Longitudinal, multimodal functional imaging of microvascular response to photothermal therapy

Abstract

Although studies have shown that photothermal therapy can coagulate selectively abnormal vasculature, the ability of this method to achieve consistent, complete removal of the vasculature is questionable. We present the use of multimodal, wide-field functional imaging to study, in greater detail, the biological response to selective laser injury. Specifically, a single-platform instrument capable of coregistered fluorescence imaging and laser speckle imaging was utilized to monitor vascular endothelial growth factor gene expression and blood flow, respectively, in a transgenic rodent model. Collectively, the longitudinal, in vivo data collected with our instrument suggest that the biological response to selective laser injury involves early-stage redistribution of blood flow, followed by increased vascular endothelial growth factor promoter activity to stimulate pro-angiogenic events.

Fig. 1

Schematic of experimental setup used to monitor VEGF promoter activity and blood flow in the dorsal window chamber microvasculature in response to selective laser injury. A, CCD camera; B, zoom lens; C, 532 nm emission filter; D, ring-light illuminator (488 nm excitation); E, window chamber; F, anesthesia nose cone; G, heating bed (to water heater/circulator); H, mirror; I, dual polarizers for attenuation; J, modular light port (633 nm He–Ne and white halogen); K, light-tight enclosure.

Fig. 2

(Color online) Representative normalized blood-flow maps derived from raw speckle images, using LSI-based analysis with a simplified speckle imaging equation. Blood-flow maps are shown at 1× (left) and 2× (right) optical magnification taken at exposure times of 10, 100, and 1000 ms. Note the increase in discernible vasculature in the blood-flow maps taken with longer exposure times.

Fig. 3

(Color online) Representative mouse dorsal window chamber imaged on Days 0 (pre-laser and post-laser irradiation), 1, 3, and 7, at 1× (top row) and 4× (middle and bottom rows) optical magnifications, to observe the vascular repair response to selective laser injury. Normalized blood-flow maps were collected using a 5000 ms exposure time. Note the presence of blood flow before laser irradiation (compared to the disruption of blood flow following laser irradiation (white arrows in middle row). Based on subsequent image sets taken on Days 1, 3, and 7, we observed a progressive increase in the number of perfused vessels surrounding the injured site. Concurrently, we observed an increase in 530 nm fluorescence emission (bottom row) immediately surrounding the postinjury site, indicating GFP/VEGF activity, with a peak signal on Day 7 (yellow arrow at bottom right). Scale bars: 1× images (2 mm), 4× images (1 mm).