Abstract 354: Improved Collagen Imaging in Large Pulsatile Vessels Using High-Resolution Second Harmonic Generation Microscopy and Retrospective Electrocardiogram Gating

Abstract

Introduction: Intravital microscopy (IVM) can provide important molecular and structural detail of the arterial wall in health and disease. However, in vivo image accuracy and resolution can be limited due to the pulsatile nature of the artery itself. Here, we developed and implemented a retrospective electrocardiogram (ECG) gating-based strategy to eliminate cardiac cycle dependent motion to improve IVM of the arterial wall. Methods: C57Bl/6 mice (n=5) were anesthetized and the right carotid artery was exposed and suffused with PBS. Second harmonic generation (SHG) micrographs of type 1 collagen in the arterial wall were collected (550m x 550m) at 30 frames/s on a custom-built multichannel microscope. Intravenously injected FITC-dextran (MW 2000kDa) provided an angiogram to identify the carotid artery. To enable gating, analog image capture and start signals were concurrently recorded with the ECG during image acquisition at a sampling rate of 100kHz. Gating of the imaging data set was compiled manually using the ADC software and ImageJ. Histopathology of the carotid confirmed the ex vivo carotid collagen thickness. Wall thickness was defined as the full width half maximum of SHG signal in each micrograph. Statistical significance was established as ANOVA P values < 0.05. Results: Retrospective ECG gating yielded images with a mean collagen wall thickness that was not significantly different than ex vivo measured samples (p>0.05), thus verifying the accuracy of the retrospective gating protocol. All ungated images showed significant differences (1.4 to 6.5 fold) in the measured wall thickness compared across various heart rates and frame averages (p<0.05). SNR measurements of gated and ungated images showed ungated images needed >200f averages to reach the SNR of a 30f gated image, which in turn needed 8-24s (240-720 frames) of image acquisition time, depending on the heart rate. Conclusion: Retrospective ECG gating enhances IVM accuracy of pulsatile tissues, as demonstrated by improved in vivo quantification of carotid arterial wall collagen in living mice. This strategy can be readily extended to multiplexed SHG and fluorescence IVM approaches to study other vascular disease models in vivo.