Three-photon Fluorescence Microscopy Research Articles

Improving Three-Photon Fluorescence of Near-Infrared Quantum Dots for Deep Brain Imaging by Suppressing Biexciton Decay.

Three-photon fluorescence microscopy (3PFM) is a promising brain research tool with submicrometer spatial resolution and high imaging depth. However, only limited materials have been developed for 3PFM owing to the rigorous requirement of the three-photon fluorescence (3PF) process. Herein, under the guidance of a band gap engineering strategy, CdTe/CdSe/ZnS quantum dots (QDs) emitting in the near-infrared window are designed for constructing 3PF probes. The formation of type II structure significantly increased the three-photon absorption cross section of QDs and caused the delocalization of electron-hole wave functions. The time-resolved transient absorption spectroscopy confirmed that the decay of biexcitons was significantly suppressed due to the appropriate band gap alignment, which further enhanced the 3PF efficiency of QDs. By utilizing QD-based 3PF probes, high-resolution 3PFM imaging of cerebral vasculature was realized excited by a 1600 nm femtosecond laser, indicating the possibility of deep brain imaging with these 3PF probes.

In vivo 3-photon fluorescence microscopy of white matter in mouse brain excited at the 1700 nm window

White matter, a densely packed collection of myelinated axons, plays an essential part in neural networks. With high spatial resolution and deep penetration, multi-photon microscopy (MPM) is promising for white matter imaging in animal models in vivo. The third harmonic generation (THG) signal can be generated from white matter, but the bottom part of the white matter layer generates weak THG due to its high scattering. Here, we demonstrate an in vivo labeling and imaging technology, capable of visualizing the white matter layer in the mouse brain, combining fluorescence labeling with MitoTracker Red and three-photon fluorescence (3PF) microscopy excited at the 1700 nm window. 3PF signals are several times higher than THG signals, resulting in deeper imaging of the white matter layer with the former. Our results indicate that 3PF microscopy is a promising technology for white matter imaging in the deep brain in vivo.

Multimodal label-free imaging of murine hepatocellular carcinoma with a subcellular resolution.

We demonstrate label-free imaging of genetically induced hepatocellular carcinoma (HCC) in a murine model provided by two- and three-photon fluorescence microscopy of endogenous fluorophores excited at the central wavelengths of 790, 980 and 1250 nm and reinforced by second and third harmonic generation microscopy. We show, that autofluorescence imaging presents abundant information about cell arrangement and lipid accumulation in hepatocytes and hepatic stellate cells (HSCs), harmonics generation microscopy provides a versatile tool for fibrogenesis and steatosis study. Multimodal images may be performed by a single ultrafast laser source at 1250 nm falling in tissue transparency window. Various grades of HCC are examined revealing fibrosis, steatosis, liver cell dysplasia, activation of HSCs and hepatocyte necrosis, that shows a great ability of multimodal label-free microscopy to intravital visualization of liver pathology development.

Band Gap Engineering Improves Three-Photon Luminescence of Quantum Dots for Deep Brain Imaging.

Three-photon fluorescence microscopy (3PFM) has emerged as a promising tool in monitoring the structures and functions of the brain. Compared to the various imaging technologies, 3PFM enables a deep-penetrating depth attributed to tighter excitation confinement and suppressed photon scattering. However, the shortage of three-photon probes with a large absorption cross section (σ3) substantially limits its uses. Herein, CdSe/CdS/ZnS quantum dots (QDs) with enhanced 3PF performance were synthesized via the band gap engineering strategy. The introduction of a CdS interlayer with optimized thickness between the emitting CdSe core and the ZnS shell significantly enhanced the 3P absorption cross section of QDs, which originated from the intrinsic piezoelectric polarization effect and the change of the core/shell structure from type-I to quasi-type-II. In addition, the outer ZnS layer compensated the poor electronic passivation of CdS, providing a high level of passivation for the improvement of quantum yield as well as the 3P action cross section of QDs. Under the excitation of a 1600 nm femtosecond laser, PEGylated CdSe/CdS/ZnS QDs were used for in vivo 3PFM imaging of cerebral vessels with high resolution. A tiny capillary with a diameter of 0.8 μm could be resolved at the imaging depth of 1550 μm in a mouse brain with an opened skull. A penetration depth of 850 μm beneath the skull was also achieved using a mouse model with an intact skull.

