in RPE cells, such as FAD, A2E and AGE etc have been obtained by the analysis of τm and
a1/a2 ratio in the whole emission spectrum.
In vivo multiphoton imaging of obstructive cholestasis in mice
Author(s):
Feng-Chieh Li;
Yu Yang Lee;
Ling-Ling Chiou;
Hsuan-Shu Lee;
Chen-Yuan Dong
Show Abstract
Combining multiphoton microscopy with a newly designed hepatic imaging window, we acquired
in vivo images of mice obstructive cholestasis. We observed that in mice with bile duct ligation, bile
canaliculi failed to appear during the whole observation period over 100 minutes following
carboxyfluorescein diacetate injection, whereas the fluorescence was retained much longer within
sinusoids. Furthermore, the fluorescence intensities in sinusoids were persistently higher than in
hepatocytes during the course.
Versatile photonic crystal fiber-enabled source for multi-modality biophotonic imaging beyond conventional multiphoton microscopy
Author(s):
Haohua Tu;
Stephen A. Boppart
Show Abstract
In a typical multiphoton microscope, the majority of the laser power from the near-IR pulsed source is thrown away in
order to prevent photo-damage on biological samples. It is thus desirable to convert this wasted power into other
wavelengths through nonlinear fiber optics to accommodate imaging modalities beyond conventional multiphoton
microscopy. Here we present a prototypical source that accommodates the requirements for laser scanning confocal
microscopy, fluorescence lifetime imaging microscopy, stimulated emission microscopy, coherent anti-Stokes Raman
microscopy/spectroscopy, transient absorption microscopy, and optical coherence tomography/microscopy. Such
versatile source consists of dispersion-engineered photonic crystal fibers simultaneously pumped by a compact ~1040
nm ultrafast ytterbium laser.
Multiplex coherent anti-Stokes Raman scattering flow cytometry for real-time classification of particles in a microfluidic channel
Author(s):
Charles H. Camp Jr.;
Siva Yegnanarayanan;
Ali A. Eftekhar;
Hamsa Sridhar;
Ali Adibi
Show Abstract
Flow cytometry is an ever-advancing, multivariate analysis tool obtaining morphological and phenotype information
from sample populations. In traditional flow cytometers, obtaining molecular information requires the
use of endogenous fluorophores, which are limited by spectral overlap, nonspecific binding, available conjugation
chemistries, and cellular toxicity. In this work, however, we apply multiplex coherent anti-Stokes Raman scattering
(MCARS), a nonlinear optical method that probes the Raman energies within a molecule, to flow cytometry
in order to demonstrate label-free, molecularly sensitive particle differentiation. To demonstrate this, we correctly
differentiate 5 μm polystyrene (PS) and poly(methyl methacrylate) (PMMA), which (linearly) optically
appear similar, but vary significantly in their MCARS spectrum.
Quantitative analysis of biological tissues using Fourier transform-second-harmonic generation imaging
Author(s):
Raghu Ambekar Ramachandra Rao;
Monal R. Mehta;
Kimani C. Toussaint Jr.
Show Abstract
We demonstrate the use of Fourier transform-second-harmonic generation (FT-SHG) imaging of collagen fibers as a
means of performing quantitative analysis of obtained images of selected spatial regions in porcine trachea, ear, and
cornea. Two quantitative markers, preferred orientation and maximum spatial frequency are proposed for differentiating
structural information between various spatial regions of interest in the specimens. The ear shows consistent maximum
spatial frequency and orientation as also observed in its real-space image. However, there are observable changes in the
orientation and minimum feature size of fibers in the trachea indicating a more random organization. Finally, the
analysis is applied to a 3D image stack of the cornea. It is shown that the standard deviation of the orientation is sensitive
to the randomness in fiber orientation. Regions with variations in the maximum spatial frequency, but with relatively
constant orientation, suggest that maximum spatial frequency is useful as an independent quantitative marker. We
emphasize that FT-SHG is a simple, yet powerful, tool for extracting information from images that is not obvious in real
space. This technique can be used as a quantitative biomarker to assess the structure of collagen fibers that may change
due to damage from disease or physical injury.
Fluorescence performance standards for confocal microscopy
Author(s):
Steffen Rüttinger;
Peter Kapusta;
Volker Völlkopf;
Felix Koberling;
Rainer Erdmann;
Rainer Macdonald
Show Abstract
State of the art confocal microscopes offer diffraction limited (or even better) spatial resolution, highest (single
molecule) sensitivity and ps-fluorescence lifetime measurement accuracy. For developers, manufacturers, as
well as users of confocal microscopes it is mandatory to assign values to these qualities. In particular for users, it
is often not easy to ascertain that the instrument is properly aligned as a large number of factors influence
resolution or sensitivity. Therefore, we aspire to design a set of performance standards to be deployed on a day-to-day fashion in order to check the instruments characteristics.
