Internal state dynamics shape brainwide activity and foraging behaviour.

The brain has persistent internal states that can modulate every aspect of an animal's mental experience1-4. In complex tasks such as foraging, the internal state is dynamic5-8. Caenorhabditis elegans alternate between local search and global dispersal5. Rodents and primates exhibit trade-offs between exploitation and exploration6,7. However, fundamental questions remain about how persistent states are maintained in the brain, which upstream networks drive state transitions and how state-encoding neurons exert neuromodulatory effects on sensory perception and decision-making to govern appropriate behaviour. Here, using tracking microscopy to monitor whole-brain neuronal activity at cellular resolution in freely moving zebrafish larvae9, we show that zebrafish spontaneously alternate between two persistent internal states during foraging for live prey (Paramecia). In the exploitation state, the animal inhibits locomotion and promotes hunting, generating small, localized trajectories. In the exploration state, the animal promotes locomotion and suppresses hunting, generating long-ranging trajectories that enhance spatial dispersion. We uncover a dorsal raphe subpopulation with persistent activity that robustly encodes the exploitation state. The exploitation-state-encoding neurons, together with a multimodal trigger network that is associated with state transitions, form a stochastically activated nonlinear dynamical system. The activity of this oscillatory network correlates with a global retuning of sensorimotor transformations during foraging that leads to marked changes in both the motivation to hunt for prey and the accuracy of motor sequences during hunting. This work reveals an important hidden variable that shapes the temporal structure of motivation and decision-making.

Quantitative absorption cytometry for measuring red blood cell hemoglobin mass and volume.

We present an optical system, called the quantitative absorption cytometer (QAC), to measure the volume and hemoglobin mass of red blood cells flowing through a microfluidic channel. In contrast to clinical hematology analyzers, where cells are sphered in order for both volume and hemoglobin to be measured accurately, the QAC measures cells in their normal physiological shape. Human red blood cells are suspended in a refractive index-matching absorbing buffer, driven through a microfluidic channel, and imaged using a transmission light microscope onto a color camera. A red and a blue LED illuminate cells and images at each color are used to independently retrieve cell volume and hemoglobin mass. This system shows good agreement with red blood cell indices retrieved by a clinical hematology analyzer and in fact measures a smaller coefficient of variation of hemoglobin concentration. In addition to cell indices, the QAC returns height and mass maps of each measured cell. These quantitative images are valuable for analyzing the detailed morphology of individual cells as well as statistical outliers found in the data. We also measured red blood cells in hypertonic and hypotonic buffers to quantify the correlation between volume and hemoglobin mass under osmotic stress. Because this method is invariant to cell shape, even extremely nonspherical cells in hypertonic buffers can be measured accurately.

Fluorescence imaging of flowing cells using a temporally coded excitation.

Imaging fluorescence in moving cells is fundamentally challenging because the exposure time is constrained by motion-blur, which limits the available signal. We report a method to image fluorescently labeled leukemia cells in fluid flow that has an effective exposure time of up to 50 times the motion-blur limit. Flowing cells are illuminated with a pseudo-random excitation pulse sequence, resulting in a motion-blur that can be computationally removed to produce near diffraction-limited images. This method enables observation of cellular organelles and their behavior in a fluid environment that resembles the vasculature.

Dye exclusion microfluidic microscopy.

We present an optical system to measure height maps of non-adherent cells as they flow through a microfluidic channel. The cells are suspended in an index-matching absorbing buffer, where cell height is evaluated by measuring the difference in absorption between the cell and the background. Unlike interferometric microscopes, the measured cell height is nearly independent of the cell's optical properties. The height maps are captured using a single exposure of a color camera, and consequently the system is capable of high-throughput characterization of large collections of cells. Using this system, we have measured more than 1600 height maps and volumes of three different leukemia cell lines.

Nanoengineering a single-molecule mechanical switch using DNA self-assembly.

The ability to manipulate and observe single biological molecules has led to both fundamental scientific discoveries and new methods in nanoscale engineering. A common challenge in many single-molecule experiments is reliably linking molecules to surfaces, and identifying their interactions. We have met this challenge by nanoengineering a novel DNA-based linker that behaves as a force-activated switch, providing a molecular signature that can eliminate errant data arising from non-specific and multiple interactions. By integrating a receptor and ligand into a single piece of DNA using DNA self-assembly, a single tether can be positively identified by force-extension behavior, and receptor-ligand unbinding easily identified by a sudden increase in tether length. Additionally, under proper conditions the exact same pair of molecules can be repeatedly bound and unbound. Our approach is simple, versatile and modular, and can be easily implemented using standard commercial reagents and laboratory equipment. In addition to improving the reliability and accuracy of force measurements, this single-molecule mechanical switch paves the way for high-throughput serial measurements, single-molecule on-rate studies, and investigations of population heterogeneity.

Highly selective oxygen reduction to hydrogen peroxide on transition metal single atom coordination.

