Volker Huck, Christian Gorzelanny, Kai Thomas, Christian Mess, Valentina Dimitrova, Martin Schwarz, Iris Riemann, Verena Niemeyer, Thomas Luger, Karsten König, Stefan Schneider
Increasing incidence of inflammatory skin diseases such as Atopic Dermatitis (AD) has been noted in the past years.
According to recent estimations around 15% of newborn subjects are affected with a disease severity that requires
medical treatment. Although its pathogenesis is multifactorial, recent reports indicate that an impaired physical skin
barrier predispose for the development of AD. The major part of this barrier is formed by the stratum corneum (SC)
wherein corneocytes are embedded in a complex matrix of proteins and lipids. Its components were synthesized in the
stratum granulosum (SG) and secreted via lamellar bodies at the SC/SG interface.
Within a clinical in vivo study we focused on the skin metabolism at the SC/SG interface in AD affected patients in
comparison to healthy subjects. Measurement of fluorescence life-time of NADH provides access to the metabolic state
of skin. Due to the application of a 5D intravital tomographic skin analysis we facilitate the non-invasive investigation of
human epidermis in the longitudinal course of AD therapy. We could ascertain by blinded analysis of 40 skin areas of 20
patients in a three month follow-up that the metabolic status at the SC/SG interface was altered in AD compromised skin
even in non-lesional, apparent healthy skin regions. This illustrates an impaired skin barrier formation even at non-affected
skin of AD subjects appearing promotive for the development of acute skin inflammation. Therefore, our
findings allow a deeper understanding of the individual disease development and the improved management of the
therapeutic intervention in clinical application.
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.
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.
We designed and constructed a two-fiber-optic two-photon endoscope with compact two axes piezo scanner system and
a miniature high 0.65 NA Grin lens objective. The high NA GRIN lens objective coupled to the fibers with a beam
splitter cube represent a rigid miniaturized optical system block which is scanned as a whole by a piezo scanner allowing
always an on-axes beam irradiation of the optical system. We have demonstrated the ability of our system to perform
good images in the micrometer field. Fluorescence detection was able at different wavelengths between 790 and 840 nm
which could allow SHG detection. The endoscope is high flexible and controllable in terms of time acquisition,
resolution and magnification. Fluorescence images were acquired over a maximal 420 μm x 420 μm field-of-view.
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.
Two-photon autofluorescence imaging offers the analysis of cells and tissues without the need of taking biopsies,
staining and complicated confocal detection systems. Therefore, it is of special interest for non- or minimal invasive
clinical diagnostics.
Until now, two-photon imaging was performed only on superficial surfaces like skin or of biopsies. To extend this
technique to deeper tissues or inside the body the optical properties have to be reduced to endoscopical sizes. This can be
achieved by tiny GRIN-optics, based on a radial gradient in the reflective index. A newly developed GRIN-lens
assembly with increased numerical aperture is of special interest which is shown by the quality of tissue constituents and
cell autofluorescence images.
A fiber directs the laser light to the specimen in an assembly like an endoscope. This well-characterized photonic crystal
fiber supports the high laser power of the femtosecond excitation impulses without the generation of non-linearities. A
sensitive PMT detector detects the fluorescence. First fluorescence images using a fiber-GRIN lens combination were
taken.
A variety of human and animal stem cells (rat and human adult pancreatic stem cells, salivary gland stem cells, dental
pulpa stem cells) have been investigated by femtosecond laser 5D two-photon microscopy. Autofluorescence and second
harmonic generation have been imaged with submicron spatial resolution, 270 ps temporal resolution, and 10 nm spectral
resolution. In particular, NADH and flavoprotein fluorescence was detected in stem cells. Major emission peaks at
460nm and 530nm with typical mean fluorescence lifetimes of 1.8 ns and 2.0 ns, respectively, were measured using time-correlated
single photon counting and spectral imaging. Differentiated stem cells produced the extracellular matrix
protein collagen which was detected by SHG signals at 435 nm.
Multiphoton optical tomography provides non-invasive optical sectioning of skin with high spatial and intracellular
resolution. The imaging system DermaInspect based on femtosecond laser pulses and equipped with a SPC 830 card for
FLIM analysis was used to perform this technique in vivo in clinical studies on patients with abnormal pigmented tissues.
After examination in vivo, biopsies were taken and analyzed again. So far it was possible to distinguish between normal
and diseased tissues mainly due to their different morphological appearances in the imaging mode: melanoma tissues
show differences in the structure of the epidermal layers and the presence of dendritic cells compared to normal skin.
The optical analysis can be improved when using the system in the time-correlated single photon counting mode.
Fluorescence lifetime analysis reveals characteristic differences between healthy and melanoma tissues even before the
visible morphological changes in human tissue in vivo.
In vivo simultaneous collagen and elastin measurements using the multiphoton tomograph DermaInspect have been
performed in skin dermis. We showed it was possible to get simultaneous measurements of autofluorescence (AF) and
Second Harmonic Generation (SHG) with a newly developed device using 2 PMTs for time-correlated single photon
counting. Unlike elastin (AF), collagen structures are able to generate second harmonics (SHG). Comparing the images
and SHG / AF ratios recorded in the depth of the outer and inner sides of the forearm of two European female volunteers
(31 and 60 years old, respectively) shows differences in collagen and elastin fibres density. It decreases with depth in the
60 years old volunteer compared to the younger one, and the skin of younger volunteer shows more collagen.
Multiphoton autofluorescence imaging offers minimal-invasive examination of cells without the need of staining and
complicated confocal detection systems. Therefore, it is especially interesting for non-invasive clinical diagnostics.
To extend this sophisticated technique from superficial regions to deep lying cell layers, internal body parts and
specimens difficult of access, the bulky optics need to be reduced in diameter. This is done by tiny GRIN-optics, based
on a radial gradient in the reflective index. Of especial interest for multi-photon applications is the newly developed
GRIN-lens assembly with increased numerical aperture. High resolution images of plant tissue, hair and cells show the
improved image quality,compared to classical GRIN-lenses.
