Nonlinear Optical Microscopy – Advancing Diagnostics and Improving Treatments

Science Highlights

Imaging Neurological Disease and Injury

Catching Unstable Plaques


Unstable fatty deposits on the inner walls of blood vessels (atherosclerotic plaques) can cause strokes and heart attacks. Some of these plaques may be missed with traditional diagnostic tools. Cheng's team and collaborators at Indiana University have recently reported the first multimodal multiphoton imaging of atherosclerotic plaques in the arteries of an obese pig. The chemical composition of plaques can be evaluated, which allows for prediction of plaque rupturing . "Right now, an unstable plaque does not show as a major flow-limiting stenosis, but if we could visualize it and put a stent there, then that would prevent a plaque from rupturing," explains Indiana University Professor of Cellular and Integrative Physiology Michael Sturek. Future applications may include examining the effect of novel drugs for atherosclerosis, evaluating the narrowing of blood vessels (stenosis) and combining NLO microscopy with existing diagnostic tools, such as intravascular ultrasound.

Nearly half a million people in the United States are living with spinal cord injury and another 400,000 are affected by multiple sclerosis (MS). These conditions are characterized by loss of myelin sheaths, a major component of a part of the nervous system called white matter. Like plastic coating around an electrical wire, myelin sheaths act as insulators, facilitating conduction of nerve impulses. Loss of myelin sheaths, termed demyelination, leads to a delay or blockage of neurological signals resulting in often-fatal impairments of movement, hearing, speech, or vision. The process of demyelination is poorly understood, in part due to an inability to image subcellular details in intact white matter.

Recently, Ji-Xin Cheng, Assistant Professor of Biomedical Engineering and Chemistry at Purdue University, identified some crucial roles of calcium and glutamate in triggering demyelination. These findings will aid researchers in understanding nerve damage and could promote better treatment of diseases such as MS. The discovery of these triggers was made using a novel imaging technique called Coherent anti-Stokes Raman scattering (CARS) microscopy. CARS detects the vibrations of chemical bonds and is particularly sensitive in imaging fatty substances, like myelin. The technique provides real-time imaging of changes in the myelin sheath following spinal cord injury and allows for measurement of dynamic parameters of demyelination, such as myelin sheath thickness.

CARS is one of several nonlinear optical (NLO) techniques, which have recently emerged as unparalleled tools for high-resolution imaging of living tissues. NLO techniques have several advantages compared with other imaging methods. For example, NLO techniques rarely require the use of dyes. Dyes provide contrast but also alter the natural state of molecules inside cells. Conventional microscopy – such as looking at cells through a microscope in a high school biology class – requires very thin tissue samples because light scattering causes thick samples to appear fuzzy. In contrast, NLO techniques are much less affected by light scattering and provide excellent three-dimensional (3D) resolution, even when imaging deep into tissues. NLO techniques allow scientists to monitor dynamic biological processes in living tissues such as immune response, brain disease, and tumor development over time. In addition to utilizing CARS microscopy, researchers at Purdue have integrated two other NLO imaging modalities – two-photon excitation fluorescence (TPEF) and sum frequency generation (SFG)—on a CARS microscope, which they termed multimodal multiphoton microscopy (MMM).

Cheng’s group has applied MMM to visualize live spinal cord and sciatic nerve in 3D. In the next few years, they will utilize MMM to study the causes of myelin degradation in MS and spinal cord injury. In collaboration with other researchers, Cheng is exploring the potential of NLO techniques in many medically important areas, including cancer, heart disease, and obesity.

Examining Tumor Architecture

A three-dimensional image of spinal cord white matter as seen using MMM.
A 3D image of spinal cord white matter as seen using MMM. The red, green, and magenta colors represent the CARS signal from myelin sheath, the SFG signal from astrocyte processes, and the TPEF signal from ethidium bromide labeled nuclei, respectively. Image courtesy of Yan Fu and Ji-Xin Cheng.

MMM offers the prospect of examining tumors in the body without the need for biopsies. Moreover, it enables investigation of the spatial organization of a tumor and its surrounding connective tissue, which cannot be visualized on thin biopsy slices. Cheng and Ignacio Camarillo, Assistant Professor of Biological Sciences at Purdue University, applied MMM to simultaneously image breast fat cells, blood capillaries, collagen fibers, and tumor cells at high 3D resolution. In conventional cancer diagnostics, physicians often introduce labels or tracers to distinguish cancer tissue from healthy tissue. For example, radioactive molecules are used in PET scans, fluorescent dyes in optical imaging, and magnetic particles in MRI. However, “In a patient, it is very difficult to label a specific cell or a tumor. One can use multimodal multiphoton microscopy to detect different types of tumors in the body, without labeling,” says Cheng.

