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.
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).
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 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.
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.
Access to the requested content is limited to institutions that have purchased or subscribe to SPIE eBooks.
You are receiving this notice because your organization may not have SPIE eBooks access.*
*Shibboleth/Open Athens users─please
sign in
to access your institution's subscriptions.
To obtain this item, you may purchase the complete book in print or electronic format on
SPIE.org.
INSTITUTIONAL Select your institution to access the SPIE Digital Library.
PERSONAL Sign in with your SPIE account to access your personal subscriptions or to use specific features such as save to my library, sign up for alerts, save searches, etc.