The rigid GRIN-endoscopes are already applied in wound healing studies. Here, the GRIN-lenses with diameters smaller
than 3 mm enter small skin depressions. They reproduce the focus of a conventional laser scanning tomograph tens of
mm apart in the specimen under study. We present first clinical measurements of elastin and SHG of collagen of in-vivo
human skin of venous ulcers (ulcer curis).
In skin, the most common form of cancer is the basal cell carcinoma, affecting about 800.000 people in US each year.
One of the treatments is photodynamic therapy (PDT) with a topical application of 5-aminolevulinic acid (ALA)
derivates. These photosensitizers accumulate mainly in cancerous cells and can be activated by light resulting in a
destruction of the cells.
We performed multiphoton tomography based on near-infrared (NIR) femtosecond laser pulses on skin biopsies in order
to study the biosynthesis of protoporphyrine IX and the effect of treatment with submicron resolution. The fluorescence
of the photosensitizer was excited using two-photon processes and monitored through the epidermis towards the basal
layer. In comparison with normal skin the cancerous cells showed different morphology and fluorescence behavior.
Multiphoton tomography might become a useful tool for high resolution imaging of cancerous tissue and to evaluate the
therapeutical effects.
Multiphoton autofluorescence imaging became an important technique for minimal invasive examination of cells in
biological tissue. Rigid and flexible endoscopes based on gradient index lenses (GRIN-lenses) extend this minimalinvasive
technique to deep lying cell layers, inner body and specimens, difficult to access.
In the rigid endoscope, a GRIN-lens overcomes the limited depth range, given by the working distance of the microscope
objective. The focus of the conventional laser scanning tomography is reproduced tens of millimeters in the specimen
under study by the GRIN-lens (diameter 1.8 and 3 &mgr;m). We will present images of fluorescent beads, proteins cells and
skin tissue, as well as first in vivo measurements on human skin. The autofluorescence signal stems from the endogenous
fluorophore elastin and SHG from collagen.
The flexible endoscope dispenses completely the need of a microscope next to the specimen of interest. The excitation
laser pulses is delivered via a well-characterized photonic crystal fiber and subsequently focused by a newly designed
GRIN-lens system. The fluorescence, also transferred by a fiber is detected by a PMT detector. We will show the
excellent imaging qualities of a newly developed GRIN-lens system with high-resolution images of proteins, cells and
plant tissue and give an out-look on multiphoton endoscopy.
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.
Confocal and multi-photon imaging systems are currently miniaturized to fit them to endoscopic size requirements of
probe diameters often less than 2.8 mm. High resolution in lateral dimensions of less than 1 micron and in axial
dimensions of less than 10 microns is desired to resolve sub-cellular details of in-vivo tissue. GRIN rod lens systems
have been widely used as high numerical aperture objectives in these applications because of their small size, good
image quality and favourable geometry with plane optical surfaces, which allows easy assembly. Image generation has
been enabled by combination with coherent imaging fiber bundles, GRIN relay lenses and scanning single fibers or
photonic band gap fibers. With previous GRIN systems of maximum NA of 0.50, a resolution limit laterally of approx.
1.0 micron and axially of 5.5 to 10 microns was obtained in the case of two-photon excitation. Here, we introduce a
novel concept of a GRIN lens objective system with significantly higher NA yielding resolution improved by a factor of
two (lateral) and four (axial). The image quality of initial 1.0 mm GRIN components will be characterized by the signal
analysis of 0.2 micron fluorescent beads in the multi-photon scheme and by conventional image tests using test grids
with transmitting illumination. Potentials for further miniaturization and for changing the direction of view will be
discussed.
Multiphoton stimulated autofluorescence microscopy and Magnetic resonance imaging (MRI) address different
molecular properties of the sample and reach to a different length scale. MRI maps density or mobility of nuclei (here:
hydrogen), and targets at whole objects from the scale of sub-millimetres to meters. Multiphoton imaging profits from
the nonlinear absorption of light in the focus of a femtosecond laser source stimulating the autofluorescence of
biomolecules. As this effect relies on a high light intensity the accessible field of view is limited, but the resolution is
very high.
Studying a plant embryo (barley) we compare the two techniques. At 770 nm excitation the cell walls of the embryo
exhibited significant autofluorescence, allowing for a subcellular resolution. While details where imaged with an
objective of N.A. 1.3, an overview was generated with a N.A. as low as 0.25. The overview image as well as merged
images and tomographical data were used to link the high-resolution optical data with the three-dimensional highresolution
MR images. There, images of the proton density were acquired using a standard 3D spin-echo imaging pulse
sequence. While the optical high-resolution data provides a field of view restricted to only a small part of the embryo,
the MR image contains the whole grain. Bridging the scales it might be possible to trace transport of e.g. nutrients from
large structure of the plant to the cellular level.
Multiphoton tomography offers a painless method to examine patients under natural physiological conditions in vivo.
Multiphoton excitation induces a weak autofluorescence of naturally endogenous fluorescent bio-molecules, such as
flavines, NAD(P)H, metal-free porphyrines, components of lipofuscin, elastin and keratin. Additionally, collagen can be
detected by second harmonic generation (SHG). Due to the nonlinearity, the effects occur only in a very tight focus,
where the photon density is high enough. This leads to high axial and lateral resolution of <1μm without any need of a
confocal detection and avoids out-of-focus damage. The limited depth range, given by the working distance of the
focusing optics, is overcome with a gradient index-lens (GRIN-lens) based endoscope.
In this work we present the first results of clinical applications in vivo of gradient-index lens endoscopes. Images of e.g.
elastin and collagen (SHG) in the dermal layer of human skin are presented.
Multiphoton tomography based on femtosecond laser NIR (near infrared) pulses was used to perform non-invasive
optical sectioning of skin with high spatial and intracellular resolution. Scar formation due to formation of collagen
fibers is an important aspect during wound healing processes in skin and tissues and was monitored in vivo using the
system DermaInspect. Multiphoton tomography was performed of a dermal wound after nevi extraction. The healing
process and the aggregation of collagen fibers could be long term monitored due to the autofluorescence of endogenous
fluorophores and SHG of collagen. The system DermaInspect might become a high resolution diagnostic tool for
dermatological diagnostics and monitoring therapeutic effects.
