Second harmonic generation (SHG) microscopy is a promising imaging technique for collagenous tissues due to its noninvasiveness and potential for 3D imaging. In tissues with densely packed and thin collagen fibrils, such as cartilage, the focal volume of the laser can comprise multiple fibrils, and the SHG from each of these fibrils can interact and contribute to the detected signal. Coherent amplification is achieved when the fibrils are aligned and oriented in the same direction. The effect of changing the size of the focal volume, as determined by the numerical aperture (NA), is therefore dependent on the length scale at which the fibrils are aligned. This effect on the image contrast is important to examine experimentally before SHG can be used in the clinic. In this study, we measured the SHG intensity and radiation direction as a function of the NA of the excitation and collecting objectives in different areas of the immature articular cartilage of young pigs. The results showed that varying the NA of the excitation objective had more effect on the SHG signals detected in the forward compared to the backward direction and that this effect varied considerably throughout the tissue. The results clearly demonstrated that SHG imaging differs from conventional histology, and the image contrast should only be interpreted in light of the imaging conditions.
The second harmonic generation from collagen is highly sensitive to what extent collagen molecules are ordered into fibrils as the SHG signal is approximately proportional to the square of the fibril thickness. This can be problematic when interpreting SHG images as thick fibers are much brighter than thinner fibers such that quantification of the amount of collagen present is difficult. On the other hand SHG is therefore also a very sensitive probe to determine whether collagen have assembled into fibrils or are still dissolved as individual collagen molecules. This information is not available from standard histology or immunohistochemical techniques. The degree for fibrillation is an essential component for proper tissue function. We will present the usefulness of SHG imaging in tissue engineering of cartilage as well as cartilage related pathologies. When engineering cartilage it is essential to have the appropriate culturing conditions which cause the collagen molecules to assemble into fibrils. By employing SHG imaging we have studied how cell seeding densities affect the fibrillation of collagen molecules. Furthermore we have used SHG to study pathologies in developing cartilage in a porcine model. In both cases SHG reveals information which is not visible in conventional histology or immunohistochemistry
Human atherosclerotic samples collected by carotid endarterectomy were investigated using electronic paramagnetic resonance imaging (EPRI) for visualization of reactive oxygen species, and nonlinear optical microscopy (NLOM) to study structural features. Regions of strong EPRI signal, indicating a higher concentration of reactive oxygen species and increased inflammation, were found to colocalize with regions dense in cholesterol crystals as revealed by NLOM.
Osteoarthritis is one of the most prevalent joint diseases in the world. Although the cause of osteoarthritis is not exactly clear, the disease results in a degradation of the quality of the articular cartilage including collagen and other extracellular matrix components. We have investigated alterations in the structure of collagen fibers in the cartilage tissue of the human knee using mulitphoton microscopy. Due to inherent high nonlinear susceptibility, ordered collagen fibers present in the cartilage tissue matrix produces strong second harmonic generation (SHG) signals. Significant morphological differences are found in different Osteoarthritic grades of cartilage by SHG microscopy. Based on the polarization analysis of the SHG signal, we find that a few locations of hyaline cartilage (mainly type II collagen) is being replaced by fibrocartilage (mainly type I cartilage), in agreement with earlier literature. To locate the different types and quantify the alteration in the structure of collagen fiber, we employ polarization-SHG microscopic analysis, also referred to as _-tensor imaging. The image analysis of p-SHG image obtained by excitation polarization measurements would represent different tissue constituents with different numerical values at pixel level resolution.
A method for measuring three-dimensional (3-D) direction images of collagen fibers in biological tissue is presented. Images of the 3-D directions are derived from the measured transmission Mueller matrix images (MMIs), acquired at different incidence angles, by taking advantage of the form birefringence of the collagen fibers. The MMIs are decomposed using the recently developed differential decomposition, which is more suited to biological tissue samples than the common polar decomposition method. Validation of the 3-D direction images was performed by comparing them with images from second-harmonic generation microscopy. The comparison found a good agreement between the two methods. It is envisaged that 3-D directional imaging could become a useful tool for understanding the collagen framework for fibers smaller than the diffraction limit.
