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3D reconstructions of the 2PM images of the brain vasculature of a transgenic mouse using a) TMR‐dextran (70 kDa) (65 nmol) and b) NE1 (30 pmol for NE1 and 65 nmol for LipoPYF5, respectively) under different excitation wavelengths. Fluorescence signals were detected at 500–550 nm (green: eYFP) and 560–690 nm (red: TMR‐dextran or NE1). All images were acquired using the same individual mouse. The excitation dwell time was 57.2 µs pixel⁻¹.
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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...
Citations
... We selected MCPY3 as a representative dye toward the application, because its red emission well matches high-sensitive region of commonly used fluorescence detector (e.g., GaAsP photomultiplier) used in confocal microscopy and two-photon microscopy (2PM). Further, MCPY3 has one-photon absorption band > 500 nm, which suggests MCPY3 would be effectively excited by two photons efficiently using a tissue-penetrative laser of which wavelength > 960 nm 25,26 . However, MCPY3 is extremely hydrophobic, and therefore, the hydrophilic analog of MCPY3, MCPY3S, was newly synthesized (Figure 4a and Figure S7). ...
Bright polymethine dyes, typified by carbocyanines, are employed in various fluorescence techniques such as the 3D visualization of living cell morphology and the tracking of extracellular vesicles in the blood vessels of a zebrafish. However, they often exhibit low photostability, particularly for dyes with red-shifted absorption/fluorescence wavelengths due to extended polymethine length, and limited photofunctionality. This limitation restricts their utility in specific applications requiring high-power excitation and/or a wash-free approach. This study introduces novel merocyanine dyes, MCPY3 and MCPY5, comprising a newly developed pyrene-fused dioxaborine and polymethine chain. Despite their minimal polymethine lengths, their absorption/fluorescence wavelengths reside in the red to near infra-red regions due to the substantial π-conjugation system of pyrene. Moreover, they exhibit a considerably superior photostability to carbocyanine dyes and fluorogenic behavior between low (ON) and high (OFF) polar solvents, while maintaining brightness comparable to carbocyanine. Leveraging these advantages, the hydrophilic analogs of MCPY3, MCPY3S, was applied to two-photon microscopy imaging of the skin tissues on the finger of a living mouse. The dye clearly visualized the individual cell morphology in the epidermis and the elastin within the dermis, highlighting the potential of the new dye as a valuable tool for fundamental dermatological and histological studies.
... In addition to these fluorescence imaging techniques, multiphoton microscopies are being developed for fine and accurate imaging of the brain. Niko et al. [402] developed high-speed two-photon microscopy to noninvasively visualize and analyze vascular dynamics in the cerebral cortex and CA1 region of the hippocampal in mouse brain. The biocompatible nanoemulsions consisting of Labrafac WL inner core and Cremophor ELP shell were prepared via spontaneous nanoemulsification to carry the LipoPYF5 fluorescent dye. ...
... (c) 3D reconstruction and 2D image of cerebral vasculature by using two-photon microscopy. Adapted with permission from Ref.[402], copyright by Wiley-VCH GmbH (2021) (color online). ...
Analyzing the complex structures and functions of brain is the key issue to understanding the physiological and pathological processes. Although neuronal morphology and local distribution of neurons/blood vessels in the brain have been known, the subcellular structures of cells remain challenging, especially in the live brain. In addition, the complicated brain functions involve numerous functional molecules, but the concentrations, distributions and interactions of these molecules in the brain are still poorly understood. In this review, frontier techniques available for multiscale structure imaging from organelles to the whole brain are first overviewed, including magnetic resonance imaging (MRI), computed tomography (CT), positron emission tomography (PET), serial-section electron microscopy (ssEM), light microscopy (LM) and synchrotron-based X-ray microscopy (XRM). Specially, XRM for three-dimensional (3D) imaging of large-scale brain tissue with high resolution and fast imaging speed is highlighted. Additionally, the development of elegant methods for acquisition of brain functions from electrical/chemical signals in the brain is outlined. In particular, the new electrophysiology technologies for neural recordings at the single-neuron level and in the brain are also summarized. We also focus on the construction of electrochemical probes based on dual-recognition strategy and surface/interface chemistry for determination of chemical species in the brain with high selectivity and long-term stability, as well as electrochemophysiological microarray for simultaneously recording of electrochemical and electrophysiological signals in the brain. Moreover, the recent development of brain MRI probes with high contrast-to-noise ratio (CNR) and sensitivity based on hyperpolarized techniques and multi-nuclear chemistry is introduced. Furthermore, multiple optical probes and instruments, especially the optophysiological Raman probes and fiber Raman photometry, for imaging and biosensing in live brain are emphasized. Finally, a brief perspective on existing challenges and further research development is provided.
... 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 We next performed mitochondria-targeting TPFI of FO2 in cortical cells in live animals. After the mice brain cortices were stereotaxically injected with a solution of FO2 in saline, the mice were re-anesthetized and visualized under a two-photon microscope with excitation at 810 nm (Fig. 3d). ...
