Abstract
Compared with visible light, near-infrared (NIR) light has deeper penetration in biological tissues. Three-photon fluorescence microscopy (3PFM) can effectively utilize the NIR excitation to obtain high-contrast images in the deep tissue. However, the weak three-photon fluorescence signals may be not well presented in the traditional fluorescence intensity imaging mode. Fluorescence lifetime of certain probes is insensitive to the intensity of the excitation laser. Moreover, fluorescence lifetime imaging microscopy (FLIM) can detect weak signals by utilizing time-correlated single photon counting (TCSPC) technique. Thus, it would be an improved strategy to combine the 3PFM imaging with the FLIM together. Herein, DCDPP-2TPA, a novel aggregation-induced emission luminogen (AIEgen), was adopted as the fluorescent probes. The three-photon absorption cross-section of the AIEgen, which has a deep-red fluorescence emission, was proved to be large. DCDPP-2TPA nanoparticles were synthesized, and the three-photon fluorescence lifetime of which was measured in water. Moreover, in vivo three-photon fluorescence lifetime microscopic imaging of a craniotomy mouse was conducted via a home-made optical system. High contrast cerebrovascular images of different vertical depths were obtained and the maximum depth was about 600 [Formula: see text]m. Even reaching the depth of 600 [Formula: see text]m, tiny capillary vessels as small as 1.9 [Formula: see text]m could still be distinguished. The three-photon fluorescence lifetimes of the capillaries in some representative images were in accord with that of DCDPP-2TPA nanoparticles in water. A vivid 3D reconstruction was further organized to present a wealth of lifetime information. In the future, the combination strategy of 3PFM and FLIM could be further applied in the brain functional imaging.
Highlights
Brain imaging has widely arisen people's attention since the last century
An aliquot of 5 mL chloroform solution containing 2.2 mg DCDPP-2TPA and 26.4 mg F-127 was added into a °askrst and the °ask was put into the ultrasonic cleaner for 5 min to ensure even mixing
The stronger laser beam was introduced into a scanning microscope (FV1200&BX61, Olympus) and re°ected by an 800 nm short-pass dichroic mirror (DC) to excite aggregation-induced emission luminogens (AIEgens) nanoparticles
Summary
Brain imaging has widely arisen people's attention since the last century. In most of the imaging techniques, optical imaging is one of the most favourable methods for its high resolution, no invasion or radiation and °exible combination with other imaging techniques.[1]. As a type of organic dyes, AIEgens have a large 3PA cross-section and enhanced °uorescence when encapsulated in nanoparticles.[14] When performing the deep-tissue 3PFM imaging, there are still some limitations, such as the small amounts of probes in the targeted samples and the weakened °uorescence intensity at a large depth. The images of these areas are lack of enough contrast This could be improved by combining the 3PFM imaging with the °uorescence lifetime imaging microscopy (FLIM), which is suitable for noninvasive study of intracellular processes,[15,16] micro°uidic systems,[17] remote sensing,[18,19] lipid order problems in physical chemistry,[20] temperature sensing[21] and clinical medicine.[22] FLIM can provide a more sensitive and precise image based on the weak signals, compared with the traditional °uorescence intensity imaging.[23] One reason is that the °uorescence lifetime of probe keeps stable at di®erent amounts or under varying intensity of the excitation laser.[24,25] Another reason is that the °uorescence lifetime of each pixel is obtained with time-correlated single photon counting (TCSPC) technique, with a signicantly increasing signal–noise ratio.[26]. The combination strategy of 3PFM imaging and FLIM could be further applied in the brain functional imaging and provide a larger detection depth
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