In vivo deep brain multiphoton fluorescence imaging emitting at NIR-I and NIR-II and excited at NIR-IV.

Multiphoton microscopy (MPM) enables deep brain imaging. Three optical windows: NIR-I, NIR-II, and NIR-III are widely used. Recently, NIR-IV (the 2200 nm window) has been demonstrated to be the last and longest window for deep tissue MPM. However, so far MPM covers only two optical windows labeled by single fluorescent probe, one for emission and one for excitation. Here we demonstrate in vivo deep brain MPM covering three optical windows, with emission at NIR-I, NIR-II, and excitation at NIR-IV, labeled by ICG. The innovations include: (1) characterizing both 3-photon excitation and emission properties of ICG emitting at both NIR-I and NIR-II, in water, plasma, and circulating blood; (2) a home-built multiphoton microscope with simultaneous dual channel detection, with which we demonstrate deep brain MPM 950 µm (NIR-I) and 850 µm (NIR-II) into the mouse brain in vivo, verifying that multi-optical window MPM is promising for deep brain imaging.

In vivo deep-brain 2-photon fluorescent microscopy labeled with near-infrared dyes excited at the 1700 nm window.

2-Photon fluorescence microscopy (2PFM) is an indispensable imaging technology for neuroscience. However, the imaging depth is usually limited to the cortical layer in mouse brain in vivo. Here, we demonstrate deep brain 2PFM in vivo excited at the 1700 nm window, using IR780 and aza-IR780 as fluorescent labels. Our detailed characterization of the multiphoton excitation and emission properties of IR780 and aza-IR780 show that: (1) IR780 or aza-IR780 generate 2-photon fluorescence excited at the 1700 nm window and are promising for 2PFM; (2) aza-IR780 exhibits a larger ησ2 with better anti-photobleaching property compared to IR780; The 2-photon action cross-sections of IR780 and aza-IR780 in plasma are an order-of-magnitude larger than those in PBS; (3) In vivo 2-photon emission spectra for both dyes show a notable red shift compared to those in vitro. Based on these characterization results, we demonstrate deep brain 2PFM labeled by them. A maximum imaging depth of 1585 µm (labeled by IR780) and 1800 µm (labeled by aza-IR780) into the mouse brain in vivo readily penetrates the subcortical region of hippocampus. Besides, a maximum of 1528 µm hemodynamic imaging depth is realized via 2PFM with aza-IR780 labeling, enabling us to measure blood flow speed in the hippocampus.

In Vivo Deep-Brain 3- and 4-Photon Fluorescence Imaging of Subcortical Structures Labeled by Quantum Dots Excited at the 2200 nm Window.

Multiphoton microscopy (MPM) is an enabling technology for visualizing deep-brain structures at high spatial resolution in vivo. Within the low tissue absorption window, shifting to longer excitation wavelengths reduces tissue scattering and boosts penetration depth. Recently, the 2200 nm excitation window has emerged as the last and longest window suitable for deep-brain MPM. However, multiphoton fluorescence imaging at this window has not been demonstrated, due to the lack of characterization of multiphoton properties of fluorescent labels. Here we demonstrate technologies for measuring both the multiphoton excitation and emission properties of fluorescent labels at the 2200 nm window, using (1) 3-photon (ησ3) and 4-photon action cross sections (ησ4) and (2) 3-photon and 4-photon emission spectra both ex vivo and in vivo of quantum dots. Our results show that quantum dots have exceptionally large ησ3 and ησ4 for efficient generation of multiphoton fluorescence. Besides, the 3-photon and 4-photon emission spectra of quantum dots are essentially identical to those of one-photon emission, which change negligibly subject to the local environment of circulating blood. Based on these characterization results, we further demonstrate deep-brain vasculature imaging in vivo. Due to the superb multiphoton properties of quantum dots, 3-photon and 4-photon fluorescence imaging reaches a maximum brain imaging depth of 1060 and 940 µm below the surface of a mouse brain, respectively, which enables the imaging of subcortical structures. We thus fill the last gap in multiphoton fluorescence imaging in terms of wavelength selection.

Resolving arteriolar wall structures in mouse brain in vivo with three-photon microscopy.

The brain arteriolar wall is a multilayered structure, whose integrity is of key significance to the brain function. However, resolving these different layers in anmial models in vivo is hampered by the lack of either labeling or imaging technology. Here, we demonstrate that three-photon microscopy (3PM) is an ideal solution. In mouse brain in vivo, excited at the 1700-nm window, label-free third-harmonic generation imaging and three-photon fluorescence (3PF) imaging with Alexa 633 labeling colocalize and resolve the internal elastic lamina. Furthermore, Alexa Fluor 594-conjugated Wheat Germ Agglutinin (WGA-594) shows time-dependent labeling behavior. As time lapses, WGA-594 first labels endothelium, and then vascular smooth muscle cells, which are readily captured and resolved with 3PF imaging. Our results show that 3PM, in combination with proper labeling, is a promising technology for investigating the structures of brain arteriolar wall in vivo.

