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The application of an exogenous polymer matrix to construct aggregation-induced emission (AIE) nanoprobes promotes the utility of AIE luminogens (AIEgens) in diagnosing brain diseases. However, the limited fluorescence (FL) and low active-targeting abilities of AIE-based nanoprobes impede their imaging application. Here, we employed endogenous albumin as an effective matrix to encapsulate AIEgens to enhance FL quantum yield (QY) and active-targeting ability. The albumin-consolidated strategy effectively inhibited the intramolecular vibration of AIEgens and enhanced endocytosis mediated by the gp60 receptor. The QYs of three kinds of albumin-based AIE nanoprobes with FL emissions ranging from the visible (400-650 nm) to the second near-infrared (NIR-II, 1000-1700 nm) region was at least 10% higher, and the tumor-targeting efficiency was ∼25% higher, compared with those of nanoprobes constructed by the exogenous polymer. Albumin-based AIE nanoprobes have achieved active-targeting NIR-II imaging of brain tumors and cerebrovascular imaging with a high signal-to-background ratio (SBR, ∼90) and high resolution (∼70 μm) in mouse models. Therefore, the albumin-based AIE nanoprobes will enable FL imaging-guided surgery of brain tumors and cerebral ischemia, which will improve surgical efficacy to prevent recurrence and side effects.

Distinguishing arteries from veins in vivo has a great significance in clinical practices and preclinical studies. Optical imaging methods such as two-photon microscopy can provide high-resolution morphological information of tissue and are therefore extremely suitable for imaging small blood vessels. However, few optical imaging methods allow in vivo identification of arteries and veins merely utilizing the autofluorescence signal of blood vessels. In this report, we found the arterial wall generates a remarkably stronger two-photon excitation autofluorescence (TPEA) signal compared with the venous wall based on BALB/c mice. According to histological analysis and fluorescence characteristic measurement, the contrast signal is confirmed to be from elastin fibers. Employing this unique feature, we propose an objective and effective artery-vein separation strategy that considers the presence of the elastin-TPEA border as the indicator of arteries. Using this strategy, the arterial and venous networks of the dorsal skin and cerebral cortex of BALB/c mice are demonstrated to be excellently mapped and accurately separated in vivo without depending on any exogenous contrast agent, empirical knowledge, and algorithm. This study may provide a novel technique for mapping arterial and venous networks for anatomic research as well as an extra aid to basic researches on the mechanism, diagnosis, and treatment of blood vessel-related diseases.

A dual modality microscopy with the highest imaging resolution reported so far based on reflection-mode photoacoustic and confocal fluorescence is presented in this study. The unique design of the imaging head of the microscope makes it highly convenient for scalable high-resolution imaging by simply switching the optical objectives. The submicron resolution performance of the system is demonstrated via in vivo imaging of zebrafish, normal mouse ear, and a xenograft tumor model inoculated in the mouse ear. The imaging results confirm that the presented dual-modality microscopy imaging system could play a vital role in observing model organism, studying tumor angiogenesis and assessment of antineoplastic drugs. MSC: 00-01, 99-00, Keywords: Photoacoustic microscopy, Confocal microscopy, Multimodal imaging

NIR-II fluorescence imaging is a promising method for visualizing biological structures in deep tissue, owing to the advantages of significantly suppressed optical scattering and diminished autofluorescence in biological tissues. However, few NIR-II fluorescence imaging approaches can simultaneously achieve a large field of view, high resolution and superior penetration depth, while exhibiting optical sectioning capability. In this paper, we present a novel NIR-II fluorescence mesoscopy system based on the f-θ scanning scheme and confocal detection to overcome these limitations. When used with NIR-II fluorescent dyes, our setup performs NIR-II fluorescence imaging on samples as large as 7.5×7.5 mm2 with a lateral resolution of 6.3 µm. In addition, our system provides a depth-resolved imaging ability and zooming function. We successfully demonstrate in vivo cerebrovascular imaging of a mouse with local ischemia. Thus, our system provides new opportunities to explore the mechanism of cerebrovascular disease.

