Clinical applications of multiphoton microscopy

Magnus Borstad Lilledahl

Nonlinear optical microscopy (NLOM) provides many opportunities for minimally invasive investigating tissues without exogenous staining. We try to integrate the process from image interpretion, through image analysis and biophysical model to maximize the information which can be presented to the clinician.

The information acquired may provide insight into diagnosis, improved therapeutics and better understanding of disease progression. Basically any tissue may be examined but we are focusing on three main areas: ostheoarthritis in cartilage, atherosclerosis in heart vessels as well as breast cancer. Nonlinear optical microscopy

Research topics

Ostheoarthritis in cartilage

Cartilage is the tissue which covers articular surfaces in the joints providing a smooth, low friction surface and cushioning for the subchondral bone. It has remarkable mechanical properties in its healthy state, but can, if damaged, lead to painful and disabling diseases like osteoarthritis.

Ostheoarthritis in cartilage

Cartilage is the tissue which covers articular surfaces in the joints providing a smooth, low friction surface and cushioning for the subchondral bone. It has remarkable mechanical properties in its healthy state, but can, if damaged, lead to painful and disabling diseases like osteoarthritis.

Cartilage consists of hydrated proteoglycan rich gel which is densely reinforced with collagen fibres. Second harmonic generation imaging can be used to image these fibres, providing information on how the structure imparts the mechanical properties on the tissue and what changes occur in the tissue in various disease states.

With the advent of in vivo NLOM it will be possible to image the structure of diseased cartilage which can be used to guide interventional procedures and give the clinician information for making the right choice for management of the patient.

NLOM using SHG can be used to visualize many different structures in cartilage. E.g. the difference in the collagen structure in the superficial and the middle layer of the cartilage can clearly be seen in figure 1 and 2. In the middle layer the collagen fibrils are too small to be resolved in the microscope. We are working with Mueller matrix ellipsometry to image these structures. The chondrocytes gives a moderately strong TPEF signal and can be visualized (fig. 1 and 2). Their size and shape can be quantified and their relationship to the pericondral matrix can be seen.

A large part of our work is aimed at finding quantitative methods for analyzing NLOM images. An example can be seen in figure 4 where the direction of the fibres have been found by a method employing Fourier analysis. We are also working towards the clinic to find areas in clinical practice where NLOM can provide a practical tool to improve the healthcare of people suffering from osteoarthritis and similar diseases.

Figure 1. Surface layer of cartilage. Collagen fibers in magenta (SHG) and chondrocytes in green (TPEF)

Figure 2. Middel layer of cartilage. Collagen fibers in magenta (SHG) and chondrocytes residing in their lacuna seenn in green (TPEF). Photo

Contact: Magnus Borstad Lilledahl

Thu, 08 Dec 2016 12:58:38 +0100

Atherosclerosis in heart vessels

Atherosclerosis is a disease which attacks the blood vessels. The disease is characterized by the formation of a plaque in the vessel wall, which is a pathological modification of the vessel wall structure. These plaques can have different sizes, shapes and morphological structures.

Atherosclerosis in heart vessels

Atherosclerosis is a disease which attacks the blood vessels. The disease is characterized by the formation of a plaque in the vessel wall, which is a pathological modification of the vessel wall structure. These plaques can have different sizes, shapes and morphological structures.

It is believed that a large part of these plaques which are called vulnerable plaques are the cause of the majority of heart attacks. These plaques consist of a thick lipid core covered by a thin fibrous cap, consisting mainly of collagen. The danger is that these plaques can rupture and cause a thrombosis which obstructs the artery, causing a heart attack.

Nonlinear optical microscopy provides (NLOM) is an ideal technique for assessing the vulnerability of these plaques. The collagen cap can easily be detect using the SHG signal of the collagen in the cap (the normal vessel has much less collagen and much more elastin). In addition the 3D dimensional imaging capability of NLOM can be used to assess the thickness of these vulnerable plaques since the thickness of the cap is an important parameter for the mechanical strength of the plaque.

We are also using NLOM to investigate the more detailed structure of the collagen fibres in the cap to see if the structure in addition to the thickness can also provide more information on the vulnerability of the plaque.

Publications

Characterization of vulnerable plaques by multiphoton microscopy. Journal of Biomedical Optics, 2007.

Collagen network in the cap of an atherosclerotic plaque. Red is SHG from collagen fibers and green is TPEF most likely due to cellular compounds or lipid deposits. Photo: Magnus Lilledahl

Contact: Magnus Borstad Lilledahl

Tue, 13 Dec 2016 14:51:46 +0100

Breast cancer

Breast cancer is a disease with a quite high incidence rate. Succesfull screening programs have fortunately led to a quite high chance of successful treatment following diagnosis. However, there is still room for enhancing diagnosis and disease stratifcation to schedule the patient for appropriate treatment.

Breast cancer

Breast cancer is a disease with a quite high incidence rate. Succesfull screening programs have fortunately led to a quite high chance of successful treatment following diagnosis. However, there is still room for enhancing diagnosis and disease stratifcation to schedule the patient for appropriate treatment.

Recently there has been increased emphasis on the extracellular matrix (ECM) component of tumors. There has been an increased understanding that the structure of the ECM can be used as an important parameter in determining the degree of malignancy.

We are using second harmonic generation (SHG) to investigate the structural alterations in tumors. Based on various image analysis algorithms we try to predict clinical outcome based on tissue structure. This work is a collaboration with Anna Bofin at the Breast cancer subtypes group.

Contact: Magnus Borstad Lilledahl

Thu, 08 Dec 2016 12:59:06 +0100

Heart valves

The valves of the heart prevent blood from flowing in the wrong direction between the ventricle and the atria. Sometimes these valves do not function exactly like they should. These problems can be visualized by ultrasound. However, to assess what is wrong, mechanical models of the valves are needed to understand the ultrasound images.

Heart valves

The valves of the heart prevent blood from flowing in the wrong direction between the ventricle and the atria. Sometimes these valves do not function exactly like they should. These problems can be visualized by ultrasound. However, to assess what is wrong, mechanical models of the valves are needed to understand the ultrasound images.

The heart valves consists mainly of collagen and elastin, both of which can be imaged by NLOM; collagen by SHG and elastin by TPEF. By imaging the structure and directionality of theses fibres, important input information for the mechanical models can be achieved.

We are also imaging these structures under various strains to better understand how the fibres impart the mechanical strength to the tissue.

Collaborators

Bjørn Helge Skallerud, Department of Structural Engineering (NTNU)

Collagen fibres in the chordae tendinae which attach the heart valve to the papillary muscles. Photo: Magnus B. Lilledahl

Contact: Magnus Borstad Lilledahl

Thu, 08 Dec 2016 12:59:19 +0100
Thu, 08 Dec 2016 12:59:33 +0100

NLOM imaging is both science and art

Photo 1: Magnus Borstad Lilledahl

Photo 2: Magnus Borstad Lilledahl

Photo 3: Magnus Borstad Lilledahl