The ability to distinguish macrophage subtypes noninvasively could have diagnostic potential in cancer, atherosclerosis, and diabetes, where polarized M1 and M2 macrophages play critical and often opposing roles. Current methods to distinguish macrophage subtypes rely on tissue biopsy. Optical imaging techniques based on light scattering are of interest as they can be translated into biopsy-free strategies. Because mitochondria are relatively strong subcellular light scattering centers, and M2 macrophages are known to have enhanced mitochondrial biogenesis compared to M1, we hypothesized that M1 and M2 macrophages may have different angular light scattering profiles. To test this, we developed an in vitro angle-resolved forward light scattering measurement system. We found that M1 and M2 macrophage monolayers scatter relatively unequal amounts of light in the forward direction between 1.6 deg and 3.2 deg with M2 forward scattering significantly more light than M1 at increasing angles. The ratio of forward scattering can be used to identify the polarization state of macrophage populations in culture.

Objectives

This study hypothesized that bright spots in intravascular optical coherence tomography (IVOCT) images may originate by colocalization of plaque materials of differing indexes of refraction. To quantitatively identify bright spots, we developed an algorithm that accounts for factors including tissue depth, distance from light source, and signal-to-noise ratio. We used this algorithm to perform a bright spot analysis of IVOCT images and compared these results with histological examination of matching tissue sections.

Background

Bright spots are thought to represent macrophages in IVOCT images, and studies of alternative etiologies have not been reported.

Methods

Fresh human coronary arteries (n = 14 from 10 hearts) were imaged with IVOCT in a mock catheterization laboratory and then processed for histological analysis. The quantitative bright spot algorithm was applied to all images.

Results

Results are reported for 1,599 IVOCT images co-registered with histology. Macrophages alone were responsible for only 23% of the bright spot-positive regions, although they were present in 57% of bright spot-positive regions (as determined by histology). Additional etiologies for bright spots included cellular fibrous tissue (8%), interfaces between calcium and fibrous tissue (10%), calcium and lipids (5%), and fibrous cap and lipid pool (3%). Additionally, we showed that large pools of macrophages in CD68(+) histology sections corresponded to dark regions in comparative IVOCT images; this is due to the fact that a pool of lipid-rich macrophages will have the same index of refraction as a pool of lipid and thus will not cause bright spots.

Conclusions

Bright spots in IVOCT images were correlated with a variety of plaque components that cause sharp changes in the index of refraction. Algorithms that incorporate these correlations may be developed to improve the identification of some types of vulnerable plaque and allow standardization of IVOCT image interpretation.