Bright Near-Infrared π-Conjugated Oligomer Nanoparticles for Deep-Brain Three-Photon Microscopy Excited at the 1700 nm Window in Vivo.

The development of three-photon fluorophores with 1700 nm excitation is pressingly desirable for in vivo imaging of tissue resided deep inside the brain. Herein, we report a designed and synthesized fluorescent molecule (OFET) for in vivo mouse brain imaging with three-photon microscopy at a record imaging depth. The OFET molecule has a relatively high fluorescence brightness and has a near-infrared (NIR) maximum emission at 820 nm after integrating as water-dispersible nanoparticles (OEFT NPs). Under 1720 nm excitation, OFET NPs show a large three-photon action cross-section of 1.06 × 10-82 cm6 s2/photon2, which is more than twice that of the commonly used sulforhodamine 101 (SR101) dye. Benefiting from the high tissue penetration depths for both the long excitation in the second NIR window of 1720 nm and the emission wavelength in the first NIR window of 820 nm, a high brightness, and a large action cross-section of three-photon, OFET NPs have good deep-brain imaging performance. Brain vasculatures of a mouse located at a depth of 1696 µm can be clearly resolved in vivo. With no observable cytotoxicity even in a high concentration, the present OFET NPs suggest that fluorescent π-conjugated oligomers are of great potential in high-resolution 3PM imaging of in vivo deep-tissue.

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-Brain Three-Photon Imaging Enabled by Aggregation-Induced Emission Luminogens with Near-Infrared-III Excitation.

Understanding the morphology and hemodynamics of cerebral vasculature at large penetration depths and microscale resolution is fundamentally important to decipher brain diseases. Among the various imaging technologies, three-photon (3P) microscopy is of significance by virtue of its deep-penetrating capability and submicron resolution, which especially benefits in vivo vascular imaging. Aggregation-induced emission luminogens (AIEgens) have been recognized to be extraordinarily powerful as 3P probes. However, systematic studies on the structure-performance relationship of 3P AIEgens have been seldom reported. Herein, a series of AIEgens has been designed and synthesized. By intentionally introducing benzene rings onto electron donors (D) and acceptors (A), the molecular distortion, conjugation strength, and the D-A relationship can be facilely manipulated. Upon encapsulation with DSPE-PEG2000, the optimized AIEgens are successfully applied for 3P microscopy with emission in the far-red/near-infrared-I (NIR-I, 700-950 nm) region under the near-infrared-III (NIR-III, 1600-1870 nm) excitation. Impressively, using mice with an opened skull, vasculature within 1700 µm and a microvessel with a diameter of 2.2 µm in deep mouse brain were clearly visualized. In addition, the hemodynamics of blood vessels were well-characterized. Thus, this work not only proposes a molecular design strategy of 3P AIEgens but also promotes the performance of 3P imaging in cerebral vasculature.

Self-phase-modulated femtosecond laser source at 1603 nm and its application to deep-brain 3-photon microscopy in vivo.

3-photon microscopy (3PM) excited at the 1700 nm window enables deep-tissue imaging in vivo, especially in brain. PC rod soliton source has previously been exclusively used as the excitation source, which is rather costly and difficult to align. Here we demonstrate a novel nonlinear optical technique to build femtosecond laser source at the 1700 nm window, based on self-phase modulation (SPM) in a short span of large-mode-area fiber. The spectral broadening experienced by the pump pulse leads to the generation of a red-shifted sidelobe at 1603 nm. After spectral filtering, this sidelobe corresponds to 170-fs, 167-nJ pulses at 1603 nm. Using this SPM source, we further demonstrate deep-brain 3 PM to a depth of 1500 µm below the mouse brain surface in vivo. Our SPM femtosecond laser source thus provides a cost effective and easy-to-align alternative excitation source to the PC rod soliton source.

Measurement of two-photon properties of indocyanine green in water and human plasma excited at the 1700-nm window.

