Fully integrated reflection-mode photoacoustic, two-photon, and second harmonic generation microscopy in vivo.

Wei Song,Yang Zhang,Qiang Xu,Wei Zheng,Yang Zhan,Liang Song

doi:10.1038/srep32240

Abstract

The ability to obtain comprehensive structural and functional information from intact biological tissue in vivo is highly desirable for many important biomedical applications, including cancer and brain studies. Here, we developed a fully integrated multimodal microscopy that can provide photoacoustic (optical absorption), two-photon (fluorescence), and second harmonic generation (SHG) information from tissue in vivo, with intrinsically co-registered images. Moreover, using a delicately designed optical-acoustic coupling configuration, a high-frequency miniature ultrasonic transducer was integrated into a water-immersion optical objective, thus allowing all three imaging modalities to provide a high lateral resolution of ~290 nm with reflection-mode imaging capability, which is essential for studying intricate anatomy, such as that of the brain. Taking advantage of the complementary and comprehensive contrasts of the system, we demonstrated high-resolution imaging of various tissues in living mice, including microvasculature (by photoacoustics), epidermis cells, cortical neurons (by two-photon fluorescence), and extracellular collagen fibers (by SHG). The intrinsic image co-registration of the three modalities conveniently provided improved visualization and understanding of the tissue microarchitecture. The reported results suggest that, by revealing complementary tissue microstructures in vivo, this multimodal microscopy can potentially facilitate a broad range of biomedical studies, such as imaging of the tumor microenvironment and neurovascular coupling.

Highlights

The ability to obtain comprehensive structural and functional information from intact biological tissue in vivo is highly desirable for many important biomedical applications, including cancer and brain studies
A representative graphite nanoparticle imaged using photoacoustic microscopy (PAM) is shown in the inset of Fig. 2(a)
The photoacoustic amplitude values along the lateral direction of the imaged nanosphere were fitted to a Gaussian function in Fig. 2(a), indicating a PAM lateral resolution of ~290.0 nm according to the full width at half maximum (FWHM)

Summary

Introduction

The ability to obtain comprehensive structural and functional information from intact biological tissue in vivo is highly desirable for many important biomedical applications, including cancer and brain studies. Over the past few decades, optical microscopy has become an indispensable technology in biophysical research It offers great advantages in investigating physiological and pathological processes by revealing the morphological, functional and molecular features of biological specimens with cellular and subcellular spatial resolution. Based on various imaging contrasts within tissues, including optical scattering, fluorescence, and polarization, several optical microscopic technologies have been established and broadly applied in neurology[1,2], oncology[3,4], vascular biology[5,6], and ophthalmology[7,8] Among all these optical microscopic technologies, multiphoton fluorescence microscopy and second harmonic generation (SHG) microscopy are capable of providing specific types of molecular imaging information at diffraction-limited spatial resolution in deep positions of solid tissues, significantly facilitating research in biological sciences and medicine[3,4,5,9]. OR-PAM has successfully extracted optical absorption-based anatomic, functional, and molecular information, and it demonstrates great potential for studying various physiologies and dynamics in living animals, including vessel anatomy, oxygen metabolism, tumor progression, and brain activities[19,20,21,22]

Methods

Results

Conclusion