Large-depth three-photon fluorescence microscopy imaging of cortical microvasculature on nonhuman primates with bright AIE probe In vivo

Multiphoton microscopy has been a powerful tool in brain research, three-photon fluorescence microscopy is increasingly becoming an emerging technique for neurological research of the cortex in depth. Nonhuman primates play important roles in the study of brain science because of their neural and vascular similarity to humans. However, there are few research results of three-photon fluorescence microscopy on the brain of nonhuman primates due to the lack of optimized imaging systems and excellent fluorescent probes. Here we introduced a bright aggregation-induced emission (AIE) probe with excellent three-photon fluorescence efficiency as well as facile synthesis process and we validated its biocompatibility in the macaque monkey. We achieved a large-depth vascular imaging of approximately 1 mm in the cerebral cortex of macaque monkey with our lab-modified three-photon fluorescence microscopy system and the AIE probe. Functional measurement of blood velocity in deep cortex capillaries was also performed. Furthermore, the comparison of cortical deep vascular structure parameters across species was presented on the monkey and mouse cortex. This work is the first in vivo three-photon fluorescence microscopic imaging research on the macaque monkey cortex reaching the imaging depth of ∼1 mm with the bright AIE probe. The results demonstrate the potential of three-photon microscopy as primate-compatible method for imaging fine vascular networks and will advance our understanding of vascular function in normal and disease in humans.

Aggregation-induced emission nanoprobe assisted ultra-deep through-skull three-photon mouse brain imaging

Optical microscopy has enabled in vivo monitoring of brain structures and functions with high spatial resolution. However, the strong optical scattering in turbid brain tissue and skull impedes the observation of microvasculature and neuronal structures at a large depth. Herein, we proposed a strategy to overcome the influence induced by the high scattering effect of both skull and brain tissue via the combination of skull optical clearing (SOC) technique and three-photon fluorescence microscopy (3PM). The visible-NIR-II compatible skull optical clearing agents (VNSOCA) we applied reduced the skull scattering and water absorption in long wavelength by refractive index matching and H2O replacement to D2O respectively. 3PM with the excitation in the 1300-nm window reached 1.5 mm cerebrovascular imaging depth in cranial window assisted by a kind of bright aggregation-induced emission (AIE) nanoprobe we developed with a large three-photon absorption cross section. Combining the two advanced technologies together, we achieved so far the largest cerebrovascular imaging depth of 1.0 mm and neuronal imaging depth of> 700 µm through intact mouse skull. Dual-channel through-skull imaging of both brain vessels and neurons was also successfully realized, giving an opportunity of non-invasively monitoring the deep brain structures and functions at single-cell level simultaneously.

A potent luminogen with NIR-IIb excitable AIE features for ultradeep brain vascular and hemodynamic three-photon imaging

Three-photon excited fluorescence microscopy (3PEFM) has emerged as a promising protocol for visualizing deep-brain vasculature and hemodynamics. However, the current situation is still far from satisfactory, due to small excitation action cross-section and short excitation wavelength of those previously reported 3PEFM luminogens. Herein, we manipulated molecular engineering by subtly regulating structural planarization/twisting to achieve ingenious integration of large three-photon absorption cross-section, high fluorescence quantum yield, ultralong near-infrared IIb excitation, and aggregation-induced emission features. The resulting molecule, namely DPCZ-BT, exhibited as high as 50.6% of fluorescence quantum yield and as large as 2.0 × 10−81 cm6s2/photon2 of three-photon absorption cross-section, which can be excited by 1665 nm fs laser and presents a recorded penetration depth of 1860 μm for deep-brain vascular structural imaging with high spatiotemporal resolution and signal-to-background ratio. Moreover, DPCZ-BT having good photostability and excellent biocompatibility is capable of impressively approaching 1600 μm depth in monitoring red blood cells flow velocity with extraordinary clarity for hemodynamics.