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).
Multiphoton tomography with the clinical femtosecond laser system DermaInspect has become an important non-invasive
high resolution imaging tool for skin research, melanoma detection, and in situ drug monitoring of
pharmaceutical and cosmetical components. The detection of endogenous fluorophores and SHG active biostructures
such as mitochondrial NAD(P)H, melanin in melancytes and basal cells, as well as the extracellular matrix components
elastin and collagen is achieved with submicron resolution when using high NA focusing optics. So far, the working
distance was limited to 200 µm. In addition, the focusing optics was large in diameter (2-3 cm).
Here we report for the first time on clinical deep-tissue high-resolution imaging with a novel high NA rigid GRIN
microendoscope which extends the potential of clinical multiphoton tomography significantly. We performed the very
first clinical in vivo measurements with two-photon endoscopes and studied wounds of patients with ulcus cruris.
Influence of laser parameters are studied and discussed in this paper dealing with safety aspects of femtosecond laser refractive surgery. Studies on transmission and energy have been done using different focusing objectives. More than 20% of NIR photons are reaching the retina using a numerical aperture of 0.3 which represent a fluence of 2 μJ/cm2 with parameters for flap procedure of about 7 μJ energy per pulse, at 10 KHz repetition rate and at a wavelength of 1040 nm. When using an objective with NA 0.9 about one order less fluence will strike the retina. To minimize bubbles expansion and side effects in tissue processing as well as to exclude any damages at retina, a compromise between ablation time, pulse energy and process strategy by the use of higher numerical aperture has to be taken into account.
KEYWORDS: Tissues, In vivo imaging, Collagen, Heart, Skin, Near infrared, Optical fibers, Second-harmonic generation, Optical tomography, In vitro testing
Multiphoton optical tomography based on NIR (near-infrared) femtosecond laser pulses provides non-invasive optical sectioning of skin with high spatial intracellular resolution and high tissue penetration. The imaging system DermaInspect was used to perform this technology in clinical studies in vivo on patients with suspicious melanoma. Pigmented cell clusters based on non-linear luminescence were clearly distinguished from non-pigmented cells in the epidermis using the autofluorescence of endogenous fluorophores like NAD(P)H, flavins, keratin, elastin, collagen and melanin. Some of the investigated tissues showed differences in the structure of the epidermal layers and the presence of dendritic cells compared to normal skin. Multiphoton laser microscopy was used to visualize extracellular matrix (ECM) structures of native and tissueengineered heart valves. The quality of the resulting 3-D images allowed an exact differentiation between collagenous and elastic fibers. The analysis of heart valve tissues of patients with cardiomyopathy revealed a dramatic loss of its capability to generate SH (second harmonic), indicating a structural deformation of the collagenous fibers, which was virtually impossible to obtain by routine histological or immunohistological staining. These results indicate that NIR femtosecond laser scanning systems can be employed as novel non-invasive optical technology for 3-D resolved ECM component imaging and in vitro and in vivo tissue diagnosis.
The multiphoton tomograph DermaInspect was used to perform first clinical studies on the early non-invasive detection of skin cancer based on non-invasive optical sectioning of skin by two-photon autofluorescence and second harmonic generation. In particular, deep-tissue pigmented lesions -nevi- have been imaged with intracellular resolution using near infrared (NIR) femtosecond laser radiation. So far, more than 250 patients have been investigated. Cancerous tissues showed significant morphological differences compared to normal skin layers. In the case of malignant melanoma, the occurrence of luminescent melanocytes has been detected. Multiphoton tomography will become a novel non-invasive method to obtain high-resolution 3D optical biopsies for early cancer detection, treatment control, and in situ drug
screening.
The two-photon-mediated autofluorescence and second harmonic generation (SHG) are acting as a novel diagnostic tool to perform tissue optical tomography with submicron resolution. The three-dimensional corneal ultrastructure of whole depth can be probed without any staining or mechanical slicing. Compared with photodisruptive surgical effects occurring at TW/cm2 light intensity, multiphoton imaging can be induced at MW-GW/cm2 photon intensity. The multiphoton microscopy based on nonlinear absorption of femtosecond laser pulses at the wavelength of 715-930nm emitted from solid-state Ti: sapphire system is being used as a precise non-invasive monitoring tool to determine the interest of region, to visualize and to verify the outcomes in the invivo intrastromal laser nanosurgery. More interesting, the activated keratocytes have been also observed in-vivo 24 hours after the laser nanosurgery with this system. Overall, these data suggest that multiphoton microscopy is a highly sensitive and promising technique for studying the morphometric properties of the microstructure of the corneal tissue and for assessing the intrastromal nanosurgery. With the help of the multiphoton-mediated imaging, the next generation of laser refractive surgery approaches based on the nonamplified femtosecond lasers with higher precision and less complications are being evaluated systematically.
Multiphoton imaging has developed into an important technique for in-vivo research in life sciences. With the laser System DermaInspect (JenLab, Germany) laser radiation from a Ti:Sapphire laser is used to generate multiphotonabsorption deep in the human skin in vivo. The resulting autofluorescence radiation arises from endogenous fluorophores such as NAD(P)H, flavines, collagen, elastin, porphyrins und melanin. Second harmonic generation (SHG) was used to detect collagen structures in the dermal layer. Femtosecond laser multiphoton imaging offers the possibility of high resolution optical tomography of human skin as well as fluorescence lifetime imaging (FLIM) with picosecond time resolution. In this work a photon detector with ultrashort rise time of less than 30ps was applied to FLIM measurements of human skin and hair with different pigmentation. Fluorescence lifetime images of different human hair types will be discussed.
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.
