Histology, biochemistry, microscopy
Deformation mechanisms in soft biological tissues often depends on density and
configuration of collagen fibers. Fibers are initially crimped and provide a
progressive contribution to the mechanical response. Other extracellular matrix
components contribute determining the elastic and dissipative deformation
behavior. Physically based models are formulated using information related to
the distribution of fibers, mechanisms of re-orientation, cross-link density,
connectivity, fibers bending and tensile stiffness, and interaction with a
matrix. We combine mechanical characterization with histological, biochemical or
microscopy based analysis of soft tissue microstructure.
In [14p],
[17p],
[5t],
[33p]
mechanical parameters obtained from in-vivo measurements on liver were
correlated with corresponding histological information. In [35p],
[18p],
mechanical data were compared with sample specific biochemical assays providing
information on content of total collagen, elastin or collagen cross-link
density. Elastin is assessed colorimetrically (using a Fastin Elastin assay kit,
Biocolor Ltd., Newton-abbey, Northern Ireland) after extraction by oxalic acid.
For the estimation of the total collagen, an acid hydrolysis method is applied
to determine the hydroxyproline. Measurement of two collagen cross-links
pyridinoline (PYD) and deoxypyridinoline (DPD) was performed by high performance
liquid chromatography (HPLC).
Microscope: Zeiss LSM 5 PASCAL
Our light microscopy system acquires images in defined optical sections of
the material sample by means of a laser beam in a confocal path, and assembles
them into a three-dimensional image stack. Our microscope is placed in a laminar
flow clean box which offers a filtered airflow across work area and allows
investigations in a dust and contamination reduced environment.
With the use of
laser light one optically resolves lateral structures down to 300nm.
Furthermore, it allows the detection and measurement of height differences from
about 20nm to the order of millimeters. In the transmission mode one may realize
for the biology and medicine typical contrast procedures as the Differential
Interference Contrast (DIC). The overlapping with a confocal fluorescence
picture of the same test place is also possible.
Technical Data:
- Resolution in xy (lateral): 300nm
- Resolution in z: 20nm
- Laser Specifications: 458, 488, 514nm, Laser Class 3 B
- Working Modes: Reflection Mode, Transmission Mode,
Fluorescence Mode
The multiphoton microscope can be combined with a in situ
stretching device submerged in saline solution. This configuration allows to
image biological materials under deformation and avoids tissue drying during
measurements. Uniaxial stress, uniaxial strain, or biaxial stress are all
possible configurations with a maximal nominal strain of 200%. The deformed
samples is imaged using a specific wavelength so that second harmonic generation
of the collagen and autofluorescence of elastin can be simultaneously detected
without staining.
The excitation through two photons has the advantage of
using long infrared wavelengths, which are able to penetrate deeper into
biological tissues. This excitation technique allows to drastically reduce the
background scattering (from out of plane fluorescence) present in conventional
confocal fluorescence microscopy.
The microscope station is controlled by Olympus FVlo-ASW
software, which allows: fast / slow 2D, 3D stacks, and time-stepping (4D)
imaging. The results data are compatible with Imaris software for image
analysis. The setup includes a pre-compensated Ti:Sapphire Laser, which supports
emission wavelength between 680 – 1050 nm. The microscope can be equipped with
an air- or water-objective. The latter can be used for imaging living tissue
immersed in physiological solution (NaCl 0.9%).
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