Indocyanine green (ICG) is a human compatible dye and is ideal for deep-tissue two-photon fluorescence (2PF) microscopy excited at the 1700-nm window in vivo. However, the two-photon excitation and emission properties of this dye remain unknown. Here we demonstrate measurement of the two-photon excitation and emission properties of ICG in both water and human plasma, using home-built two-photon action cross-sectional measurement and two-photon emission spectrum measurement systems. Our results show that excited from 1600 to 1800 nm, 2PF can be generated from ICG dissolved in both water and human plasma. The measured two-photon action cross-sectional ησ2 of ICG dissolved in human plasma is an order-of-magnitude larger than that dissolved in water. The measured two-photon emission spectrum overlaps with the one-photon emission spectrum for ICG dissolved in both human plasma and water. Our results will provide key two-photon parameters for the clinical use of ICG.

3-photon microscopy of myelin in mouse digital skin excited at the 1700-nm window.

Myelin is a key component of the peripheral nervous system, whose structure anomaly in the digital skin is implicated in neuropathy. Here we demonstrate an in vivo labeling and imaging technique, capable of visualizing myelin sheaths deep in the mouse digital skin. Through material characterization, we verify that 3-photon fluorescence (3PF) can be generated from a commonly used dye- FluoroMyelin Red for labeling myelin, excited at the 1700-nm window. Topical injection of FluoroMyelin Red in the mouse digit leads to bright labeling of myelin sheaths. Harnessing the deep-penetration capability of 3-photon microscopy excited at the 1700-nm window, we demonstrate that 3PF imaging of FluoroMyelin Red-labeled myelin sheaths in the mouse digit in vivo can be achieved to a depth 340 µm below the skin surface, revealing both branching bundle of and individual myelin sheaths.

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.

In vivo deep-brain blood flow speed measurement through third-harmonic generation imaging excited at the 1700-nm window.

Measurement of the hemodynamic physical parameter blood flow speed in the brain in vivo is key to understanding brain physiology and pathology. 2-photon fluorescence microscopy with single blood vessel resolution is typically used, which necessitates injection of toxic fluorescent dyes. Here we demonstrate a label-free nonlinear optical technique, third-harmonic generation microscopy excited at the 1700-nm window, that is promising for such measurement. Using a simple femtosecond laser system based on soliton self-frequency shift, we can measure blood flow speed through the whole cortical grey matter, even down to the white matter layer. Together with 3-photon fluorescence microscopy, we further demonstrate that the blood vessel walls generate strong THG signals, and that plasma and circulating blood cells are mutually exclusive in space. This technique can be readily applied to brain research.

Deep-brain 2-photon fluorescence microscopy in vivo excited at the 1700 nm window.

Here we demonstrate deep-brain 2-photon fluorescence microscopy in mouse in vivo, excited at the 1700 nm window. Through signal versus power measurement, we show that indocyanine green (ICG) is a promising 2-photon fluorescent dye excitable at the 1700 nm window. In order to excite ICG circulating in the vasculature in the deep brain, we employ a circular-polarization soliton self-frequency shift technique to generate energetic femtosecond pulses at 1617 nm. Combining the labeling and laser technologies, we achieve a record 2-photon fluorescence brain vasculature imaging depth of 2000 µm in vivo. Both the effective attenuation length measurement and signal-to-background ratio measurement indicate that we have reached the theoretical depth limit in 2-photon fluorescence microscopy.

3-photon fluorescence imaging of sulforhodamine B-labeled elastic fibers in the mouse skin in vivo.

Elastic fibers are key constituents of the skin. The commonly adopted optical technique for visualizing elastic fibers in the animal skin in vivo is 2-photon microscopy (2 PM) of autofluorescence, which typically suffers from low signal level. Here we demonstrate a new optical methodology to image elastic fibers in animal models in vivo: 3-photon microscopy (3 PM) excited at the 1700-nm window combining with preferential labeling of elastic fibers using sulforhodamine B (SRB). First, we demonstrate that intravenous injection of SRB can circumvent the skin barrier (encountered in topical application) and preferentially label elastic fibers, as verified by simultaneous 2 PM of both autofluorescence and SRB fluorescence from skin structures. Then through 3-photon excitation property characterization, we show that 3-photon fluorescence can be excited from SRB at the 1700-nm window, and 1600-nm excitation is most efficient according to our 3-photon action cross section measurement. Based on these results and using our developed 1600-nm femtosecond laser source, we finally demonstrate 3 PM of SRB-labeled elastic fibers through the whole dermis in the mouse skin in vivo, with only 3.7-mW optical power deposited on the skin surface. We expect our methodology will provide novel optical solution to elastic fiber research.

In Vivo Deep-Brain Structural and Hemodynamic Multiphoton Microscopy Enabled by Quantum Dots.

