If no fresh skin samples can be obtained or used, it is important for research and industries to have models and stored
tissue samples as close to the native state as possible at disposal. One way to preserve tissues for a longer timeframe is to
use deep freezing cryo-techniques. Unfortunately much damage can be induced during the cooling and the thawing
processes like disruption of cells and extra-cellular matrices due to the formation of ice crystals. This could lead to a
disturbance of the united cell structure up to the point of a loss of cell viability. Two-photon microscopy is able to gather
information about cells and tissue components via excitation of the autofluorescence deep inside the sample with a high
resolution in both, frozen and thawed states. It is possible to monitor the samples before and after and, important,
observe events during the freezing process like the formation of ice crystals.
To determine the state of skin tissues after slow rate freezing and the quick process of vitrification, the samples were
examined with two-photon microscopy. To establish an optimized freezing-protocol for skin tissues, morphological
changes, changes in autofluorescence of endogenous fluorophores (NADH, keratin, flavins, elastin) or changes in second
harmonic generation of collagen fibres could provide information about the quality of the used freezing parameters and
protective additives and lead to an optimized freezing-protocol with a new set of parameters to obtain mostly intact
tissue samples. Multiphoton microscopy has been established as a useful tool for optical in situ quality control of frozen
tissues.
Photoacoustic imaging exploits contrast mechanisms that depend on optical and thermomechanical properties of
optical absorbers. The photoacoustic signal bandwidth is dictated by the absorber size and the laser pulse width. In this
work we demonstrate that photoacoustic signals can be detected from micron and sub-micron particles. We anticipate
applications to include cellular imaging with nanometer sized contrast agents such as gold nanoshells, nanorods, and
nanocages.
An existing acoustic microscopy system was used (the SASAM 1000, kibero GmbH). This platform is developed on
an Olympus IX81 optical microscope with a rotating column that has an optical condenser for transmission optical
microscopy and an acoustic module for the acoustic microscopy. The adapted optoacoustic module consists of a Qswitched
Nd:YAG solid-state-laser (Teem Photonics, France) generating
sub-nanosecond pulses. Scans were acquired of
microparticles (1 μm black Toner particles) and cells.
The confocal arrangement allowed high signal to noise ratio photoacoustic signals (>30 dB) to be detected at
approximately 400 MHz. The particles of various sizes produced signals of different frequency content. In imaging
mode, the full width half maximum (FWHM) was measured to be 3.6 μm for the 400 MHz transducer which is in
general agreement theory for a 0.3 NA objective (4.3μm). Moreover, images are generated from single melanoma cells,
generated by the endogenous contrast from the intracellular melanin.
Multiphoton optical tomography or intravital tomography (IVT) provides non-invasive optical sectioning of biological
specimens, e.g. skin, with subcellular spatial resolution without any need of contrast agents. It can be used to distinguish
between normal and diseased tissue due to the differences in morphological appearance. Additional information beyond
morphology can be obtained by analyzing the collected fluorescence light spectroscopically and by means of its
fluorescence decay time. This is frequently termed spectral fluorescence lifetime imaging (SFLIM) or 5D-intravital
tomography (5D-IVT). Spectral and temporal resolution scales with the number of detection increments (i.e. spectral
channels and time bins). 5D-IVT enables us to detect new physiological parameters, however accompanied by a decrease
in intensity per channel. Moreover, the increase of data requests a higher need of software skills.
In this study we investigate and evaluate different technical modes of 5D-IVT with respect to their clinical relevance: (1)
a multichannel photomultiplier tube (PMT) array coupled to a diffraction grating, each channel being analyzed by timecorrelated
single photon counting (TCSPC), (2) three separate PMTs in spectral separation path using dichroic mirrors,
each channel being analyzed by TCSPC and (3) a single PMT TCSPC setup in combination with a high-resolution CCDspectrograph
for pointwise microspectroscopy.