Mitochondria, key organelles which keep in tune with energy demands for eukaryotic cells, are firmly associated with neurological conditions and post-traumatic rehabilitation. In vivo fluorescence imaging of mitochondria, especially with deep tissue penetration, would open a window to investigate the actual context of the brain. However, the depth of traditional two-photon mitochondrial fluorescence imaging is still limited due to the poor biological compatibility or low two-photon absorption cross-sections. A biocompatible mitochondria-targeted two-photon fluorescent dye (FO2) with an excellent two-photon absorption cross-section (the maximum of 1184 GM at 790 nm) and low cellular toxicity was designed and synthesized to overcome this problem. With this dye, we reached an imaging depth of ca. 640 μm during mitochondrial imaging of cortical cells in live animals. FO2 could be an excellent mitochondrial probe for live animal neural imaging to investigate the function and dysfunction of mitochondria in the brain.
... 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 We next performed mitochondria-targeting TPFI of FO2 in cortical cells in live animals. After the mice brain cortices were stereotaxically injected with a solution of FO2 in saline, the mice were re-anesthetized and visualized under a two-photon microscope with excitation at 810 nm (Fig. 3d). ...
Correction for ‘A biocompatible two-photon absorbing fluorescent mitochondrial probe for deep in vivo bioimaging’ by Lingmin Lin et al. , J. Mater. Chem. B , 2022, DOI: 10.1039/d1tb02040d.
... The newly developed EMCCD-gaincontrolled 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. ...
Fluorescence imaging is a noninvasive and dynamic real-time imaging technique; however, it exhibits poor spatial resolution in centimeter-deep tissues because biological tissues are highly scattering media for optical radiation. The recently developed ultrasound-controlled fluorescence (UCF) imaging is a novel imaging technique that can overcome this bottleneck. Previous studies suggested that the effective contrast agent and sensitive imaging system are the two pivotal factors for generating high-resolution UCF images ex vivo and/or in vivo. Here, this review highlights the recent advances (2015–2020) in the design and synthesis of contrast agents and the improvement of imaging systems to realize high-resolution UCF imaging of deep tissues. The imaging performances of various UCF systems, including the signal-to-noise ratio, imaging resolution, and imaging depth, are specifically discussed. In addition, the challenges and prospects are highlighted. With continuously increasing research interest in this field and emerging multidisciplinary applications, UCF imaging with higher spatial resolution and larger imaging depth may be developed shortly, which is expected to have a far-reaching impact on disease surveillance and/or therapy.
... Short-pulse lasers operating in the wavelength region of ∼ 900 nm show significant potential for use in applications like biomedical imaging [1] and entangled-photon generation. The main benefit for entangled-photon generation is high sensitivity of silicon-based photodetectors in this wavelength range [2]. ...
In this work, we present a fiber-based setup for femtosecond pulse generation at $\sim910 - 940\;{\rm nm}$ ∼ 910 − 940 n m wavelength. Pump pulses from a Mamyshev oscillator with center wavelength at 1036 nm 1 ps duration are amplified and coupled into a photonic crystal fiber forming a soliton at 1051 nm. Due to the nonlinear four-wave mixing process, soliton pulses are converted to 910–940 nm with a few hundred femtosecond pulses with energy up to 0.7 nJ. The proposed design utilizes features of the Mamyshev oscillator, enabling a robust way of generating femtosecond pulses at $\sim910 - 940\;{\rm nm}$ ∼ 910 − 940 n m .
Flexible crystals have gained significant attention owing to their remarkable pliability, plasticity, and adaptability, making them highly popular in various research and application fields. The main challenges in developing flexible crystals lie in the rational design, preparation, and performance optimization of such crystals. Therefore, a comprehensive understanding of the fundamental origins of crystal flexibility is crucial for establishing evaluation criteria and design principles. This Perspective offers a retrospective analysis of the development of flexible crystals over the past two decades. It summarizes the elastic standards and possible plastic bending mechanisms tailored to diverse flexible crystals and analyzes the assessment of their theoretical basis and applicability. Meanwhile, the compatibility between crystal elasticity and plasticity has been discussed, unveiling the immense prospects of elastic/plastic crystals for applications in biomedicine, flexible electronic devices, and flexible optics. Furthermore, this Perspective presents state-of-the-art experimental avenues and analysis methods for investigating molecular interactions in molecular crystals, which is vital for the future exploration of the mechanisms of crystal flexibility.
Distinguishing the luminescence contribution from the surface and bulk of a crystal is a long-standing challenge in crystal materials. Herein, three-dimensional, multiphoton, luminescence microscope imaging of the elastic molecular single crystal 1,4-bis(4-methylthien-2-yl)-2,3,5,6-tetrafluorobenzene, was conducted. Further, the luminescence contribution from the surface and bulk of the crystal was experimentally distinguished. Strong luminescence was observed only from the surface of the crystal, while the bulk did not emit strongly. Furthermore, the surface and bulk luminescence behavior responded well to the mechanical shape change of the crystal; i.e., strong luminescence was observed for the elongated side of the crystal.