One of the main advantages of nonlinear microscopy is that it provides 3D imaging capability. Second harmonic generation is widely used to image the 3D structure of collagen fibers, and several works have highlighted the modification of the collagen fiber fabric in important diseases. By using an ellipsoidal specific fitting technique on the Fourier transformed image, we show, using both synthetic images and SHG images from cartilage, that the 3D direction of the collagen fibers can be robustly determined.
The collagen meshwork in articular cartilage of chicken knee is characterized using Mueller matrix imaging and multiphoton microscopy. Direction and degree of dispersion of the collagen fibers in the superficial layer are found using a Fourier transform image-analysis technique of the second-harmonic generated image. Mueller matrix images are used to acquire structural data from the intermediate layer of articular cartilage where the collagen fibers are too small to be resolved by optical microscopy, providing a powerful multimodal measurement technique. Furthermore, we show that Mueller matrix imaging provides more information about the tissue compared to standard polarization microscopy. The combination of these techniques can find use in improved diagnosis of diseases in articular cartilage, improved histopathology, and additional information for accurate biomechanical modeling of cartilage.
Cartilage from the medial femoral condyle of chicken was sectioned and imaged using second harmonic generation
microscopy. Using image analysis techniques based on the Fourier transform we derived quantitative threedimensional
data of the fiber direction and dispersion of the collagen fiber network in the superficial layer. These
data can be used directly in biomechanical models to enhance the fidelity of these models.
Mechanical models are important in many areas of medicine and physiological research. These are usually based
on continuum mechanics using macroscopic mechanical parameters. However, knowledge of the microscopic
structure of tissue, that is, the organization of structural proteins, gives useful information for improving such
models for a given tissue. In this paper we image the structural changes in these proteins under strain by using
multiphoton microscopy. This gives insight into the response to the tissue at microscopic level and can be used
to modify existing mechanical models of tissue. Specifically we imaged the straightening of collagen fibers in
bovine chordae tendinae and related this to the macroscopic strain applied to the tissue.
Cardiovascular disease is the primary cause of death in the United States; the majority of these deaths are caused by the rupture of vulnerable plaques. An important feature of vulnerable plaques is the thickness of the fibrous cap that covers the necrotic core. A thickness of less than 65 µm has been proposed as a value that renders the plaque prone to rupture. This work shows that multiphoton microscopy (MPM) can image the plaque with µm resolution to a depth deeper than 65 µm. The fibrous cap emits primarily second harmonic generation due to collagen, in contrast to the necrotic core and healthy artery, which emits primarily two-photon excited fluorescence from elastin. This gives a good demarcation of the fibrous cap from underlying layers, facilitating the measurement of the fibrous cap thickness. Based on a measure of the collagen/elastin ratio, plaques were detected with a sensitivity of 65% and specificity of 81%. Furthermore, the technique gives detailed information on the structure of the collagen network in the fibrous cap. This network ultimately determines the mechanical strength of the plaque. A mechanical model based on this information could yield a measure of the propensity of the plaque to rupture.
Multiphoton microscopy is a techniques that fascilitates three dimensional imaging of intact, unstained tissue.
Especially connective tissue has a relatively strong nonlinear optical response and can easily be imaged.
Atherosclerosis is a disease where lipids accumulate in the vessel wall and there is a thickening of the intima by
growth of a cap of connective tissue. The mechanical strength of this fibrous cap is of clinically importance. If
the cap ruptures a thrombosis forms which can block a coronary vessel and therby causing myocardial infarction.
Multiphoton microscopy can be used to image the fibrous cap and thereby determine the thickness of the cap
and the structure of the connective fibres. This could possibly be developed into a diagnostic and clincal tool to
monitor the vulnerability of a plaque and also to better understand the development of a plaque and effects of
treatment. We have collected multiphoton microscopy images from atherosclerotic plaque in human aorta, both
two photon excited fluorescens and second harmonic generated signal. The feasability of using this technique to
determine the state of the plaque is explored.
Determining the cause of an injury and the force behind the impact may be of crucial importance in a court case.