Karsten König, Martin Weinigel, Hans Breunig, Axel Gregory, Peter Fischer, Marcel Kellner-Höfer, Rainer Bückle, Martin Schwarz, Iris Riemann, Frank Stracke, Volker Huck, Christian Gorzelanny, Stefan Schneider
Some years ago, CE-marked clinical multiphoton systems for 3D imaging of human skin with subcellular resolution have
been launched. These tomographs provide optical biopsies with submicron resolution based on two-photon excited
autofluorescence (NAD(P)H, flavoproteins, keratin, elastin, melanin, porphyrins) and second harmonic generation by
collagen. The 3D tomograph was now transferred into a 5D imaging system by the additional detection of the emission
spectrum and the fluorescence lifetime based on spatially and spectrally resolved time-resolved single photon counting.
The novel 5D intravital tomograph (5D-IVT) was employed for the early detection of atopic dermatitis and the analysis
of treatment effects.
Two-photon imaging of human skin using ultra short laser pulses can be used to obtain information about the state of
cells and tissues by means of their natural autofluorescence. Using this method, it is possible to determine whether the
normal cell pattern is disturbed or the autofluorescence is influenced by internal or external stimuli. Two-photon
fluorescence lifetime imaging (FLIM) can further enhance this providing information about physiological processes,
fluorophores (like NAD(P)H, collagen, keratin, elastin, flavins, melanin,...) and external applied probes inside cells and
tissue parts. For example the part of the cells metabolism and energy level can be determined by analyzing the NADH
regarding its free / bound state and its oxidized / reduced state. The combination of two-photon imaging with FLIM may
lead to a better understanding and diagnosis of skin reactions and disorders. We also present some results of in vivo
simultaneous collagen and elastin measurements in skin dermis. Changes of dermal collagen and elastin content are
characteristic for skin aging as well as for pathological skin conditions.
The trans-cutaneous pathway for drug delivery is of particular interest since it allows a simple and non-invasive
administration of pharmaceutically relevant compounds. As the skin is an effective barrier for many of these compounds,
various strategies have been developed to enable and control the trans-cutaneous transport. Here we discuss, how
multiphoton microscopy and spectral imaging can be valuable tools for the analysis of the penetration pathways of
topically applied drugs. A time dependent study of the cutaneous penetration of a fluorescent drug model released from a
nano-particular carrier is presented. The localization of single nano-particles in human skin (ex vivo) and the
discrimination of different fluorescent compounds, as the drug model, the particle's label and the cutaneous endofluorescence
by spectral imaging and selective excitation is shown. Multiphoton imaging techniques were found to be
excellent methods for the non-invasive evaluation of cutaneous drug delivery strategies and analysis of dermal
penetration pathways down to the sub-cellular level.
With increasing demand for in-vivo observation of living cells, microscope techniques that do not need staining become
more and more important. In this talk we present a combined multi-photon-acoustic microscope with the possibility to
measure synchronously properties addressed by ultrasound and two-photon fluorescence. Ultrasound probes the local
mechanical properties of a cell, while the high resolution image of the two-photon fluorescence delivers insight in cell
morphology and activity. In the acoustic part of the microscope an ultrasound wave, with a frequency of GHz, is
focused by an acoustic sapphire lens and detected by a piezo electric transducer assembled to the lens. The achieved
lateral resolution is in the range of 1&mgr;m. Contrast in the images arises mainly from the local absorption of sound in the
cells, related to properties, such as mass density, stiffness and viscose damping. Additionally acoustic microscopy can
access the cell shape and the state of the cell membrane as it is a intrinsic volume scanning technique.The optical part
bases on the emission of fluorescent biomolecules naturally present in cells (e.g. NAD(P)H, protophorphyrin IX,
lipofuscin, melanin). The nonlinear effect of two-photon absorption provides a high lateral and axial resolution without
the need of confocal detection. In addition, in the near-IR cell damages are drastically reduced in comparison to direct
excitation in the visible or UV. Both methods can be considered as minimal invasive, as they relay on intrinsic contrast
mechanisms and dispense with the need of staining. First results on living cells are presented and discussed.
