Martin Stark, Daniel Dörr, Alexander Ehlers, Daniel Sauer, Rainer Bückle, Sven Martin, Friederike Ehrhart, Jennifer Baunach, Alisa Katsen-Globa, Heiko Zimmermann, Karsten König
Monitoring the functional status of cryo-preserved cells and tissue in-situ, i.e. in the frozen state, might allow for optimal
adjustment of preservation conditions and might provide the information necessary to predict a functionality recovery
rate. Here, an imaging approach with compositional sensitivity seems favourable.
In our approach we use multiphoton microscopy in combination with fluorescence lifetime imaging to investigate cells,
human and plant tissue at cryogenic conditions. By the non-linearity of multiphoton excitation we largely suppress image
distortions attributed to scattering of incoming light. Only where the intensity of the pulsed near-infrared laser beam is
sufficiently large, significant fluorescence is excited. This allows reaching penetration depth in ice comparable to the
liquid state. As additional information we use the fluorescence decay to assign compositional entities. Results obtained
on cells and tissues are discussed with respect to temperature dependencies and the related use for applications.
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.
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