Microscopic chemical mapping of living plant tissues without the use of extrinsic labels would represent a major
advance in analytical capability for many areas of biological research; Coherent Raman Scattering (CRS) microscopy
offers label-free chemical imaging based on vibrational spectroscopy and is an obvious solution. However, due to the
high levels of optical absorption and fluorescent emission in plant tissues the technique is severely limited for in-vivo
plant imaging. This paper reports preliminary results regarding the technical issues associated with performing label-free
imaging in plant tissues with CRS and discusses how they may be mitigated in future applications.
Second harmonic generation (SHG) and two-photon fluorescence (TPF) microscopy is used to image the intercellular and pericellular matrix in normal and degenerate equine articular cartilage. The polarization sensitivity of SHG can be used directly to determine fiber orientation in the superficial 10 to 20 µm of tissue, and images of the ratio of intensities taken with two orthogonal polarization states reveal small scale variations in the collagen fiber organization that have not previously been reported. The signal from greater depths is influenced by the birefringence and biattenuance of the overlying tissue. An assessment of these effects is developed, based on the analysis of changes in TPF polarization with depth, and the approach is validated in tendon where composition is independent of depth. The analysis places an upper bound on the biattenuance of tendon of 2.65×10−4. Normal cartilage reveals a consistent pattern of variation in fibril orientation with depth. In lesions, the pattern is severely disrupted and there are changes in the pericellular matrix, even at the periphery where the tissue appears microscopically normal. Quantification of polarization sensitivity changes with depth in cartilage will require detailed numerical models, but in the meantime, multiphoton microscopy provides sensitive indications of matrix changes in cartilage degeneration.
Articular cartilage possesses an extensive extracellular matrix consisting of a highly organised network of collagen
fibres embedded in a much finer mesh of proteoglycans and other glycoproteins. Many fundamental issues of cartilage
biomechanics, its ageing and the development of osteoarthritis concern the detailed organisation of this matrix. Here we
investigate the application of multi-photon microscopy to characterise the structure of the extracellular matrix. In
reflection mode both second harmonic Generation (SHG) and two photon fluorescence (TPF) imaging modalities reveal
differences in the pericellular and inter-territorial matrix in normal tissue and additional changes in degenerative lesions.
The SHG signal from the surface zone is dependent on the direction of polarization of the laser excitation beam but the
TPF signal is not. The former can be quantified to determine fibre orientation although the pattern is less well resolved
than in tendon, reflecting the less regular orientation of the finer fibres. Nevertheless, previously unreported subtle
variations in fibre orientation over the surface of the cartilage can be observed. In order to characterise variations with
depth we carried out polarization sensitivity experiments at depths up to 180 microns into the tissue. At greater depths
the polarization sensitivity is affected by the birefringence and dichroism of the overlying tissue and we have quantified
these effects to allow correction of the data.
Equine articular cartilage has been imaged using both polarization-sensitive optical coherence tomography (PS-OCT)
and non-linear microscopy. PS-OCT has been used to spatially map the birefringence in the cartilage and we have found
that in the vicinity of the lesion the images display a characteristic disruption in the regular birefringence bands shown by
normal cartilage. We also note that significant (e.g. x2) variations in the apparent birefringence of samples taken from
young (18 month) animals that otherwise appear visually homogeneous are found over spatial scales of a few
millimeters. We have also imaged the cartilage using non-linear microscopy and compare the scans taken with second
harmonic generation (SHG) light and the two photon fluorescence (TPF) light. SHG images collected using 800 nm
excitation reveals the spatial distribution of collagen fibers, whilst TPF images clearly shows the distribution of
intracellular and pericellular fluorophores.
Sub cellular resolution images of equine articular cartilage have been obtained using both second harmonic generation microscopy (SHGM) and two-photon fluorescence microscopy (TPFM). The SHGM images clearly map the distribution of the collagen II fibers within the extracellular matrix while the TPFM images show the distribution of
endogenous two-photon fluorophores in both the cells and the extracellular matrix, highlighting especially the pericellular matrix and bright 2-3μm diameter features within the cells. To investigate the source of TPF in the extracellular matrix experiments have been carried out to see if it may originate from the proteoglycans. Pure solutions of the following proteoglycans hyaluronan, chondroitin sulfate and aggrecan have been imaged, only the aggrecan produced any TPF and here the intensity was not great enough to account for the TPF in the extracellular matrix. Also cartilage samples were subjected to a process to remove proteoglycans and cellular components. After this process the TPF from the samples had decreased by a factor of two, with respect to the SHG intensity.
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