Adult human and rat pancreas stem cells as well as tumor spheroids were irradiated with femtosecond laser pulses in the
near infrared (NIR) spectral range at high transient GW/cm2 and TW/cm2 intensities. The cellular response to the laser
exposure was probed by the detection of modifications of NAD(P)H autofluorescence, the formation of reactive oxygen
species (ROS) and DNA strand breaks (TUNEL-assay), and viability (live/dead test). The results confirm that long-term
scanning of stem cells can be performed at appropriate laser exposure parameters without a measurable impact on the
cellular metabolism and vitality. In addition, it was proven that a targeted inactivation of a particular single stem cells or
a single tumour cell inside a 3D cell cluster using single point illumination at TW/cm2 laser intensities can be performed
without affecting neighbouring cells.
Therefore multiphoton microscopes can be considered as biosafe tools for long-term analysis of stem cells as well as
highly precise optical knocking out of single cells within cell clusters and tissues.
Multiphoton optoporation of vital cells was performed using a femtosecond pulsed laser in the near infrared (NIR).
Exogenous materials such as macromolecules and exogenes were transported into the targets via laser assisted transient
opening of the cell membrane. This method is also appropriate for nanoprocessing and optoporation inside 3D stem cell
structures without photodestructive collateral effects which was confirmed with TUNEL-assay (DNA strand breaks) and
tests for reactive oxygen species (ROS).
Sub-80nm, sub-wavelength multiphoton nanoprocessing of silicon wafers as well as 3D maskless lithography by two
two-photon polymerization in combination with five-dimensional (x,y,z, λ, τ) multiphoton analysis have been
performed with the compact near infrared MHz femtosecond laser galvoscanning microscope FemtoCut (JenLab
GmbH) as well as a modified ZEISS LSM510-NLO system. Laser excitation radiation was provided by a tuneable turnkey,
one-box Chameleon as well as a MaiTai Ti:sapphire laser oscillator. Nanostructuring of silicon wafers with oil
immersion objectives was based on NIR laser-induced periodic surface structures (LIPPS) likely due to selforganization
processes. For the first time, periodic 70nm nanogrooves have been generated in wafers which is one order
below the 800 nm laser wavelength by multiphoton phenomena at TW/cm2 transient intensities and low sub-3nJ pulse
energies. Three-dimensional two-photon polymerization in SU-8 photoresists at GW/cm2 allowed rapid prototyping
with sub-200nm precision. The same intensities have been used to image endogenous and exogenous fluorophores in a
variety of materials for target finding and the evaluation of the nanoprocessing procedures.
Near infrared (NIR) femtosecond laser microscopes enable the user to perform highly precise nanosurgery. Tissue components, cells and single organelles of cells inside tumor-sphaeroids and tissues can be precisely manipulated and optically knocked out without collateral damage. In addition, the monitoring effects of nanosurgery in situ using two photon excitation of auto fluorescence of endogenous fluorophores can be performed quite easily with a sub-cellular resolution. This method may become a useful instrument for nano manipulation and nano-surgery in several fields of life sciences.
Multiphoton Microscopy with a femtosecond pulsed Ti:sapphire laser in the near infrared (NIR) enables the user not only to image cells and tissues with a subcellular resolution but also to perform highly precise nanosurgery. Intratissue compartments, single cells and even cell organelles like mitochondria, membranes or chromosomes can be manipulated and optically knocked out. Working at transient TW/cm2 laser intensities, single cells of tumor-sphaeroids were eliminated efficiently inside the sphaeroid without damaging the neighbour cells. Also single organelles of cells inside tissues could be optically knocked out with the nanoscalpel without collateral damage. Tissue structures inside a human tooth have been ablated with sizes below 1 μm. This method may become a useful instrument for nano-manipulating and surgery in several fields of science, including targeted transfection.
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