Deep-Tissue Three-Photon Fluorescence Microscopy in Intact Mouse and Zebrafish Brain

Multiphoton microscopy techniques, such as two-photon microscopy (2PM) and three-photon microscopy (3PM), are powerful tools for deep-tissue in vivo imaging with subcellular resolution. 3PM has two major advantages for deep-tissue imaging over 2PM that has been widely used in biology laboratories: (i) longer attenuation length in scattering tissues by employing ~1,300 nm or ~1,700 nm excitation laser; (ii) less background fluorescence generation due to higher-order nonlinear excitation. As a result, 3PM allows high-contrast structural and functional imaging deep within scattering tissues such as intact mouse brain from the cortical layers to the hippocampus and the entire forebrain of adult zebrafish. Today, laser sources suitable for 3PM are commercially available, enabling the conversion of an existing two-photon (2P) imaging system to a three-photon (3P) system. Additionally, multiple commercial 3P microscopes are available, which makes this technique readily available to biology research laboratories. This paper shows the optimization of a typical 3PM setup, particularly targeting biology groups that already have a 2P setup, and demonstrates intravital 3D imaging in intact mouse and adult zebrafish brains. This protocol covers the full experimental procedure of 3P imaging, including microscope alignment, prechirping of ~1,300 and ~1,700 nm laser pulses, animal preparation, and intravital 3P fluorescence imaging deep in adult zebrafish and mouse brains.

Deep-Tissue Three-Photon Fluorescence Microscopy in Intact Mouse and Zebrafish Brain.

Multiphoton microscopy techniques, such as two-photon microscopy (2PM) and three-photon microscopy (3PM), are powerful tools for deep-tissue in vivo imaging with subcellular resolution. 3PM has two major advantages for deep-tissue imaging over 2PM that has been widely used in biology laboratories: (i) longer attenuation length in scattering tissues by employing ~1,300 nm or ~1,700 nm excitation laser; (ii) less background fluorescence generation due to higher-order nonlinear excitation. As a result, 3PM allows high-contrast structural and functional imaging deep within scattering tissues such as intact mouse brain from the cortical layers to the hippocampus and the entire forebrain of adult zebrafish. Today, laser sources suitable for 3PM are commercially available, enabling the conversion of an existing two-photon (2P) imaging system to a three-photon (3P) system. Additionally, multiple commercial 3P microscopes are available, which makes this technique readily available to biology research laboratories. This paper shows the optimization of a typical 3PM setup, particularly targeting biology groups that already have a 2P setup, and demonstrates intravital 3D imaging in intact mouse and adult zebrafish brains. This protocol covers the full experimental procedure of 3P imaging, including microscope alignment, prechirping of ~1,300 and ~1,700 nm laser pulses, animal preparation, and intravital 3P fluorescence imaging deep in adult zebrafish and mouse brains.

Boundary-Preserved Deep Denoising of Stochastic Resonance Enhanced Multiphoton Images.

Objective: With the rapid growth of high-speed deep-tissue imaging in biomedical research, there is an urgent need to develop a robust and effective denoising method to retain morphological features for further texture analysis and segmentation. Conventional denoising filters and models can easily suppress the perturbative noise in high-contrast images; however, for low photon budget multiphoton images, a high detector gain will not only boost the signals but also bring significant background noise. In such a stochastic resonance imaging regime, subthreshold signals may be detectable with the help of noise, meaning that a denoising filter capable of removing noise without sacrificing important cellular features, such as cell boundaries, is desirable. Method: We propose a convolutional neural network-based denoising autoencoder method — a fully convolutional deep denoising autoencoder (DDAE) — to improve the quality of three-photon fluorescence (3PF) and third-harmonic generation (THG) microscopy images. Results: The average of 200 acquired images of a given location served as the low-noise answer for the DDAE training. Compared with other conventional denoising methods, our DDAE model shows a better signal-to-noise ratio (28.86 and 21.66 for 3PF and THG, respectively), structural similarity (0.89 and 0.70 for 3PF and THG, respectively), and preservation of the nuclear or cellular boundaries (F1-score of 0.662 and 0.736 for 3PF and THG, respectively). It shows that DDAE is a better trade-off approach between structural similarity and preserving signal regions. Conclusions: The results of this study validate the effectiveness of the DDAE system in boundary-preserved image denoising. Clinical Impact: The proposed deep denoising system can enhance the quality of microscopic images and effectively support clinical evaluation and assessment.

Imaging deeper than the transport mean free path with multiphoton microscopy.