We report on sub-μm structuring of semiconductors, dielectrics, polymers and metals using a laser scanning microscope. A commercial Ti:Sapphire oscillator laser (20 nJ/pulse; 90 MHz; 150 fs) was coupled into a laser scanning microscope FemtOcut (JenLab GmbH). High numerical aperture objectives were applied to obtain fluences in the range of a few J/cm2 which are well above the ablation threshold. Such a cost-effective and reliable system compared to amplified lasers systems (μJ or mJ/pulse) is adapted for material manufacturing and can be of prime interest for specific applications in security like counterfeiting.
KEYWORDS: Skin, Tissues, Luminescence, Tissue optics, In vivo imaging, Near infrared, Femtosecond phenomena, Second-harmonic generation, Ultraviolet radiation, Imaging systems
High resolution non-invasive 3D imaging devices are required to detect pathogenic microorganisms such as Anthrax spores, bacteria, viruses, fungi and chemical agents entering biological tissues such as the epidermis. Due to the low light penetration depth and the biodamage potential, ultraviolet light sources can not be employed to realize intratissue imaging of bio- and chemohazards. We report on the novel near infrared laser technology multiphoton tomography and the high resolution 4D imaging tool DermaInspect for non-invasive detection of intratissue agents and their influence on cellular metabolism based on multiphoton autofluorescence imaging (MAI) and second harmonic generation (SHG). Femtosecond laser pulses in the spectral range of 750 nm to 850 nm have been used to image in vivo human skin with subcellular spatial and picosecond temporal resolution. The non-linear induced autofluorescence of both, skin tissues and microorganisms, originates mainly from naturally endogenous fluorophores/protein structures like NAD(P)H, flavins, keratin, collagen, elastin, porphyrins and melanin. Bacteria emit in the blue/green spectral range due to NAD(P)H and flavoproteins and, in certain cases, in the red spectral range due to the biosynthesis of Zn-porphyrins, coproporphyrin and protoporphyrin. Collagen and exogenous non-centrosymmetric molecules can be detected by SHG signals. The system DermaInspect consists of a wavelength-tunable compact 80/90 MHz Ti:sapphire laser, a scan module with galvo scan mirrors, piezo-driven objective, fast photon detector and time-resolved single photon counting unit. It can be used to perform optical sectioning and 3D autofluorescence lifetime imaging (τ-mapping) with 1 μm spatial resolution and 270 ps temporal resolution. The parameter fluorescence lifetime depends on the type of fluorophore and its microenvironment and can be used to distinguish bio- and chemohazards from cellular background and to gain information for pathogen identification. The novel in vivo non-invasive imaging system offers the possibility to detect and to localize CB agents in tissues and to gain information on their impact on respiratory chain activity, cell division and metabolism. The system DermaInspect can also be used to detect food and water contamination.
The intratissue multiphoton autofluorescence imaging (MAI) and the second harmonic generation (SHG) based on nonlinear process of femtosecond nanojoule laser pulses at wave length of 750-850 nm emitted from solid-state Titanium: Sapphire Chameleon have been used as a highly precise non-destructive tool to realize the in-vivo differentiation of corneal layers with the assistance of intratissue optical tomography and to visualize the keratocyte structures and collagen lamellas with submicron resolution. Multiphoton nonlinear imaging occurs only with high light intensity on an order of MG-GW/cm2 and photon flux density of more than 1024 photons cm-2s-1 in a 0.1femtoliter intrastromal focus volume obtained by diffraction-limited focussing with high-numerical objectives. This technique, acting as a novel diagnostic tool, proved to be essential for femtosecond (fs) nanojoule (nJ) cornea surgery to determine the interest of region preoperation, to visualize and verify the outcomes immediately after the laser surgery and has potential to become a powerful tool in advancing understanding of corneal biomechnics and cellular reactions after laser induced lesion.
A diode-pumped Yb:glass femtosecond laser oscillator with electro-optical cavity-dumping has been applied for nonlinear laser-scanning microscopy and processing of biomaterials. The high-energy pulses delivered by this source in combination with its unique parameters proved very efficient for micro-processing of biomaterials.
Multiphoton optical tomography provides the capability of non-invasive optical sectioning of skin with high spatial and intracellular resolution as well as high NIR (near infrared) light penetration into pigmented skin areas. The imaging system DermaInspect based on femtosecond laser pulses was used to perform multiphoton optical tomography in clinical studies. Patients with abnormal pigmented tissues were imaged in vivo. After the multiphoton imaging procedure, biopsies were taken, imaged again and further processed with standard histological methods. We report on preliminary results. The visualization of pigmented cell clusters based on non-linear luminescence using the novel multiphoton device was possible. These clusters could be clearly distinguished from non-pigmented cells. Cancerous tissues showed significant differences in the cell structure of the epidermal layers. The system DermaInspect might become a high resolution diagnostic tool for melanoma diagnostics.
High resolution optical tomography studies on topically applied probes and UVA exposed skin were performed with the novel imaging system DermaInspect. 3D images with submicron resolution based on the two-photon excitation of the probes and the autofluorescence of epidermal cells and structures with femtosecond laser pulses in the near infrared (NIR) spectral range between 750 nm and 850 nm were obtained. The distribution of natural endogenous fluorophores, mainly keratin and NAD(P)H, as well as of the exogenous probe molecules was detected in situ. The high resolution imaging system DermaInspect offers an unique chance of in situ, non-invasive drug screening in skin on a molecular level and to study the effect of UV exposure.
We report on the histological results of in-vivo animal follow-up studies on refractive femtosecond laser surgery. Non-invasive flap-free intrastromal ablation as well as flap generation has been performed with MHz nanojoule near infrared femtosecond laser pulses. In particular, the dynamics of corneal wound healing have been studied. Wound-healing effects could be detected up to 90 days post-operation in the case of lasermediated flap generation. The flap-free intrastromal cavity was identified until the 28th day post-treatment. Interestingly, eosinophil granulocytes were observed. The follow-up studies confirmed that the near infrared femtosecond laser at near-nanojoule pulse energy is a highly precise and an attractive tool for intraocular refractive surgery, especially for flap-free intrastromal surgery.
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.