The main quantities such performance standard must address are:
• Spatial resolution
• Sensitivity
• Fluorescence lifetime
To facilitate the deployment and thus promote wide range adoption in day-to-day performance testing the
corresponding standards have to be ready made, easy to handle and to store. The measurement procedures
necessary should be available on as many different setups as possible and the procedures involved in their
deployment should be as easy as possible.
To this end, we developed two performance standards to accomplish the mentioned goals:
• Resolution reference
• Combined molecular brightness and fluorescence lifetime reference
The first one is based on sub-resolution sized Tetra-SpeckTM fluorescent beads or alternatively on single
molecules on a glass surface to image and to determine quantitatively the confocal volume, while the latter is a
liquid sample containing fluorescent dyes of different concentrations and spectral properties. Both samples are
sealed in order to ease their use and prolong their storage life. Currently long-term tests are performed to
ascertain durability and road capabilities.
Fiber-based multiphoton system
Author(s):
Gangjun Liu;
Khanh Kieu;
Frank W. Wise;
Zhongping Chen
Show Abstract
A fiber based multiphoton microscopy (MPM) system is designed and demonstrated.
An all normal dispersion fiber laser with central wavelength around 1um was used as laser
source. A double clad photonic crystal fiber (DCPCF), and galvanometer mirror scanner based
handheld probe is designed. Second harmonic generation (SHG) images and two photon excited
fluorescence (TPEF) images of biological tissue were demonstrated by the system.
In vivo deep tissue imaging with long wavelength multiphoton excitation
Author(s):
Demirhan Kobat;
Michael E. Durst;
Nozomi Nishimura;
Angela W. Wong;
Chris B. Schaffer;
Chris Xu
Show Abstract
As a result of the large difference between scattering mean free paths and absorption lengths in brain tissue, scattering
dominates over absorption by water and intrinsic molecules in determining the attenuation factor for wavelengths
between 350 nm and 1300 nm. We propose using longer wavelengths for two-photon excitation, specifically the 1300-nm region, in order to reduce the effect of scattering and thereby increase imaging depth. We present two photon
fluorescence microscopy images of cortical vasculature in in vivo mouse brain beyond 1 mm. We also explore the
capabilities of the 1300-nm excitation for third harmonic generation microscopy of red blood cells in in vivo mouse
brain.
Coherent anti-Stokes Raman scattering microscopy using photonic crystal fibers
Author(s):
Majid Naji;
Sangeeta Murugkar;
Kaisar R. Khan;
Hanan Anis
Show Abstract
The performance of two different photonic crystal fibers (PCF) of identical lengths for implementation of the Stokes
source in a multimodal CARS microscopy and spectroscopy setup is compared. RIN measurements are performed to
experimentally determine the noise in the supercontinuum from the two fibers as well as in the CARS signal under
similar excitation conditions of the input pulse into the PCF. The RIN of the CARS signal is found to be higher than
the RIN of the corresponding Stokes signal, in both fibers. The implications for CARS microscopy of the SC spectrum
and its noise dependence on input pulse conditions in both fibers, are discussed.
High-resolution imaging of a corneal incision by second- and third-harmonic generation microscopy
Author(s):
L. Jay;
C. Dion;
A. Brocas;
K. Singh;
J.-C. Kieffer;
I. Brunette;
T. Ozaki
Show Abstract
We demonstrate high spatial resolution imaging of a stromal cut in the ex-vivo pig
cornea, using second- and third-harmonic generation microscopy. From these images,
we see in detail how the cut affects the corneal layers. In the beginning of the cut, the
anterior layers, in which the blade is passing through, are disorganized, which could
explain the shadows observed on the images. In the stroma, the cut can be imaged by
third harmonic microscopy, probably due to the χ3 contrast. Although the current
results were obtained from the healthy ex-vivo cornea, it already allows one to
understand the effects of the cut on the tissue characteristics (such as scattering).
Broadband multiplex CARS microspectroscopy in the picosecond regime
Author(s):
Sebastien Michel;
Antoine Courjaud;
John Dudley;
Christophe Finot;
Eric Mottay;
Herve Rigneault
Show Abstract
We investigate a compact, stable and broadband multiplex Coherent anti-Stokes Raman Scattering (CARS) source for
micro-spectroscopy. By pumping an adapted photonic crystal fiber, we generate the broadband Stokes pulses required for
multiplex CARS measurements. The CARS signal stability is provided by an active fiber coupling, avoiding therefore
the thermal or mechanical drifts. With only a few nanojoule for pump and Stokes pulses energies, we demonstrate on test
liquids the capability of the source to generate multiplex CARS spectra in the 600-2000 cm-1 spectral range.