For non-penetrating soft tissue injuries there is a lack of information available in the literature. In this study
controlled bruises were inflicted on an anesthetized pig by high speed, low-weight paintball projectiles (diameter
17.1 mm, weight 3.15 g). The speed of the object and the impact itself were monitored using a high speed
camera. Punch biopsies (5 mm) were collected from the injury sites. A red and purple ring with a diameter of
1.5 cm appeared on the skin within 30 seconds after the paintball impact. The ring was visually fully established
after 5-10 minutes. Numerical finite element simulations performed with ABAQUS\Explicit showed a build up of
shear stresses in the skin where the ring formed. Biopsies demonstrated severe dermal vessel damage in the same
area. It is concluded that considerable shear stresses during the impact will create dermal vessel damage and
thereby cause a visible bruise. Larger forces are required for compressive stresses to inflict equivalent vascular
damage.
Heart disease is the primary cause of death in the western world. Many of these deaths are caused by the rupture of vulnerable plaque. Vulnerable plaques are characterized by a large lipid core covered by a thin fibrous cap. One method for detecting these plaques is reflection spectroscopy. Several studies have investigated this method using statistical methods. A more analytic and quantitative study might yield more insight into the sensitivity of this detection modality. This is the approach taken in this work. Reflectance spectra in the spectral region from 400 to 1700 nm are collected from 77 measurement points from 23 human aortas. A measure of lipid content in a plaque based on reflection spectra is presented. The measure of lipid content is compared with the thickness of the lipid core, determined from histology. Defining vulnerable plaque as having a lipid core >500 µm and fibrous cap <500 µm, vulnerable plaques are detected with a sensitivity of 88% and a specificity of 94%. Although the method can detect lipid content, it is not very sensitive to the thickness of the fibrous cap. Another detection modality is necessary to detect this feature.
When performing intravascular spectroscopy there is always the possibility that there will be a thin layer of blood between the probe and the vessel wall. This will affect measurements espescially below 600 nm where blood absorption is strong (important for fluorescence and Raman measurements) and above 1400 nm where the water absorption is strong. Between these two regions (the theraputic window) the absorption is fairly low and can be neglected. This article explores the possibility of performing measurements outside the optical window by minimizing blood layer and increasing the excitation signal.
An objective method for dating of soft tissue injuries is highly desired in forensic medicine. To achieve this, obtaining a fundamental understanding of extravascular blood behavior is necessary. Porcine blood mixed with fluorescein was inserted subcutaneously on an anaesthetized pig. The fluorescence build-up was measured as a function of time, using a fiber probe setup. Fluorescence measurements proved to be a suitable tool for
monitoring the temporal distribution of fluid in a subcutaneous hemorrhage. Reasonable values for the fluid velocity was obtained by monitoring the fluorescein distribution around the infusion point. Further theoretical work and development of the experimental technique is necessary to obtain a good understanding of subcutaneous transport of blood.
Many methods for detecting and measuring vulnerable atherosclerotic plaques have been proposed. These include reflection spectroscopy, thermography, ultrasound, computed tomography (CT) and magnetic resonance imaging (MRI). This paper presents an analysis and a comparison of two of these methods, near-infrared reflection spectroscopy (NIRS) and thermography.
Most of the published literature evaluate methods statistically. A more analytic approach will make it easier to compare the different methods and determine if the measured signal will be strong enough in a real measurement situation. This is the approach taken in this article.
Eight samples of human aorta were examined by NIRS and subsequently prepared for histology. A total of 28 measurement points were selected. A measure of the lipid content based on reflection spectra is proposed. Comparisons of this lipid measure with histology show that the lipid content in the plaques yields relatively small changes in the value of this lipid-index. Reflectance spectra from models based on the diffusion approximation for total reflectance were simulated.
Temperature measurements were performed on three Watanabe heritable hyperlipidemic (WHHL) rabbits and one New Zealand white (NZW) rabbit with a thermistor-type intravascular temperature sensor. The measurements gave no significant signals which correlated with the subsequent histology. A simple analytic model was developed which indicates that a temperature increase of more than 0.01-0.04 °C at the surface of a vessel wall, due to inflammation in a plaque, is unlikely. Such a small temperature difference will probably be obscured by normal variation in the vessel wall temperature.
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.