Shifting electrochemical oxygen reduction towards 2e- pathway to hydrogen peroxide (H2O2), instead of the traditional 4e- to water, becomes increasingly important as a green method for H2O2 generation. Here, through a flexible control of oxygen reduction pathways on different transition metal single atom coordination in carbon nanotube, we discovered Fe-C-O as an efficient H2O2 catalyst, with an unprecedented onset of 0.822 V versus reversible hydrogen electrode in 0.1 M KOH to deliver 0.1 mA cm-2 H2O2 current, and a high H2O2 selectivity of above 95% in both alkaline and neutral pH. A wide range tuning of 2e-/4e- ORR pathways was achieved via different metal centers or neighboring metalloid coordination. Density functional theory calculations indicate that the Fe-C-O motifs, in a sharp contrast to the well-known Fe-C-N for 4e-, are responsible for the H2O2 pathway. This iron single atom catalyst demonstrated an effective water disinfection as a representative application.

Dictionary-enhanced imaging cytometry.

State-of-the-art high-throughput microscopes are now capable of recording image data at a phenomenal rate, imaging entire microscope slides in minutes. In this paper we investigate how a large image set can be used to perform automated cell classification and denoising. To this end, we acquire an image library consisting of over one quarter-million white blood cell (WBC) nuclei together with CD15/CD16 protein expression for each cell. We show that the WBC nucleus images alone can be used to replicate CD expression-based gating, even in the presence of significant imaging noise. We also demonstrate that accurate estimates of white blood cell images can be recovered from extremely noisy images by comparing with a reference dictionary. This has implications for dose-limited imaging when samples belong to a highly restricted class such as a well-studied cell type. Furthermore, large image libraries may endow microscopes with capabilities beyond their hardware specifications in terms of sensitivity and resolution. We call for researchers to crowd source large image libraries of common cell lines to explore this possibility.

Microscopy, Meet Big Data.

The analysis of massive microscopy datasets using deep neural networks provides an alternative to molecular labeling to characterize cellular states.

Nanoplasmonic fiber tip probe detects significant reduction of intracellular Alzheimer's disease-related oligomers by curcumin.

Considerable evidence shows critical roles of intracellular pathogenic events of Alzheimer's disease (AD). In particular, intracellular amyloid-ß accumulation and oligomerization are early AD pathologic processes, which may lead to changes in inflammatory molecules and other AD-related pathological components. Curcumin and its analogs have been identified as potential drug candidates for AD. However, the effects of curcumin on intracellular AD pathologic processes remain largely unknown. Here we utilized a recently developed nanoplasmonic fiber tip probe (nFTP) technology and investigated whether curcumin leads to intracellular AD pathologic changes. We showed that our nFTP technology could robustly detect intracellular AD-related protein changes caused by a well-known inflammation inducer and a familial AD mutation. Intriguingly, curcumin remarkably reduced the level of intracellular oligomers while modestly reduced the level of an inflammatory cytokine. Thus, our results provided evidence that curcumin's mechanism of action in attenuating AD pathology is through a major role of decreasing oligomerization.

Nanoscale label-free bioprobes to detect intracellular proteins in single living cells.

Fluorescent labeling techniques have been widely used in live cell studies; however, the labeling processes can be laborious and challenging for use in non-transfectable cells, and labels can interfere with protein functions. While label-free biosensors have been realized by nanofabrication, a method to track intracellular protein dynamics in real-time, in situ and in living cells has not been found. Here we present the first demonstration of label-free detection of intracellular p53 protein dynamics through a nanoscale surface plasmon-polariton fiber-tip-probe (FTP).

Hyperspectral fluorescence microfluidic (HFM) microscopy.

We present an imaging system that collects hyperspectral images of cells travelling through a microfluidic channel. Using a single monochrome camera and a linear variable bandpass filter (LVF), the system captures a bright field image and a set of hyperspectral fluorescence images for each cell. While the bandwidth of the LVF is 20 nm, we have demonstrated that we can determine the peak wavelength of a fluorescent object's emission spectrum with an accuracy of below 3 nm. In addition, we have used this system to capture fluorescence spectra of individual spatially resolved cellular organelles and to spectrally resolve multiple fluorophores in individual cells.

Microfabricated multiple field of view imaging flow cytometry.

The combination of microscopy and flow cytometry enables image based screening of large collections of cells. Despite the proposition more than thirty years ago, adding high resolution wide-field imaging to flow cytometers remains challenging. The velocity of cells in flow cytometry can surpass a meter per second, requiring either sub-microsecond exposure times or other sophisticated photodetection techniques. Instead of faster detectors and brighter sources, we demonstrate that by imaging multiple channels simultaneously, a high throughput can be maintained with a flow velocity reduced in proportion to the degree of parallelization. The multi-field of view imaging flow cytometer (MIFC) is implemented with parallel arrays of microfluidic channels and diffractive lenses that produce sixteen wide field images with a magnification of 45 and submicron resolution. Using this device, we have imaged latex beads, red blood cells, and acute myeloid leukemia cells at rates of 2,000-20,000 per second.