Visualizing deep-brain vasculature and hemodynamics is key to understanding brain physiology and pathology. Among the various adopted imaging modalities, multiphoton microscopy (MPM) is well-known for its deep-brain structural and hemodynamic imaging capability. However, the largest imaging depth in MPM is limited by signal depletion in the deep brain. Here we demonstrate that quantum dots are an enabling material for significantly deeper structural and hemodynamic MPM in mouse brain in vivo. We characterized both three-photon excitation and emission parameters for quantum dots: the measured three-photon cross sections of quantum dots are 4-5 orders of magnitude larger than those of conventional fluorescent dyes excited at the 1700 nm window, while the three-photon emission spectrum measured in the circulating blood in vivo shows a slight red shift and broadening compared with ex vivo measurement. On the basis of these measured results, we further demonstrate both structural and hemodynamic three-photon microscopy in the mouse brain in vivo labeled by quantum dots, at record depths among all MPM modalities at all demonstrated excitation wavelengths.

High-energy polarized soliton synthesis and its application to deep-brain 3-photon microscopy in vivo.

Here, we demonstrate a polarized high-energy soliton synthesis technique for deep-brain 3-photon microscopy (3PM) excited at the 1700-nm window. Through coherent combining, we generate linearly polarized high-energy solitons whose energy is twice as high than those of each linearly polarized solitons. Due to the nonlinear origin of signals, both measured 3-photon fluorescence signal and third-harmonic signals are thus boosted by ~8 times in a tissue phantom. Using this technique, we further demonstrate 3PM of sulforhodamine 101 labeled vasculature 1600 µm in the mouse brain in vivo, which cannot be achieved by single-polarized soliton excitation.

Measurement of 3-photon excitation and emission spectra and verification of Kasha's rule for selected fluorescent proteins excited at the 1700-nm window.

Fluorescent proteins are widely used to visualize structures and dynamics in various biological samples. Multiphoton microscopy is especially suitable for imaging structures expressing fluorescent proteins with subcellular resolution. 3-photon microscopy (3PM) excited at the 1700-nm window has proven to be promising for deep-tissue (such as brain) imaging expressing red fluorescent proteins. However, the 3-photon excitation and emission spectra of fluorescent proteins suitable at this window remain largely unknown, hampering protein selection and detection optimization. Here we demonstrate detailed measurement of 3-photon excitation and emission spectra for selected fluorescent proteins, suitable for 3-photon excitation at the 1700-nm window. The measured 3-photon excitation spectra will provide guidelines for protein and excitation wavelength selection. The measured 3-photon emission spectra and comparison with the 1-photon emission spectra, on one hand proves that the fundamental Kasha's rule is still valid for 3-photon fluorescence in these fluorescent proteins, on the other hand will be helpful for efficient fluorescence signal detection.

A near-infrared I emissive dye: toward the application of saturable absorber and multiphoton fluorescence microscopy in the deep-tissue imaging window.

A boron-dipyrromethene (BODIPY) dye emitting in the near-infrared (NIR) I region (723 nm) exhibits strong saturable absorption at 680 nm. Its multiphoton absorption spectra in the NIR II and III regions are determined. Three-photon fluorescence imaging of the BODIPY-labeled cells excited at 1665 nm is also demonstrated.

Propagation dynamics of a circular Airy beam in a uniaxial crystal.

This paper investigates theoretically and numerically the propagation characteristics of a circular Airy beam (CAB) in a uniaxial crystal in detail. The beam loses its boundary cylindrical symmetry during propagation because of the medium anisotropy, although it propagates along the optical axis. This effect of anisotropy on the propagating beam becomes increasingly evident with the increase of the propagation distance. Another main influential factor of the propagation characteristics is the ratio of the extraordinary refractive index to the ordinary refractive index (ne/no). The more the value deviates from 1, the worse the symmetry of the beam intensity distribution becomes. The polarization becomes notably complicated, but possesses a vortex state with a topological charge of 2 during propagation. The abruptly autofocusing characteristic, the most important property of CABs, also appears when the crystal length is long enough, which is greatly different from that characteristic in isotropic media. This work is helpful for the design of optical devices based on uniaxial crystals for beams with some special wavefronts.

Terahertz photodetector based on double-walled carbon nanotube macrobundle-metal contacts.

We report on the characterization of a terahertz (THz) photodetector with an extremely simple structure consisting of only a macroscopic bundle of double-walled carbon nanotubes (DWCNTs) suspended between two metal electrodes. Polarization-sensitive, broadband, and significant photoresponse occurring at the DWCNT-metal contacts under THz illumination are observed with room-temperature photocurrent and photovoltage responsivities up to ∼16 mA/W and ∼0.2 V/W at 2.52 THz, respectively. Scanning photocurrent measurements provide evidence that the photothermoelectric mechanism dominates the detector response. The simple geometry and compact nature of our device make it suitable for integration and show promising applications for THz detection.