Multiphoton fluorescence microscopy enables deep in vivo imaging by using long excitation wavelengths to increase the penetration depth of ballistic photons and nonlinear excitation to suppress the out-of-focus fluorescence. However, the imaging depth of multiphoton microscopy is limited by tissue scattering and absorption. This fundamental depth limit for two-photon microscopy has been studied theoretically and experimentally. Long wavelength three-photon fluorescence microscopy was developed to image beyond the depth limit of two-photon microscopy and has achieved unprecedented in vivo imaging depth. Here we extend the theoretical framework for characterizing the depth limit of two-photon microscopy to three-photon microscopy. We further verify the theoretical predictions with experimental results from tissue phantoms. We demonstrate experimentally that high spatial resolution diffraction-limited imaging at a depth of 10 scattering mean free paths, which is nearly twice the transport mean free path, is possible with multiphoton microscopy. Our results indicate that the depth limit of three-photon microscopy is significantly beyond what has been achieved in biological tissues so far, and further technological development is required to reach the full potential of three-photon microscopy.

One wavelength to excite them all: deep tissue imaging going multicolor

Three-photon fluorescence microscopy has emerged as one of the leading tools for deep tissue imaging in opaque biological specimens. A recent study by Hontani et al. demonstrated a new strategy for multicolor imaging deep in the mouse brain using a single excitation wavelength.

Three-dimensional characterization of collagen remodeling in cell-seeded collagen scaffolds via polarization second harmonic generation.

In this study, we use non-linear imaging microscopy to characterize the structural properties of porous collagen-GAG scaffolds (CGS) seeded with human umbilical vein endothelial cells (HUVECs), as well as human mesenchymal stem cells (hMSCs), a co-culture previously reported to form vessel-like structures inside CGS. The evolution of the resulting tissue construct was monitored over 10 days via simultaneous two- and three-photon excited fluorescence microscopy. Time-lapsed 2- and 3-photon excited fluorescence imaging was utilized to monitor the temporal evolution of the vascular-like structures up to 100 µm inside the scaffold up to 10 days post-seeding. 3D polarization-dependent second harmonic generation (PSHG) was utilized to monitor collagen-based scaffold remodeling and determine collagen fibril orientation up to 200 µm inside the scaffold. We demonstrate that polarization-dependent second harmonic generation can provide a novel way to quantify the reorganization of the collagen architecture in CGS simultaneously with key biomechanical interactions between seeded cells and CGS that regulate the formation of vessel-like structures inside 3D tissue constructs. A comparison between samples at different days in vitro revealed that gradually, the scaffolds developed an orthogonal net-like architecture, previously found in real skin.

In vivo deep-brain imaging of microglia enabled by three-photon fluorescence microscopy.

Microglia act as the first and main form of active immune defense in brain. However, in animal models, research on these cells is limited to the superficial layer of the brain, due to the lack of a deep-imaging technique. Here we break this depth limit using three-photon fluorescence (3PF) microscopy excited at the 1700-nm window. Three-photon action cross-section (ησ3) measurement lays the basis for dye selection and the resultant maximization of 3PF generation. 3PF imaging suppresses the surface background, leading to a much improved signal-to-background ratio compared to the commonly used two-photon microscopy (2PM). We can image microglia 1124 µm below the brain surface in vivo, 3.7 times deeper than previous results using 2PM for microglia imaging. This technique enables us to visualize microglia in the white matter layer in vivo for the first time.

Facile Synthesis of Efficient Luminogens with AIE Features for Three-Photon Fluorescence Imaging of the Brain through the Intact Skull.

Visualization of the brain in its native environment is important for understanding common brain diseases. Herein, bright luminogens with remarkable aggregation-induced emission (AIE) characteristics and high quantum yields of up to 42.6% in the solid state are synthesized through facile reaction routes. The synthesized molecule, namely BTF, shows ultrabright far-red/near-infrared emission and can be fabricated into AIE dots by a simple nanoprecipitation procedure. Due to their high brightness, large Stokes shift, good biocompatibility, satisfactory photostability, and large three-photon absorption cross section, the AIE dots can be utilized as efficient fluorescent nanoprobes for in vivo brain vascular imaging through the intact skull by a three-photon fluorescence microscopy imaging technique. This is the first example of using AIE dots for the visualization of the cerebral stroke process through the intact skull of a mouse with high penetration depth and good image contrast. Such good results are anticipated to open up a new venue in the development of efficient emitters with strong nonlinear optical effects for noninvasive bioimaging of living brain.