For the first time, sub-100 nm nanostructuring as well as five-dimensional (5D) multiphoton analysis with submicron spatial resolution, 270 ps temporal resolution and 10 nm spectral resolution have been performed on metal films, semiconductors, polymers and biological tissues using near infrared, 80/90 MHz femtosecond laser pulses at <3nJ pulse energy. A compact (65x62x48 cm3) multiport laser scanning microscope FemtoCut (JenLab GmbH) equipped with fast detectors for time-correlated single photon counting and a Sagnac interferometer for spectral imaging as well as the ZEISS laser scanning microscope LSM 510 Meta-NLO have been employed. Laser excitation radiation was provided by a tuneable turn-key, one-box Chameleon as well as a MaiTai Ti:sapphire laser oscillator. We were able to perform precise multiphoton nanopatterning of gold films, photoresists and polymers with submicron (cut) sizes. Interestingly, sub-80 nm laser-induced periodic surface structures (LIPPS) have been created in silicon wafers and used to produce nanostructured 2D silicon arrays.
Multiphoton imaging has developed into an important technique for in-vivo research in life sciences in the last few years. A near-infrared laser beam is focused into a sample such that multiphoton-absorption can be generated which stimulates a fluorescence signal as well as second harmonic generation (SHG). Recently it has been shown
that it is possible to image the epidermis in vivo with a resolution of about 1 μm. It was possible to produce 3-dimensional autofluorescence maps of the investigated tissue. However, the depth range of this technique is limited through the working distance of the focusing optics mostly to the epidermal part of the skin. Gradient index lenses offer possibilities to expand the imaging depth into the dermal layer. With typical diameters of up to 2mm and lengths between 2 and 6 cm they are capable of transmitting the laser, fluorescence and SHG radiation and to be integrated into an imaging system. First results of the applicability of gradient index lenses for imaging of skin are presented.
Multi-dimensional time-correlated single photon counting (TCSPC) is based on the excitation of the sample by a high-repetition rate laser and the detection of single photons of the fluorescence signal in several detection channels. Each photon is characterised by its time in the laser period, its detection channel number, and several additional variables such as the coordinates of an image area, or the time from the start of the experiment. Combined with a confocal or two-photon laser scanning microscope and a pulsed laser, multi-dimensional TCSPC makes a fluorescence lifetime technique with multi-wavelength capability, near-ideal counting efficiency, and the capability to resolve multi-exponential decay functions. We show that the same technique and the same hardware can be used to for precision fluorescence decay analysis, fluorescence correlation spectroscopy (FCS), and fluorescence intensity distribution analysis (FIDA and FILDA) in selected spots of a sample.
Multiphoton imaging represents a novel and very promising medical diagnostic technology for the high-resolution analysis of living biological tissues. We performed multiphoton imaging to analyzed structural features of extracellular matrix (ECM) components, e.g., collagen and elastin, of vital pulmonary and aortic heart valves. High-resolution autofluorescence images of collagenous and elastic fibers were demonstrated using multifluorophore, multiphoton excitation at two different wavelengths and optical sectioning, without the requirement of embedding, fixation, or staining. Collagenous structures were selectively imaged by detection of second harmonic generation (SHG). Additionally, routine histology and electron microscopy were integrated to verify the observed results. In comparison with pulmonary tissues, aortic heart valve specimens show very similar matrix formations. The quality of the resulting three-dimensional (3-D) images enabled the differentiation between collagenous and elastic fibers. These experimental results indicate that multiphoton imaging with near-infrared (NIR) femtosecond laser pulses may prove to be a useful tool for the nondestructive monitoring and characterization of cardiovascular structures.
Near infrared (NIR) ultrashort laser pulses of 780 nm have been used to induce intracellular photodynamic reactions by nonlinear excitation of porphyrin photosensitizers. Intracellular accumulation and photobleaching of the fluorescent photosensitizers protoporphyrin IX and Photofrin (PF) have been studied by non-resonant two-photon fluorescence excitation of PF and aminolevulinic acid (ALA)-labeled Chinese hamster ovary (CHO) cells. To testify the efficacy of both substrates to induce irreversible destructive effects, the cloning efficiency (CE) of cells exposed to femtosecond pulses of a multiphoton laser scanning microscope (40x/1.3) was determined. In the case of Photofrin accumulation, CEs of 50% and 0% were obtained after 17 laserscans (2 mW?, 16 s/ frame) and 50 scans, respectively. All cells exposed to 50 scans died within 48h after laser exposure. 100 scans were required to induce lethal effects in ALA labeled cells.
Sensitizer-free control cells could be scanned 250 times (1.1 h) and more without impact on the reproduction behavior, morphology, and vitality. In addition to the slow phototoxic effect by photooxidation processes, another destructive but immediate effect based on optical breakdown was induced when employing high intense NIR femtosecond laser beams. This was used to optically knock out single tumor cells in living mice (solid Ehrlich-Carcinoma) in a depth of 10 to 100 μm.
Sequence specific cutting of DNA is a standard method in molecular biology. This cutting is realized with enzymes which have a defined recognition sequence and cutting sequence. Therefore one can manipulate only sequences for which an enzyme is available. With current physical methods (AFM) any sequences can be cut, but the precise sequence specific and highly parallel cutting is not possible.
Near infrared (NIR) femtosecond laser systems have been used to optically knock out genomic regions of highly condensed DNA in human chromosomes as well as of single expanded (stretched) DNA molecules. Working with 80 MHz laser pulses at 800 nm of low 2 nJ pulse energy but at high TW/cm2 light intensities, multiphoton ionization and optical breakdown (OB) resulted in highly precise material ablation with sub-100 nm cut sizes. This is far below the diffraction-limited spot size. A minimum FWHM cut size of 65 nm was achieved in the case of the nanodissection of a laser-treated stretched λ-DNA (48kb) molecule which corresponded to 200 optically knocked out bases.
By the use of metal nanoparticles as energy coupling objects for fs laser radiation we expect a specific highly local destruction effect within the DNA molecule (cut). Thereby, a sequence-specific binding of DNA nanoparticle complexes along the target DNA is a fundamental condition. The effect of laser exposure on DNA and DNA-nanoparticle complexes are presented.
