Integrated Fluorescent Nanoprobe Design for High‐Speed In Vivo Two‐Photon Microscopic Imaging of Deep‐Brain Vasculature in Mice

Abstract

High‐speed two‐photon microscopy can be used to analyze vascular dynamics in living animals and is essential for the understanding of brain diseases. Recent advances in fluorescent probes/optical systems have allowed successful imaging of the hippocampal vasculature in the deep brain of mice (1 mm from the brain surface) under low‐speed conditions (1–2 fps); however, using high‐speed techniques (>30 fps), observation of the deep‐brain vasculature is still challenging. Here, a new nanoemulsion that encapsulates thousands of red‐emissive pyrene dye molecules while maintaining their high two‐photon brightness [1.5 × 102 GM (GM = 10−50 cm4·s·photon−1·molecule−1) at 960 nm excitation] and delivers a large amount of such pyrene dyes (65 nmol) into the blood vessels of mice is developed. Remarkably, the nanoprobe is found to exploit the inherent performance of a commonly used Ti:sapphire excitation laser and a sensitive gallium arsenide phosphide nondescanned fluorescence detector to the limit, enabling visualization of the brain vasculature under the cortex region of mice (up to 1.5 mm) under very low‐speed conditions. As a highlight, such a nanoprobe is successfully used to directly observe the blood flow in the hippocampal CA1 region (1.1 mm) through high‐speed resonant scanning (120 fps).

A biocompatible two-photon absorbing fluorescent mitochondrial probe for deepin vivobioimaging

“…These results showed the superior ability of FO2 in visualizing brain blood vessels, which is of vital importance for better understanding of the CNS injury, such as cerebral vascular occlusion, cerebral hemorrhage, and leukocyte extravasation. 8,40…”

Synthesis and photophysical properties of a new push–pull pyrene dye with green-to-far-red emission and its application to human cellular and skin tissue imaging

“…42 After washing to remove excess PC and LUCID, images were acquired under 960 nm excitation light, which is relatively tissue-penetrative among the wavelengths that a conventional titan-sapphire laser can operate (typically 700–1040 nm) with sufficient output power. 43–45 The images were obtained using four channels: cyan (492 nm, for second harmonic generation), green (500–550 nm), orange (560–593 nm), and red (593–690 nm), and were then reconstructed to create 3D image stacks. The near surfaces of tissue blocks ( z < 0.1 mm, 2D images) were also visualized using a spectral imaging technique.…”

Recent advances in ultrasound-controlled fluorescence technology for deep tissue optical imaging

“…The newly developed EMCCD-gain-controlled UCF imaging system with high resolution, large scan speed, excellent SNR, and better imaging depth exhibits immense potential for in vivo UCF imaging and investigation on deep-tissue lesions. It will be useful for examining the vascular dynamics in animal models and understanding the cerebral diseases in deep tissues [ 55 ]. From the perspective of ultrasonic control conditions, the optimized parameters of the given HIFU conditions are also useful for improving the performance of the ex vivo or/and in vivo UCF imaging.…”