Cells in the brain act as components of extended networks. Therefore, to understand neurobiological processes in a physiological context, it is essential to study them in vivo. Super-resolution microscopy has spatial resolution beyond the diffraction limit, thus promising to provide structural and functional insights that are not accessible with conventional microscopy. However, to apply it to in vivo brain imaging, we must address the challenges of 3D imaging in an optically heterogeneous tissue that is constantly in motion. We optimized image acquisition and reconstruction to combat sample motion and applied adaptive optics to correcting sample-induced optical aberrations in super-resolution structured illumination microscopy (SIM) in vivo. We imaged the brains of live zebrafish larvae and mice and observed the dynamics of dendrites and dendritic spines at nanoscale resolution.

While calcium imaging has become a mainstay of modern neuroscience, the spectral properties of current fluorescent calcium indicators limit deep tissue imaging as well as simultaneous use with other probes. Using two monomeric near-infrared fluorescent proteins, we engineered a near-infrared FRET-based genetically encoded calcium indicator (iGECI). iGECI exhibits high brightness, high photostability, and up to 600% increase in fluorescence response to calcium. In dissociated neurons, iGECI detects spontaneous neuronal activity, and electrically and optogenetically induced firing. We validated iGECI performance up to a depth of almost 400 μm in acute brain slices using one-photon light-sheet imaging. Applying hybrid photoacoustic and fluorescence microscopy, we simultaneously monitored neuronal and hemodynamic activities in the mouse brain through an intact skull, with ~3 μm lateral and ~25–50 μm axial resolution. Using two-photon imaging, we detected evoked and spontaneous neuronal activity in the mouse visual cortex, with fluorescence changes of up to 25%. iGECI allows biosensors and optogenetic actuators to be multiplexed without spectral crosstalk.

Optical microscopy, owing to its noninvasiveness and subcellular resolution, enables in vivo visualization of neuronal structure and function in the physiological context. Optical-sectioning structured illumination microscopy (OS-SIM) is a widefield fluorescence imaging technique that uses structured illumination patterns to encode in-focus structures and optically sections 3D samples. However, its application to in vivo imaging has been limited. In this study, we optimized OS-SIM for in vivo neural imaging. We modified OS-SIM reconstruction algorithms to improve signal-to-noise ratio and correct motion-induced artifacts in live samples. Incorporating an adaptive optics (AO) module to OS-SIM, we found that correcting sample-induced optical aberrations was essential for achieving accurate structural and functional characterizations in vivo. With AO OS-SIM, we demonstrated fast, high-resolution in vivo imaging with optical sectioning for structural imaging of mouse cortical neurons and zebrafish larval motor neurons, and functional imaging of quantal synaptic transmission at Drosophila larval neuromuscular junctions.

Based on a molecular-mechanism-based anticancer drug discovery program enabled by an innovative femtomedicine approach, we have found a previously unknown class of non-platinum-based halogenated molecules (called FMD compounds) as potent antitumor agents for effective treatment of cancers. Here, we present in vitro and in vivo studies of the compounds for targeted chemotherapy of cervical, breast, ovarian, and lung cancers. Our results show that these FMD agents led to DNA damage, cell cycle arrest in the S phase, and apoptosis in cancer cells. We also observed that such a FMD compound caused an increase of reduced glutathione (GSH, an endogenous antioxidant) levels in human normal cells, while it largely depleted GSH in cancer cells. We correspondingly found that these FMD agents exhibited no or little toxicity toward normal cells/tissues, while causing significant cytotoxicity against cancer cells, as well as suppression and delay in tumor growth in mouse xenograft models of cervical, ovarian, breast and lung cancers. These compounds are therefore a previously undiscovered class of potent antitumor agents that can be translated into clinical trials for natural targeted chemotherapy of multiple cancers.

Estimation of optical aberrations from volumetric intensity images is a key step in sensorless adaptive optics for 3D microscopy. Recent approaches based on deep learning promise accurate results at fast processing speeds. However, collecting ground truth microscopy data for training the network is typically very difficult or even impossible thereby limiting this approach in practice. Here, we demonstrate that neural networks trained only on simulated data yield accurate predictions for real experimental images. We validate our approach on simulated and experimental datasets acquired with two different microscopy modalities and also compare the results to non-learned methods. Additionally, we study the predictability of individual aberrations with respect to their data requirements and find that the symmetry of the wavefront plays a crucial role. Finally, we make our implementation freely available as open source software in Python.

Significance Characterizing blood flow by tracking individual red blood cells as they move through vessels is essential for understanding vascular function. With high spatial resolution, two-photon fluorescence microscopy is the method of choice for imaging blood flow at the cellular level. However, its application is limited to a low flow speed regimen in anesthetized animals by its slow focus scanning mechanism. Using an ultrafast scanning module, we demonstrated two-photon fluorescence imaging of blood flow at 1,000 two-dimensional frames and 1,000,000 one-dimensional line scans per second in the brains of awake mice. These ultrafast measurements enabled us to study hemodynamic and fluid mechanical regimens beyond the reach of conventional methods.

Studying neuronal activity at synapses requires high spatiotemporal resolution. For high spatial resolution in vivo imaging at depth, adaptive optics (AO) is required to correct sample-induced aberrations. To improve temporal resolution, Bessel focus has been combined with two-photon fluorescence microscopy (2PFM) for fast volumetric imaging at subcellular lateral resolution. To achieve both high-spatial and high-temporal resolution at depth, we develop an efficient AO method that corrects the distorted wavefront of Bessel focus at the objective focal plane and recovers diffraction-limited imaging performance. Applying AO Bessel focus scanning 2PFM to volumetric imaging of zebrafish larval and mouse brains down to 500 µm depth, we demonstrate substantial improvements in the sensitivity and resolution of structural and functional measurements of synapses in vivo. This enables volumetric measurements of synaptic calcium and glutamate activity at high accuracy, including the simultaneous recording of glutamate activity of apical and basal dendritic spines in the mouse cortex.

Cisplatin is the first and most widely used platinum-based chemotherapy drug and is the cornerstone agent in treating a broad spectrum of cancers. However, its clinical application is often limited by severe toxic side effects and drug resistance. Based on the discovered dissociative electron transfer mechanism of cisplatin, a novel combination of cisplatin with [9-(2-carboxyphenyl)-6-diethylamino-3-xanthenylidene]-diethylammonium chloride (basic violet 10, BV10) is proposed to potentiate the chemotherapeutic effect of cisplatin. Here, we show that this combination enhances the anti-cancer effect of cisplatin in both in vitro cell lines and in vivo xenograft mouse models of cisplatin-sensitive and -resistant lung, ovarian and cervical cancers while introducing minimal additional toxic side effects. Furthermore, femtosecond time-resolved laser spectroscopic measurements demonstrate that cisplatin reacts with BV10 via an electron transfer mechanism. These results indicate that the combination of cisplatin with BV10 is promising for improving the chemotherapy of cancers with various extents of cisplatin resistance.