Complementary equipment for controlling multiple laser beams on single scanner MPLSM systems
Author(s):
P. Johannes Helm;
Gabriele Nase;
Paul Heggelund;
Trond Reppen
Show Abstract
Multi-Photon-Laser-Scanning-Microscopy (MPLSM) now stands as one of the most powerful experimental tools in
biology. Specifically, MPLSM based in-vivo studies of structures and processes in the brains of small rodents and
imaging in brain-slices have led to considerable progress in the field of neuroscience.
Equipment allowing for independent control of two laser-beams, one for imaging and one for photochemical
manipulation, strongly enhances any MPLSM platform. Some industrial MPLSM producers have introduced double
scanner options in MPLSM systems.
Here, we describe the upgrade of a single scanner MPLSM system with equipment that is suitable for independently
controlling the beams of two Titanium Sapphire lasers. The upgrade is compatible with any actual MPLSM system and
can be combined with any commercial or self assembled system.
Making use of the pixel-clock, frame-active and line-active signals provided by the scanner-electronics of the MPLSM,
the user can, by means of an external unit, select individual pixels or rectangular ROIs within the field of view of an
overview-scan to be exposed, or not exposed, to the beam(s) of one or two lasers during subsequent scans. The switching
processes of the laser-beams during the subsequent scans are performed by means of Electro-Optical-Modulators
(EOMs).
While this system does not provide the flexibility of two-scanner modules, it strongly enhances the experimental
possibilities of one-scanner systems provided a second laser and two independent EOMs are available.
Even multi-scanner-systems can profit from this development, which can be used to independently control any number
of laser beams.
Investigating the protective properties of milk phospholipids against ultraviolet light exposure in a skin equivalent model
Author(s):
Ashley Russell;
Andrea Laubscher;
Rafael Jimenez-Flores;
Lily H. Laiho
Show Abstract
Current research on bioactive molecules in milk has documented health advantages of bovine milk and its components.
Milk Phospholipids, selected for this study, represent molecules with great potential benefit in human health and
nutrition. In this study we used confocal reflectance and multiphoton microscopy to monitor changes in skin
morphology upon skin exposure to ultraviolet light and evaluate the potential of milk phospholipids in preventing
photodamage to skin equivalent models. The results suggest that milk phospholipids act upon skin cells in a protective
manner against the effect of ultraviolet (UV) radiation. Similar results were obtained from MTT tissue viability assay
and histology.
In-vivo tissue imaging using a compact mobile nonlinear microscope
Author(s):
Riccardo Cicchi;
Dimitrios Kapsokalyvas;
Despoina Stampouli;
Vincenzo De Giorgi;
Daniela Massi;
Torello Lotti;
Francesco S. Pavone
Show Abstract
We have built a compact flexible non-linear microscope equipped with a combination of different non-linear laser
imaging techniques including two-photon fluorescence, second-harmonic generation, fluorescence lifetime imaging
microscopy, and multispectral two-photon emission detection. The system is composed of a microscope head, containing
both scanning and detection system, as well as the electronic and electro-mechanical devices, optically relayed to the
laser source with a seven-mirror articulated arm. The particular mirror positioning inside the arm allows to move the
microscope head maintaining the optical alignment of the system. The microscope head is composed by two ErGaAl
anodized boards, one for laser scanning and the other for signal detection. System performances were characterized by
means of point spread function and instrument response function measurements as well as by spatial, temporal, and
spectral calibration. The instrument, offering high spatial (up to 300 nm) and temporal (up to 300 ps) resolution, was
tested on in-vivo skin imaging of both cellular epidermis and connective dermis. Lifetime and spectral features of
fluorescence were used for differentiating epidermal layers by means of fluorescence lifetime and for scoring skin ageing
through spectral detection of both second-harmonic and two-photon fluorescence.
