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.

Multiphoton imaging of hippocampal neural circuits: techniques and biological insights into region-, cell-type-, and pathway-specific functions.

Significance: The function of the hippocampus in behavior and cognition has long been studied primarily through electrophysiological recordings from freely moving rodents. However, the application of optical recording methods, particularly multiphoton fluorescence microscopy, in the last decade or two has dramatically advanced our understanding of hippocampal function. This article provides a comprehensive overview of techniques and biological findings obtained from multiphoton imaging of hippocampal neural circuits. Aim: This review aims to summarize and discuss the recent technical advances in multiphoton imaging of hippocampal neural circuits and the accumulated biological knowledge gained through this technology. Approach: First, we provide a brief overview of various techniques of multiphoton imaging of the hippocampus and discuss its advantages, drawbacks, and associated key innovations and practices. Then, we review a large body of findings obtained through multiphoton imaging by region (CA1 and dentate gyrus), cell type (pyramidal neurons, inhibitory interneurons, and glial cells), and cellular compartment (dendrite and axon). Results: Multiphoton imaging of the hippocampus is primarily performed under head-fixed conditions and can reveal detailed mechanisms of circuit operation owing to its high spatial resolution and specificity. As the hippocampus lies deep below the cortex, its imaging requires elaborate methods. These include imaging cannula implantation, microendoscopy, and the use of long-wavelength light sources. Although many studies have focused on the dorsal CA1 pyramidal cells, studies of other local and inter-areal circuitry elements have also helped provide a more comprehensive picture of the information processing performed by the hippocampal circuits. Imaging of circuit function in mouse models of Alzheimer's disease and other brain disorders such as autism spectrum disorder has also contributed greatly to our understanding of their pathophysiology. Conclusions: Multiphoton imaging has revealed much regarding region-, cell-type-, and pathway-specific mechanisms in hippocampal function and dysfunction in health and disease. Future technological advances will allow further illustration of the operating principle of the hippocampal circuits via the large-scale, high-resolution, multimodal, and minimally invasive imaging.

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.

Three-Photon Adaptive Optics for Mouse Brain Imaging.

Three-photon microscopy (3PM) was shown to allow deeper imaging than two-photon microscopy (2PM) in scattering biological tissues, such as the mouse brain, since the longer excitation wavelength reduces tissue scattering and the higher-order non-linear excitation suppresses out-of-focus background fluorescence. Imaging depth and resolution can further be improved by aberration correction using adaptive optics (AO) techniques where a spatial light modulator (SLM) is used to correct wavefront aberrations. Here, we present and analyze a 3PM AO system for in vivo mouse brain imaging. We use a femtosecond source at 1300 nm to generate three-photon (3P) fluorescence in yellow fluorescent protein (YFP) labeled mouse brain and a microelectromechanical (MEMS) SLM to apply different Zernike phase patterns. The 3P fluorescence signal is used as feedback to calculate the amount of phase correction without direct phase measurement. We show signal improvement in the cortex and the hippocampus at greater than 1 mm depth and demonstrate close to diffraction-limited imaging in the cortical layers of the brain, including imaging of dendritic spines. In addition, we characterize the effective volume for AO correction within brain tissues, and discuss the limitations of AO correction in 3PM of mouse brain.

Recent advances in intravital microscopy for preclinical research.

Intravital microscopy (IVM) has revolutionized our understanding of single-cell behavior in complex tissues by enabling real-time observation of molecular and cellular processes in their natural environment. In preclinical research, IVM has emerged as a standard tool for mechanistic studies of therapy response and the rational design of new treatment strategies. Technological developments keep expanding the imaging depth and quality that can be achieved in living tissue, and the maturation of imaging modalities such as fluorescence and phosphorescence lifetime imaging facilitates co-registration of individual cell dynamics with metabolic tissue states. Correlation of IVM with mesoscopic and macroscopic imaging modalities further promotes the translation of mechanistic insights gained by IVM into clinically relevant information. This review highlights some of the recent advances in IVM that have made the transition from experimental optical techniques to practical applications in basic and preclinical research.