Background: Multicolor fluorescence microscopy has proved essential in biological studies. However, the application of conventional multicolor microscopy to imaging subcellular organelles is restricted by its diffraction-limited spatial resolution. Re-scan confocal microscopy (RCM), a novel super-resolution imaging technique, can effectively address this problem. However, previous multicolor RCM imaging methods usually led to spatial mismatch in images due to the sequential scanning of the sample with multiple excitation lasers. Methods: We present a new RCM system to achieve multicolor super-resolution imaging. A spectrograph was used as the multicolor detection system, and a linear spectral unmixing algorithm was applied to separate different fluorophores in the spectral image. Moreover, since the image reconstruction process induced an artificial resolution improvement, a gamma correction was introduced to restore the multicolor super-resolution image. Results: By imaging phalloidin-labeled F-actin in breast cancer cells, we found that the lateral resolution of our system is approximately 171 nm, which is a 1.8-fold improvement over that of wide-field imaging. The successful identification of three types of fluorescent beads indicated that our multicolor RCM can resolve different fluorophores whose spectra largely overlap with each other. Finally, we demonstrated that our method is suitable for imaging multicolor-labeled organelles of live cells. Conclusions: Our novel RCM system can acquire multicolor super-resolution images of live cells without spatial mismatch, obvious photobleaching or photodamage. This system may provide a new imaging tool for monitoring dynamic events involving interactions between multiple molecules and organelles in cells.

Characterizing an ischemic brain injury at its early stage is critical to biological research and the clinical diagnosis of cerebral ischemia-related diseases. However, approaches with intravital, label-free, and real-time characterization capabilities are scarce. Two-photon excitation fluorescence lifetime imaging microscopy (FLIM) can detect variations in energy metabolism based on the autofluorescence of reduced nicotinamide adenine dinucleotide (NADH). Using this unique feature, we proposed a novel approach for cerebral ischemia characterization. From investigating cell and animal models, the cerebral NADH fluorescence lifetime was observed to be sensitive to metabolic changes caused by ischemia and consistent with ischemic time. A comparison with standard blood flow imaging and neuronal injury assessment further suggests that, the two-photon FLIM, using NADH as an indicator, can characterize degrees of cerebral ischemia and related injuries, particularly at the early stage. These findings demonstrate that NADH FLIM is promising for providing intravital, label-free, and real-time assessment of cerebral ischemia and ischemic brain injury that will be significant to the study and diagnosis of related diseases.

Optical clearing methods reduce the optical scattering of biological samples and thereby extend optical imaging penetration depth. However, refractive index mismatch between the immersion media of objectives and clearing reagents induces spherical aberration (SA), causing significant degradation of fluorescence intensity and spatial resolution. We present an adaptive optics method based on pupil ring segmentation to correct SA in optically cleared samples. Our method demonstrates superior SA correction over a modal-based adaptive optics method and restores the fluorescence intensity and resolution at high imaging depth. Moreover, the method can derive an SA correction map for the whole imaging volume based on three representative measurements. It facilitates SA correction during image acquisition without intermittent SA measurements. We applied this method in mouse brain tissues treated with different optical clearing methods. The results illustrate that the synaptic structures of neurons within 900 μm depth can be clearly resolved after SA correction.

We present confocal fluorescence lifetime imaging microscopy in the second near-infrared (NIR-II) window to assess the morphological and biochemical information of live samples. A home-built superconducting single-photon detector (SSPD) was used to facilitate the NIR-II fluorescence lifetime measurement. The SSPD has many advantages, including high sensitivity to NIR-II signals (detection efficiency > 50 % ), fast temporal response ( ∼ 109 p s ), low timing jitter ( ∼ 50 p s ), and low dark count rate ( < 100 c p s ). We demonstrate the feasibility of the developed microscopy system by comparing fluorescence lifetimes of a range of fluorophores with emission in the NIR-II window and by performing multicolor three-dimensional fluorescence lifetime imaging of a mouse ear in vivo. The biochemical properties of the cells and tissues probed by the fluorescence lifetimes of the fluorophores provide complementary information for biomedical studies, significantly benefiting diverse applications in life science.

Observing microvasculature in its native environment provides invaluable information to understand the initiation and development of microcirculatory related diseases. However, the lack of a high-resolution three-dimensional (3D) imaging technique hinders in vivo investigation of the microvasculature. Recently, we found that the red blood cells can emit autofluorescence signals with short-wavelength two-photon excitation. In this study, we exploited this property and developed a time-resolved two-photon excitation microscopy system using a homemade 520 nm femtosecond fiber laser as the excitation source. Using this system, we could achieve intravital high-resolution 3D imaging of a microvascular network noninvasively. In a mouse tumor model, tumorous blood vessels could be observed and distinguished clearly from the normal vessels.