Fluorescent nanobeads embedded in agarose and skin biopsies were used to optically characterize spatial and temporal resolution of multiphoton laser scanning devices (MPLSD). Optical sections based on two-photon excited bead fluorescence have been performed at various sample depths. Three-dimensional reconstruction of the image stacks allowed determination of the point spread function. Using calculated point spread functions to apply deconvolution procedures (e.g. Huygens software), the visualization and hence the interpretation of intradermal structures, such as extracellular matrix components in 150 μm tissue depth, was improved.
Near infrared (NIR) femtosecond laser imaging systems represent a novel and very promising diagnostic technology for non-invasive cross-sectional analysis of living biological tissues. In this study 3D multiphoton imaging with submicron resolution has been performed for non-invasive analysis of living native and tissue-engineered (TE) heart valves and blood vessels. High-resolution autofluorescence and second harmonic generation (SHG) images of collagenous structures and elastic fibers were demonstrated using multiphoton excitation at two different wavelengths. Non-invasive optical sections have been obtained without the need of staining or embedding. The quality of the resulting three-dimensional images allowed exact differentiation between collagenous structures and elastic fibers. These experimental results are very encouraging for NIR femtosecond laser scanning microscopy as a useful tool for future non-destructive monitoring and characterization of vital and intact TE cardiovascular structures.
The novel compact femtosecond NIR (near infrared) laser imaging system DermaInspect was used to perform for the first time in vivo high resolution non-invasive 4D tomography of human skin based on multiphoton autofluorescence imaging and second harmonic generation (SHG). Using fast galvoscan mirrors, a time correlated single photon counting (TCSPC) module and femtosecond 80 MHz laser pulses in the spectral range of 750 nm-850 nm human skin was analyzed with subcellular spatial resolution (3D) and 250 ps temporal resolution (4D). The non-linear induced autofluorescence originates from naturally endogenous fluorophores and protein structures like NAD(P)H, flavins, phorphyrins, melanin, elastin and collagen. Collagenous structures were detected using SHG. Tissues of patients with dermatological disorders like nevi and melanoma have been investigated with a clear visualization of cells and intratissue
structures. Further characterization of those components was performed by the fluorescence lifetime imaging (FLIM) and the determination of two photon excitation spectra. This method of non invasive high resolution optical biopsy provides a painless diagnostic tool for dermatological applications.
Near infrared (NIR) femtosecond laser scanning microscopy represents a novel and very promising medical diagnostic imaging technology for non-invasive cross-sectional analysis of living biological tissues. In this study multiphoton imaging has been performed to analyze the structural features of extracellular matrix (ECM) components, e.g. collagen and elastin, of living pulmonary and aortic heart valves. High-resolution autofluorescence and second harmonic generation (SHG) images of collagenous and elastic fibers were demonstrated using multifluorophore, multiphoton excitation at two different wavelengths and non-invasive optical sectioning, without the need of embedding or staining. The quality of the resulting three-dimensional images allowed exact differentiation of the ECM components. These experimental results indicated that NIR femtosecond laser scanning microscopy may prove to be a useful tool for the non-destructive monitoring and characterization of cardiovascular structures.
The novel femtosecond NIR (near infrared) laser based high resolution imaging system DermaInspect was used for non-invasive diagnostics of pigmented skin. The system provides fluorescence and SHG images of high spatial submicron resolution (3D) and 250 ps temporal resolution (4D) based on time resolved single photon counting (TCSPC). Pigmented tissue biopsies from patients with nevi and melanoma have been investigated using the tunable 80 MHz femtosecond laser MaiTai with laser wavelengths in the range of 750 - 850 nm. The autofluorescence patterns of different intratissue cell types and structures were determined. The non-linear induced autofluorescence originates from naturally endogenous fluorophores and protein structures like NAD(P)H, flavins, elastin, collagen, phorphyrins and melanin. In addition to autofluorescence, SHG (second harmonic generation) was used to detect dermal collagen structures. Interestingly, pigmented cells showed intense luminescence signals. Further characterization of tissue components was performed via 4D measurements of the fluorescence lifetime (x, y, z, τ). The novel multiphoton technique offers the possibility of a painless high resolution non invasive diagnostic method (optical biopsy), in particular for the early detection of skin cancer.
We report on a method for refractive laser surgery based on low-energy femtosecond laser pulses provided by ultracompact turn-key non-amplified laser systems. An additional excimer laser is not required for ablation of the stroma. The novel method has the potential to be used for (i) optical flap creation as well as stroma ablation and (ii) for non-invasive flap-free intrastromal ablation. In addition, 3D multiphoton imaging of the cornea can be performed. In particular, we used sub-nanojoule near infrared 80 MHz femtosecond laser pulses for multiphoton imaging of corneal structures with ultrahigh resolution (< 1μm) as well as for highly precise intraocular refractive surgery. Imaging based on two-photon excited cellular autofluorescence and SHG formation in collagen structures was performed at GW/cm2 intensities, whereas destructive optical breakdown for nanoprocessing occurred at TW/cm2 light intensities. These high intensities were realized with sub-nJ pulses within a subfemtoliter intrastromal volume by diffraction-limited focussing with high NA objectives and beam scanning 50 to 140 μm below the epithelial surface. Multiphoton tomography of the cornea was used to determine the target of interest and to visualize intraocular post-laser effects. Histological examination with light- and electron microscopes of laser-exposed porcine and rabbit eyes reveal a minimum intratissue cut size below 1 μm without destructive effects to surrounding collagen structures. LASIK flaps and intracorneal cavities could be realized with high precision using 200 fs, 80 MHz, sub-nanojoule pulses at 800 nm. First studies on 80 MHz femtosecond laser surgery on living rabbits have been performed.