Two- and three-photon absorption cross-section characterization for high-brightness, cell-specific multiphoton fluorescence brain imaging.

We demonstrate an accurate quantitative characterization of absolute two- and three-photon absorption (2PA and 3PA) action cross sections of a genetically encodable fluorescent marker Sypher3s. Both 2PA and 3PA action cross sections of this marker are found to be remarkably high, enabling high-brightness, cell-specific two- and three-photon fluorescence brain imaging. Brain imaging experiments on sliced samples of rat's cortical areas are presented to demonstrate these imaging modalities. The 2PA action cross section of Sypher3s is shown to be highly sensitive to the level of pH, enabling pH measurements via a ratiometric readout of the two-photon fluorescence with two laser excitation wavelengths, thus paving the way toward fast optical pH sensing in deep-tissue experiments.

Slide-free imaging of hematoxylin-eosin stained whole-mount tissues using combined third-harmonic generation and three-photon fluorescence microscopy.

Intraoperative margin assessment of surgical tissues during cancer surgery is clinically important, especially in the case of tissue conserving surgery like Mohs micrographic surgery in which minimization of the surgical area is considered crucial. Frozen pathology is the gold standard of assessing excised tissues for signs of remaining cancerous lesions. The current protocol, however, is time-consuming and labor-intensive. Instead of the complex frozen sectioning, staining, and traditional white light microscopy imaging protocol, optically sectioned histopathological imaging of hematoxylin-eosin stained whole-mount skin tissues with a subfemtoliter resolution is demonstrated by using nonlinear microscopy in this study. With our proposed method, the reagents of staining and the contrast of imaging are fully consistent with the current clinical standard of frozen pathology, thus facilitating rapid intraoperative assessment of surgical tissues for future applications. Image: Slide-free nonlinear microscopy imaging of H&E stained whole-mount skin tissue showing the morphology of sweat glands.

Deep-brain three-photon microscopy excited at 1600 nm with silicone oil immersion.

Three-photon microscopy excited at the 1700-nm window (roughly covering 1600-1840 nm) is especially suitable for deep-brain imaging in living animals. To match the brain refractive index, D2 O has been exclusively used as the immersion medium. However, the hygroscopic property of D2 O leads to a decrease of transmittance of the excitation light and as a result a decrease in three-photon signals over time. Solutions such as replacing D2 O from time to time, wrapping both the objective lens and the immersion D2 O, and sealing D2 O with paraffin liquid have all been demonstrated, which add to the system complexity. Based on our recent characterization of immersion oils, we propose using silicone oil as a potential alternative to D2 O for deep-brain imaging. Excited at 1600 nm, our comparative deep-brain imaging using both D2 O and silicone oil immersion show that silicone oil immersion yields 17% higher three-photon signal in third-harmonic generation imaging within the white matter. Besides, silicone oil immersion also enables three-photon fluorescence imaging of vasculature up to 1460 µm (mechanical depth) into the mouse brain in vivo acquired at 2 seconds/frame. Together with the nonhygroscopic physical property, silicone oil is promising for long-span three-photon brain imaging excited at the 1700-nm window.

Long-term in vivo single-cell lineage tracing of deep structures using three-photon activation.

Genetic labeling techniques allow for noninvasive lineage tracing of cells in vivo. Two-photon inducible activators provide spatial resolution for superficial cells, but labeling cells located deep within tissues is precluded by scattering of the far-red illumination required for two-photon photolysis. Three-photon illumination has been shown to overcome the limitations of two-photon microscopy for in vivo imaging of deep structures, but whether it can be used for photoactivation remains to be tested. Here we show, both theoretically and experimentally, that three-photon illumination overcomes scattering problems by combining longer wavelength excitation with high uncaging three-photon cross-section molecules. We prospectively labeled heart muscle cells in zebrafish embryos and found permanent labeling in their progeny in adult animals with negligible tissue damage. This technique allows for a noninvasive genetic manipulation in vivo with spatial, temporal and cell-type specificity, and may have wide applicability in experimental biology.