Aggregation-induced emission nanoparticles for in vivo three-photon fluorescence microscopic rat brain angiography

Rodents are popular biological models for physiological and behavioral research in neuroscience and rats are better models than mice due to their higher genome similarity to human and more accessible surgical procedures. However, rat brain is larger than mice brain and it needs powerful imaging tools to implement better penetration against the scattering of the thicker brain tissue. Three-photon fluorescence microscopy (3PFM) combined with near-infrared (NIR) excitation has great potentials for brain circuits imaging because of its abilities of anti-scattering, deep-tissue imaging, and high signal-to-noise ratio (SNR). In this work, a type of AIE luminogen with red fluorescence was synthesized and encapsulated with Pluronic F-127 to make up form nanoparticles (NPs). Bright DCDPP-2TPA NPs were employed for in vivo three-photon fluorescent laser scanning microscopy of blood vessels in rats brain under 1550[Formula: see text]nm femtosecond laser excitation. A fine three-dimensional (3D) reconstruction up to the deepness of 600[Formula: see text][Formula: see text]m was achieved and the blood flow velocity of a selected vessel was measured in vivo as well. Our 3PFM deep brain imaging method simultaneously recorded the morphology and function of the brain blood vessels in vivo in the rat model. Using this angiography combined with the arsenal of rodent’s brain disease, models can accelerate the neuroscience research and clinical diagnosis of brain disease in the future.

Aggregation-induced emission luminogen for in vivo three-photon fluorescence lifetime microscopic imaging

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.

Three-photon light-sheet fluorescence microscopy

We present the first demonstration of three-photon excitation light-sheet fluorescence microscopy. Light-sheet fluorescence microscopy in single- and two-photon modes has emerged as a powerful wide-field, low-photodamage technique for fast volumetric imaging of biological samples. We extend this imaging modality to the three-photon regime, enhancing its penetration depth. Our present study uses a conventional femtosecond pulsed laser at 1000nm wavelength for the imaging of 450μm diameter cellular spheroids. In addition, we show, experimentally and through numerical simulations, the potential advantages in three-photon light-sheet microscopy of using propagation-invariant Bessel beams in preference to Gaussian beams.

Tunable Aggregation-Induced Emission Nanoparticles by Varying Isolation Groups in Perylene Diimide Derivatives and Application in Three-Photon Fluorescence Bioimaging.

The development of fluorogens with deep-red emission is one of the hottest topics of investigation in the field of bio/chemosensors and bioimaging. Herein, the tunable fluorescence of perylene diimide (PDI) derivatives was achieved by the incorporation of varied isolation groups linked on the PDI core. With the enlarged sizes of isolation groups, the conversion from aggregation caused quenching to aggregation-induced emission was obtained in their fluorescence variations from solutions to nanoparticles, as the result of the efficient inhibition of π-π stacking by the larger isolation groups. Accordingly, DCzPDI bearing 1,3-di(9H-carbazol-9-yl)benzene as the biggest isolation group exhibited the bright deep-red emission in the aggregated state with a quantum yield of 12.3%. Combined with the three-photon excited fluorescence microscopy (3PFM) technology, through-skull 3PFM imaging of mouse cerebral vasculature can be realized by DCzPDI nanoparticles with good biocompatibility, and the penetration depth can be as deep as 450 μm.

Three-photon fluorescence microscopy with an axially elongated Bessel focus.

Volumetric imaging tools that are simple to adopt, flexible, and robust are in high demand in the field of neuroscience, where the ability to image neurons and their networks with high spatiotemporal resolution is essential. Using an axially elongated focus approximating a Bessel beam, in combination with two-photon fluorescence microscopy, has proven successful at such an endeavor. Here, we demonstrate three-photon fluorescence imaging with an axially extended Bessel focus. We use an axicon-based module that allowed for the generation of Bessel foci of varying numerical apertures and axial lengths, and apply this volumetric imaging tool to image mouse brain slices and for in vivo imaging of the mouse brain.