Polymer dots enable deep in vivo multiphoton fluorescence imaging of microvasculature.

Ahmed M Hassan,David R Miller,Andrew K Dunn,Jiangbo Yu,Xu Wu,Shihan Xu,Evan P Perillo,Hsin-Chih Yeh,Jeremy W Jarrett,Daniel T Chiu,Yen-Liang Liu

doi:10.1364/boe.10.000584

Abstract

Deep in vivo imaging of vasculature requires small, bright, and photostable fluorophores suitable for multiphoton microscopy (MPM). Although semiconducting polymer dots (pdots) are an emerging class of highly fluorescent contrast agents with favorable advantages for the next generation of in vivo imaging, their use for deep MPM has never before been demonstrated. Herein, we characterize the multiphoton properties of three pdot variants and perform deep in vivo MPM imaging of cortical rodent microvasculature. We find pdot brightness exceeds conventional fluorophores, including quantum dots, and their broad multiphoton absorption spectrum permits imaging at wavelengths better-suited for biological imaging and confers compatibility with a range of longer excitation wavelengths. This results in substantial improvements in signal-to-background ratio (>3.5-fold) and greater cortical imaging depths (z = 1,300 µm). Ultimately, pdots are a versatile tool for MPM due to their extraordinary brightness and broad absorption, enabling interrogation of deep structures in vivo.

Highlights

In vivo imaging with multiphoton fluorescence microscopy (MPM) is widely used due to its ability to provide diffraction-limited, three-dimensional images of intact tissue at depths ranging from a few hundred microns to more than 1 mm [1,2,3,4,5]
We tested the excitation power dependence of various pdots including a CNPPV and two fluorene-based copolymers, PFBT and PFPV, at wavelengths ranging from 790 - 850 nm, 1,060 nm, and 1,200 – 1,350 nm (Appendix A)
The polymer dots had the additional advantage of a long shelf life and easy storage, characteristics that allow them to be disseminated for broad use [41]

Summary

Introduction

In vivo imaging with multiphoton fluorescence microscopy (MPM) is widely used due to its ability to provide diffraction-limited, three-dimensional images of intact tissue at depths ranging from a few hundred microns to more than 1 mm [1,2,3,4,5]. Recent reports of deep tissue, in vivo imaging with two- and three-photon excitation have demonstrated the potential of imaging structures in the brain beyond the cortex, including the corpus callosum and hippocampus [6,7]. These advances have been driven in part by the availability of new ultrafast laser sources at longer wavelengths (λex = 1,000 – 1,600 nm) than those traditionally used in two-photon fluorescence imaging (λex = 700 – 900 nm). Traditional exogenous contrast agents for in vivo MPM include organic dyes such as dextran-conjugated fluorescein and indocyanine green or inorganic semiconductor quantum dots [9,10]. The biological imaging community is eager for a safer, brighter, and more stable probe for deep, high-resolution in vivo imaging

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