In this study, we describe a time-correlated single photon counting (TCSPC) technique for multi-wavelength lifetime imaging in laser-scanning microscopes. The technique is based on a four-dimensional histogramming process that records the photon density versus the time in the fluorescence decay, the x-y coordinates of the scanning area and the wavelength. It avoids any time gating or wavelength scanning and, therefore, yields a near-ideal counting efficiency. The decay functions are recorded in a large number of time channels, and the components of a multi-exponential decay can be resolved down to less than 30 ps. A single TCSPC imaging channel works with a high detection efficiency up to a photon count rate of about 5•106s-1. A modified version of the TCSPC fluorescence lifetime imaging (FLIM) technique uses several fully parallel detector and TCSPC channels. It operates at a count rate of more than 107 photons per second and records double-exponential FLIM data within less than 10 seconds.
Five-dimensional (5D) multiphoton measurements with submicron spatial resolution, 270 ps temporal
resolution and 5 nm spectral resolution have been performed on living cells and tissues at 750 nm - 850
nm laser excitation. A compact (65x62x48 cm3) multiport laser scanning microscope TauMap (JenLab
GmbH) equipped with fast PMT and CCD camera, SPC 830 time-correlated single photon counting
board and Sagnac interferometer was used. Laser excitation radiation was provided by a tuneable
MaiTai Ti:sapphire femtosecond laser as well as by a 405 nm 50 MHz picosecond laser diode. The
spectral and temporal fluorescence behaviour of intratissue chloroplasts of water plant leafs, of a variety
of exogenous fluorophores as well as of fluorescent proteins in transfected brain cells have been studied.
When calculating fluorescence lifetime images (FLIM) we found differences in intracellular twophoton
fluorescence lifetimes vs. one-photon fluorescence lifetimes.
Multiphoton FLIM-FRET and multiphoton spectral FRET studies have been performed in living
HBMEC brain cells using CFP and YFP fusion proteins. It was shown that FLIM-FRET data depend on
laser power due to photodestructive multiphoton effects. This has to be considered in long-term
fluorescence resonance energy transfer studies of dynamic protein-protein interactions.
Targeted transfection of cells is an important technique for gene therapy and related biomedical applications. We delineate how high-intensity (1012 W/cm2) near-infrared (NIR) 80 MHz nanojoule femtosecond laser pulses can create highly localised membrane perforations within a minute focal volume, enabling non-invasive direct transfection of mammalian cells with DNA. We suspended Chinese hamster ovarian (CHO), rat kangaroo kidney epithelial (PtK2) and rat fibroblast cells in 0.5 ml culture medium in a sterile miniaturized cell chamber (JenLab GmbH, Jena, Germany) containing 0.2 μg plasmid DNA vector pEGFP-N1 (4.7 kb), which codes for green fluorescent protein (GFP). The NIR laser beam was introduced into a femtosecond laser scanning microscope (JenLab GmbH, Jena, Germany) and focussed on the edge of the cell membrane of a target cell for 16 ms. The integration and expression efficiency of EGFP were assessed in situ by two-photon fluorescence-lifetime imaging using time-correlated single photon counting. The unique capability to transfer foreign DNA safely and efficiently into specific cell types (including stem cells), circumventing mechanical, electrical or chemical means, will have many applications, such as targeted gene therapy and DNA vaccination.
We describe the novel high resolution imaging tool DermaInspect 100 for non-invasive diagnosis of dermatological disorders based on multiphoton autofluorescence imaging (MAI)and second harmonic generation. Femtosecond laser pulses in the spectral range of 750 nm to 850 nm have been used to image in vitro and in vivo human skin with subcellular spatial and picosecond temporal resolution. The non-linear induced autofluorescence originates mainly from naturally endogenous fluorophores/protein structures like NAD(P)H, flavins, keratin, collagen, elastin, porphyrins and melanin. Second harmonic generation was observed in the stratum corneum and in the dermis. The system with a wavelength-tunable compact 80 MHz Ti:sapphire laser, a scan module with galvo scan mirrors, piezoelectric objective positioner, fast photon detector and time-resolved single photon counting unit was used to perform optical sectioning and 3D autofluorescence lifetime imaging (t-mapping). In addition, a modified femtosecond laser scanning microscope was involved in autofluorescence measurements. Tissues of patients with psoriasis, nevi, dermatitis, basalioma and melanoma have been investigated. Individual cells and skin structures could be clearly visualized. Intracellular components and connective tissue structures could be further characterized by tuning the excitation wavelength in the range of 750 nm to 850 nm and by calculation of mean fluorescence lifetimes per pixel and of particular regions of interest. The novel non-invasive imaging system provides 4D (x,y,z,t) optical biopsies with subcellular resolution and offers the possibility to introduce a further optical diagnostic method in dermatology.
Nanojoule and sub-nanojoule 80 MHz femtosecond laser pulses at 750-850 nm of a compact titanium:sapphire laser have been used for highly precise nanoprocessing of DNA as well as of intracellular and intratissue compartments. In particular, a mean power between 15 mW and 100 mW, 170 fs pulse width, submicron distance of illumination spots and microsecond beam dwell times on spots have been used for multiphoton- mediated nanoprocessing of human chromosomes, brain and ocular intrastromal tissue. By focusing the laser beam with high numerical aperture focusing optics of the laser scan system femt-O-cut and of modified multiphoton scanning microscopes to diffraction-limited spots and TW/cm2 light intensities, precise submicron holes and cuts have been processed by single spot exposure and line scans. A minimum FWHM cut size below 70 nm during the partial dissection of the human chromosome 3 was achieved. Complete chromosome dissection could be performed with FWHM cut sizes below 200 nm. Intracellular chromosome dissection was possible. Intratissue processing in depths of 50 - 100micrometers and deeper with a precision of about 1micrometers including cuts through a nuclei of a single intratissue cell without destructive photo-disruption effects to surrounding tissue layers have been demonstrated in brain and eye tissues. The femt-O-cut system includes a diagnostic system for optical tomography with submicron resolution based on multiphoton- excited autofluorescence imaging (MAI) and second harmonic generation. This system was used to localize the intracellular and intratissue targets and to control the effects of nanoprocessing. These studies show, that in contrast to conventional approaches of material processing with amplified femtosecond laser systems and (mu) J pulse energies, nanoprocessing of materials including biotissues can be performed with nJ and sub-nJ high repetition femtosecond laser pulses of turn-key compact lasers without collateral damage. Potential applications include highly precise cell and embryo surgery, gene diagnostics and gene therapy, intrastromal refractive surgery, cancer therapy and brain surgery.