“Obesity increases the risk of several types of cancer, including breast, uterine, prostate and colon. Obese patients have higher morbidity rates and increased [cancer] drug resistance. Many people are not aware of this relationship,” cautions Camarillo. He and Cheng used MMM to compare tissues from lean and obese rats and determined structural differences that may be an underlying factor in the link between obesity and cancer. Camarillo’s research group is planning to use MMM to monitor tissue changes in response to therapy over time and characterize different obesity-associated tumors. “Imaging the 3D architecture of tissues can provide significant new insights into tumor formation and changes that lead to tumor progression. Standard histology cannot provide that,” concludes Camarillo.

Future Directions

Cheng anticipates that this state-of-the-art imaging technology will find many applications in the coming decades. The cosmetics and pharmaceutical industries are both eager to embrace these new imaging techniques. Some drugs are imbedded in biodegradable materials, termed polymers, which control the timing of drug release and delivery to tissues. Although drug molecules per se are not visible under a fluorescence microscope, their polymer coatings can be readily visualized using CARS imaging. “CARS will be a wonderful system to study drug release from polymer matrix and drug distribution in a tissue,” said Cheng.

“Another key direction will be developing endoscopy for in vivo diagnosis of tumors as well as atherosclerotic lesions,” indicates Cheng. As a first step, Cheng’s group recently tested a miniature lens that can be inserted into an animal through the skin to image deep tissues in real time.

A final area for future application is individualized medicine. Some patients do not respond to standard medications for certain diseases. For example, in up to 70 percent of people, widely used cholesterol-lowering drugs – statins such as Lipitor and Crestor – do not prevent heart attacks. By providing a very close and detailed look at the site of disease, MMM will help researchers understand the architecture of diseased tissues. In turn, this understanding will provide a basis for developing new, more effective drugs. Application of MMM in the clinic will offer a promise of improved diagnostics and treatments tailored to individual needs.

This research is funded in part by the National Institute of Biomedical Imaging and Bioengineering, the National Heart, Lung, and Blood Institute, the National Cancer Institute, and the National Center for Research Resources.

Cheng JX. Coherent anti-Stokes Raman scattering microscopy. Appl Spectrosc. 2007;61:197-208.

Fu Y, Wang H, Huff TB, Shi R, Cheng JX. Coherent anti-Stokes Raman scattering imaging of myelin degradation reveals a calcium dependent pathway in Lyso-PtdCho induced demyelination. J Neurosci Res. 2007;85:2871-81.

Kang E, Wang H, Kwon IK, Robinson J, Park K, Cheng JX. In situ visualization of paclitaxel distribution and release by Coherent anti-Stokes Raman scattering microscopy. Anal Chem. 2006;78:8036-43.

Le TT, Langohr IM, Locker MJ, Sturek M, Cheng JX. Label-free molecular imaging of atherosclerotic lesions using multimodal nonlinear optical microscopy. J Biomed Opt. 2007;12:054007.

Le TT, Rehrer CW, Huff TB, Nichols MB, Camarillo IG, Cheng JX. Nonlinear optical imaging to evaluate the impact of obesity on mammary gland and tumor stroma. Mol Imaging. 2007;6:205-11.

Wang H, Huff TB, Fu Y, Jia KY, Cheng JX. Increasing the imaging depth of Coherent anti-Stokes Raman scattering microscopy with a miniature microscope objective. Opt Lett. 2007;32:2212-4.

Cross-section view of an atherosclerotic plague in an artery imaged by CARS, TPEF, and SFG on the same platform.
(a) Cross-section view of an atherosclerotic plaque in an artery imaged by epi-detected CARS, TPEF, and SFG on the same platform. The plaque is enriched in lipids (grey) and auto fluorescence (green) but devoid of collagen fibrils (blue). (b) Characteristics of different types of plaque defined by American Heart Association Lesion Committee. Image courtesy of Han-Wei Wang and Ji-Xin Cheng.


CARS imaging of fat cells (red) and SHG (Second Harmonic Generation - another NLO technique) imaging of collagen fibrils (green) used to evaluate the imapct of obesity on breast tumors.
CARS imaging of fat cells (red) and SHG (Second Harmonic Generation - another NLO technique) imaging of collagen fibrils (green) used to evaluate the impact of obesity on breast tumors. Images in the top row are from lean rats, while those in the bottom row are from obese rats. The first image in each row (A and E) is of mammary glands, while the remaining images (B-D and F-H) are of the tissue surrounding the tumors (stroma). These images indicate that obesity is associated with decreased collagen content in mammary glands and increased collagen content and aggressiveness in breast tumors. Scale bars = 25 um. Image courtesy of Ignacio Camarillo.
Health Terms
Breast Cancer
Heart Disease
Prostate Cancer