Many imaging technologies, such as two photon microscopy (TPM), second generation microscopy (SHG) and photoacoustic microscopy (PAM), have successfully demonstrated the ability to extract anatomical, functional and molecular information of biological samples. However, they always fail to obtain comprehensive information from biological tissue due to single imaging modality. To address this limitation, we developed a multimodal microscopy with fully integrated PAM, TPM and SHG. The home-built multimodal microscopy system enable label-free imaging of biomedical tissue with sub-micron resolution in vivo and in vitro. The results may offer a new tool to provide the capable of fast comprehensive information capturing for biological studies.

The kidneys are essential regulatory organs whose main function is to regulate the balance of electrolytes in the blood, along with maintaining pH homeostasis. The study of the microscopic structure of the kidney will help identify kidney diseases associated with specific renal histology change. Spectrally encoded microscopy (SEM) is a new reflectance microscopic imaging technique in which a grating is used to illuminate different positions along a line on the sample with different wavelengths, reducing the size of system and imaging time. In this paper, a SEM device is described which is based on a super luminescent diode source and a home-built spectrometer. The lateral resolution was measured by imaging the USAF resolution target. The axial response curve was obtained as a reflect mirror was scanned through the focal plane axially. In order to test the feasibility of using SEM for depth-section imaging of an excised swine kidney tissue, the images of the samples were acquired by scanning the sample at 10 μm per step along the depth direction. Architectural features of the kidney tissue could be clearly visualized in the SEM images, including glomeruli and blood vessels. Results from this study suggest that SEM may be useful for locating regions with probabilities of kidney disease or cancer.

DLC films can play an important role in tribological properties of Al-alloys. A gradient layer of AlN/Ti/TiN/DLC film has been prepared by plasma-based ion implanted N, followed by Ti, and N and Ti, and finally C on 2024 Al-alloy. Emphasis has been placed on the tribological properties of the gradient layer. Its composition depth profile and chemical structure were characterized using X-ray photoelectron spectroscopy (XPS), the surface C-layer and the wear tracks were analyzed by laser Raman spectroscopy. The morphologies of the C-layer were observed by atomic force microscopy (AFM), the hardness of the gradient layer was measured with the mechanical property microprobe and the dry wear tests against AISI steel ball at different sliding loads were performed with a ball-on-disk wear tester in ambient environment. The results indicate that the gradient layer shows a gradual change in hardness, compact surface appearance and good tribological properties owing to the gradient structure. As the number of the sliding cycles or the load is increasing, the tribological properties decrease due to the graphitization of the DLC films. Meanwhile, the gradient layer can be controlled by PBII processing parameters, thus an optimized gradient layer can be obtained to offer the possibility of making aluminum alloys and other soft alloys qualified candidates for particular engineering applications.

Using plasma-based ion implantation, two types of gradient layers have been prepared on 2024 Al alloy. One is prepared by N-implantation then C-deposition, the other adds an interlayer composed of a Ti layer and a Ti–N layer between N-implantation and C-deposition. C-deposition is carried out at various implanting voltages or C2H2/H2 ratios. The composition depth profiles of these layers were characterized by x-ray photoelectron spectroscopy. The structure, morphologies and microstructure of the C layers were studied using Raman spectroscopy, atomic force microscope and transmission electron microscope, respectively. The surface hardness was measured with a Knoop tester and a mechanical property microprobe. The dry ball-on-disc wear tests were performed in ambient air. The gradient layer without interlayer is composed of an N-implanted layer rich in AlN and a diamond-like carbon (DLC) layer (film), and the two layers are connected with a C–Al transition layer containing Al4C3. The Ti layer rich in α -Ti and the N-implanted layer are connected by a Ti–Al transition layer containing TiAl3, while the Ti–N layer rich in TiN and the DLC film are connected by a C–Ti transition layer containing TiC, TiCN, etc. Thus, the gradient layer with interlayers has optimized the gradient structure. DLC films are compact and amorphous, contain high sp3/sp2 ratios and depend on the implanting voltage and the C2H2/H2 ratio. Similarly, these gradient layers exhibit significant improvement in morphologies, surface hardness and tribological properties; the interlayer, the implanting voltage and the C2H2/H2 ratio all have prominent effects on these properties.