Fast rasterscanning enables FLIM in macroscopic samples up to several centimeters
Author(s):
F. Koberling;
V. Buschmann;
C. Hille;
M. Patting;
C. Dosche;
A. Sandberg;
A. Wheelock;
R. Erdmann
Show Abstract
Fluorescence Lifetime Imaging (FLIM) based on Time-Correlated Single Photon Counting (TCSPC) is nowadays a well
established technique that is very often realised as an add-on for confocal laser scanning microscopes. However, the
standard laser scanning technique limits the maximum scan range in these setups to a few millimetre, making it therefore
unsuited for e.g. fluorescence multiplexing in multi well plate based assays or for macroscopic material science studies
on solar cells, wafers and similar material. In order to also realize larger scanning ranges, we have developed a sample
scanning approach based on a xy-cross stage equipped with piezo linear motors. Using online position monitoring, this
approach permits fast acceleration and scanning as well as precise positioning and features scan ranges from 100×100
microns up to 80×80 mm with submicron positioning accuracy. Standard upright and inverse microscope bodies can
easily be equipped with this scanning device. Along with the necessary excitation and detection components "largearea"
FLIM thus becomes possible. We will show new results obtained with a modified MicroTime 100 (PicoQuant
GmbH) illustrating the system capabilities for lifetime based imaging in macroscopic samples such as the improvement
of the fluorescence sensitivity in 2D gel electrophoresis or the possibility to perform lifetime based fluorescence
multiplexing in μ-well plate based assays. Even Two Photon Excitation (TPE) imaging is possible with this widerange
sample scanning approach and first FLIM results on cockroach salivary glands, loaded with a chloride sensitive dye
(MQAE) will be presented.
Extracellular oxygen concentration mapping with a confocal multiphoton laser scanning microscope and TCSPC card
Author(s):
Neveen A. Hosny;
David A. Lee;
Martin M. Knight
Show Abstract
Extracellular oxygen concentrations influence cell metabolism and tissue function. Fluorescence Lifetime Imaging
Microscopy (FLIM) offers a non-invasive method for quantifying local oxygen concentrations. However, existing
methods show limited spatial resolution and/or require custom made systems. This study describes a new optimised
approach for quantitative extracellular oxygen detection, providing an off-the-shelf system with high spatial resolution
and an improved lifetime determination over previous techniques, while avoiding systematic photon pile-up.
Fluorescence lifetime detection of an oxygen sensitive fluorescent dye, tris(2,2'-bipyridyl)ruthenium(II) chloride
hexahydrate [Ru(bipy)3]2+, was measured using a Becker&Hickl time-correlated single photon counting (TCSPC) card
with excitation provided by a multi-photon laser. This technique was able to identify a subpopulation of isolated
chondrocyte cells, seeded in three-dimensional agarose gel, displaying a significant spatial oxygen gradient. Thus this
technique provides a powerful tool for quantifying spatial oxygen gradients within three-dimensional cellular models.
A multimodal multiphoton microscope for biological imaging
Author(s):
Rabah Mouras;
Andrew Downes;
Grigore Rischitor;
Meropi Mari;
Alistair Elfick
Show Abstract
We report on the construction of a highly flexible system for advanced biological imaging, where all the following
imaging techniques are integrated into the same microscope: Coherent anti-Stokes Raman scattering (CARS), two
photon excitation fluorescence (TPEF), second harmonic generation (SGH), sum frequency generation (SFG),
fluorescence lifetime imaging (FLIM) and differential interference contrast (DIC). The system employs a Nd:YVO4 laser
as pump (7 ps, 1064 nm), and two tunable OPOs (6 ps, 700 - 1000 nm). Our microscope comprises a heater stage and
perfusion cell for imaging of live cells, and features an atomic force microscope (AFM) which enables optical imaging at
10 nm resolution. Multimodal imaging of breast cancer cells and tissue will be demonstrated as well as imaging of anticancer
drugs in living cells.
Nonlinear 3D microscopy of ex vivo corneas
Author(s):
Juan M. Bueno;
Emilio J. Gualda;
Pablo Artal
Show Abstract
A multiphoton microscope has been developed to investigate the sources of nonlinear fluorescence (TPEF) and second
harmonic generation (SHG) in non-stained samples of ex-vivo corneas. Stacks of images from different depths are
recorded to reconstruct high-resolution 3D (volume) images of the cornea. The corneal epithelium and endothelium
provide significant TPEF signal, while the only source of SHG is the stroma. Within the stroma, the keratocytes can also
be visualized. Volumetric 3D images of the cornea combining TPEF and SHG signals are useful to characterize the
organization of the corneal collagen and to describe the distribution of keratocytes. These images will help to better
understand how different pathologies modify the corneal structure and to control the changes produced by surgical or
healing processes.
Scheme for efficient fiber-based CARS probe
Author(s):
Mihaela Balu;
Gangjun Liu;
Zhongping Chen;
Bruce J. Tromberg;
Eric O. Potma
Show Abstract
We demonstrate a fiber-based probe for maximum collection of the Coherent
anti-Stokes Raman Scattering (CARS) signal in biological tissues. We discuss the design
challenges including capturing the back-scattered forward generated CARS signal in the
sample and the effects of fiber nonlinearities on the propagating pulses. Three different
biological tissues were imaged in vitro in order to assess the performance of our fiberdelivered
probe for CARS imaging, a tool which we consider an important advance
towards label-free, in vivo probing of superficial tissues.