Integrated photoacoustic and fluorescence confocal microscopy

Abstract

We have developed a dual-modality imaging system by integrating optical-resolution photoacoustic microscopy and fluorescence confocal microscopy to provide optical absorption and fluorescence contrasts simultaneously. By sharing the same laser source and objective lens, intrinsically registered photoacoustic and fluorescence images are acquired in a single scan. The micrometer resolution allows imaging of both blood and lymphatic vessels down to the capillary level. Simultaneous photoacoustic angiography and fluorescence lymphangiography were demonstrated, presenting more information to study tumor angiogenesis, vasculature, and microenvironments in vivo.

Fig. 1

Schematic of the dual-modality PA-FCM system.

Fig. 2

Spatial resolutions of the dual-modality PA-FCM. (a) The lateral edge spread function of the PAM. (b) The axial resolution of the PAM. (c) The lateral edge spread function of the confocal microscope. (d) The axial resolution of the confocal microscope.

Fig. 3

Combined functional vascular and lymphatic images of a mouse ear in vivo. (a) PAM image of microvasculature acquired at 570 nm. (b) PA sO₂ mapping based on dual-wavelength (570 nm and 593 nm) measurements. (c) FCM image of the lymphatic system acquired at 570 nm after the injection of 20% RITC-dextran. (d) Fused mapping of total hemoglobin concentration and fluorescent lymphatics. (e) Fused mapping of sO₂ and fluorescent lymphatics. (f) Optical microscopic image acquired within the same region. FL: fluorescence. OM: optical microscopy.