We describe a novel method of 3D imaging of specific regions of DNA in interphase nuclei and tissues based on multiphoton microscopy and multicolor fluorescence in situ hybridization (M-FISH). Multiphoton Multicolor FISH (MM-FISH) combines the advantages of (i) using a single NIR excitation wavelength for the simultaneous excitation of multiple FISH fluorophores, (ii) absence of fading in out-of-focus regions, (iii) intrinsic 3D imaging capability and (iv) high light penetration depth. Detection of chromosomal aberrations in amniocytes and tumor cells as well as imaging of FISH fluorophores in biopsies using femtosecond laser pulses at 780 nm and 800 nm are described. First two-photon excited fluorescence decay curves of FISH fluorophores are presented. The fluorophores have been excited via non- resonant two-photon excitation with 150 fs pulses of 0.1 to 8 mW mean laser power of a frequency doubled ultra compact 50 MHz fiber laser and with 80 fs pulses of a compact 80 MHz Ti:sapphire laser. MM-FISH may become an interesting tool in preimplantation diagnosis and molecular pathology.
We developed a nanotechnology for highly localized and ultraprecise non-contact laser surgery within living cells, in particular for nanoprocessing of intranuclear structure.
Intracellular photodynamic reactions by nonlinear excitation of porphyrin photosensitizers have been induced using near infrared ultrashort laser pulses at 200 fs pulse width, 80 MHz pulse repetition rate and 2 mW mean laser power. In particular, a highly focused 780 nm pulsed laser scanning beam was employed at a frame rate of 1/16 s-1 (60 microsecond(s) pixel dwell time) to expose Photofrin-labeled and aminolevulinic acid (ALA)-labeled Chinese hamster ovary cells. Intracellular accumulation and photobleaching of the fluorescent photosensitizers protoporphyrin IX and Photofrin have been studied by non-resonant two-photon fluorescence excitation. Subsequent scanning of the sensitizer-labeled cells resulted in reduced cloning efficiency of 50% and 0% after about 13 scans (approximately equals 10 mJ) and 50 scans, respectively, in the case of Photofrin accumulation (5 (mu) g/ml) and after about 24 scans and 100 scans in the case of ALA administration (1.5 mg/ml). Live/dead assays revealed the loss of vitality of most of cells after 50 scans for Photofrin-labeled cells and 100 scans for ALA-labeled cells. Sensitizer-free control cells could be scanned more than 250 times (1.1 h) without impact on the reproduction behavior, morphology, and vitality.
The influence of the peak power, laser wavelength and the pulse duration of near infrared ultrashort laser pulses on the reproduction behavior of Chinese hamster ovary (CHO) cells has been studied. In particular, we determined the cloning efficiency of single cell pairs after exposure to ultrashort laser pulses with an intensity in the range of GW/cm2 and TW/cm2. A total of more than 3500 non- labeled cells were exposed to a highly focused scanning beam of a multiphoton laser microscope with 60 microsecond(s) pixel dwell time per scan. The beam was provided by a tunable argon ion laser pumped mode-locked 76 MHz Titanium:Sapphire laser as well as by a compact solid-state laser based system (Vitesse) at a fixed wavelength of 800 nm. Pulse duration (tau) was varied in the range of 100 fs to 4 ps by out-of- cavity pulse-stretching units consisting of SF14 prisms and blazed gratings. Within an optical (laser power) window CHO cells could be scanned for hours without severe impact on reproduction behavior, morphology and vitality. Ultrastructural studies reveal that mitochondria are the major targets of intense destructive laser pulses. Above certain laser power P thresholds, CHO cells started to delay or failed to undergo cell division and, in part, to develop uncontrolled cell growth (giant cell formation). The damage followed a P2/(tau) relation which is typical for a two- photon excitation process. Therefore, cell damage was found to be more pronounced at shorter pulses. Due to the same P2/(tau) relation for the efficiency of fluorescence excitation, two-photon microscopy of living cells does not require extremely short femtosecond laser pulses nor pulse compression units. Picosecond as well as femtosecond lasers can be used as efficient light sources in safe two photon fluorescence microscopy. Only in three photon fluorescence microscopy, femtosecond laser pulses are advantageous over picosecond pulses.
The influence of the peak power, laser wavelength and the pulse duration of near infrared (NIR) ultrashort laser pulses on the reproduction behavior of Chinese hamster ovary (CHO) cells has been studied. In particular we determined the cloning efficiency of single cell pairs after exposure to ultrashort laser pulses with an intensity in the range of GW/cm2 and TW/cm2. A total of more than 3500 non- labeled cells were exposed to a highly focused scanning beam of a multiphoton laser microscope with 60 microsecond pixel dwell time per scan. The beam was provided by a tunable argon ion laser pumped mode-locked 76 MHz Titanium:Sapphire laser as well as by a compact solid-state laser based system (Vitesse) at a fixed wavelength of 800 nm. Pulse duration (tau) was varied in the range of 100 fs to 4 ps by out-of-cavity pulse- stretching units consisting of SF14 prisms and blazed gratings. Within an optical (laser power) window CHO cells could be scanned for hours without severe impact on reproduction behavior, morphology and vitality. Ultrastructural studies reveal that mitochondria are the major targets of intense destructive laser pulses. Above certain laser power P thresholds, CHO cells started to delay or failed to undergo cell division and, in part, to develop uncontrolled cell growth (giant cell formation). The damage followed a P2/(tau) relation which is typical for a two-photon excitation process. Therefore, cell damage was found to be more pronounced at shorter pulses. Due to the same P2/(tau) relation for the efficiency of fluorescence excitation, two- photon microscopy of living cells does not require extremely short femtosecond laser pulses nor pulse compression units. Picosecond as well as femtosecond layers can be used as efficient light sources in safe two photon fluorescence microscopy. Only in three photon fluorescence microscopy, femtosecond laser pulses are advantageous over picosecond pulses.
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