Ultrafast real-time optical imaging is an indispensable tool for studying dynamical events such as shock waves, chemical dynamics in living cells, neural activity, laser surgery and microfluidics. However, conventional CCDs (charge-coupled devices) and their complementary metal-oxide-semiconductor (CMOS) counterparts are incapable of capturing fast dynamical processes with high sensitivity and resolution. This is due in part to a technological limitation-it takes time to read out the data from sensor arrays. Also, there is the fundamental compromise between sensitivity and frame rate; at high frame rates, fewer photons are collected during each frame-a problem that affects nearly all optical imaging systems. Here we report an imaging method that overcomes these limitations and offers frame rates that are at least 1,000 times faster than those of conventional CCDs. Our technique maps a two-dimensional (2D) image into a serial time-domain data stream and simultaneously amplifies the image in the optical domain. We capture an entire 2D image using a single-pixel photodetector and achieve a net image amplification of 25 dB (a factor of 316). This overcomes the compromise between sensitivity and frame rate without resorting to cooling and high-intensity illumination. As a proof of concept, we perform continuous real-time imaging at a frame speed of 163 ns (a frame rate of 6.1 MHz) and a shutter speed of 440 ps. We also demonstrate real-time imaging of microfluidic flow and phase-explosion effects that occur during laser ablation.

Understanding information processing in the brain requires us to monitor neural activity at high spatiotemporal resolution. Using an ultrafast two-photon fluorescence microscope (2PFM) empowered by all-optical laser scanning, we imaged neural activity in vivo at up to 3,000 frames per second and submicron spatial resolution. This ultrafast imaging method enabled monitoring of both supra- and sub-threshold electrical activity down to 345 μm below the brain surface in head-fixed awake mice.

Amplified dispersive Fourier transformation ͑ADFT͒ is a powerful technique that maps the spectrum of an optical pulse into a time-domain waveform using group-velocity dispersion ͑GVD͒ and simultaneously amplifies it in the optical domain. It replaces a diffraction grating and detector array with a dispersive fiber and single photodetector, greatly simplifying the system and, more importantly, enabling ultrafast real-time spectroscopic measurements. Here we present a theory of ADFT by deriving the general equation and spectral resolution for ADFT and studying the evolution of the pulse spectrum into time, the effect of GVD coefficients on ADFT, and the requirement for dispersion. This theory is expected to lend valuable insights into the process and implementation of ADFT.

Optical time-stretch imaging enables the continuous capture of non-repetitive events in real time at a line-scan rate of tens of MHz—a distinct advantage for the ultrafast dynamics monitoring and high-throughput screening that are widely needed in biological microscopy. However, its potential is limited by the technical challenge of achieving significant pulse stretching (that is, high temporal dispersion) and low optical loss, which are the critical factors influencing imaging quality, in the visible spectrum demanded in many of these applications. We present a new pulse-stretching technique, termed free-space angular-chirp-enhanced delay (FACED), with three distinguishing features absent in the prevailing dispersive-fiber-based implementations: (1) it generates substantial, reconfigurable temporal dispersion in free space (>1 ns nm−1) with low intrinsic loss (<6 dB) at visible wavelengths; (2) its wavelength-invariant pulse-stretching operation introduces a new paradigm in time-stretch imaging, which can now be implemented both with and without spectral encoding; and (3) pulse stretching in FACED inherently provides an ultrafast all-optical laser-beam scanning mechanism at a line-scan rate of tens of MHz. Using FACED, we demonstrate not only ultrafast laser-scanning time-stretch imaging with superior bright-field image quality compared with previous work but also, for the first time, MHz fluorescence and colorized time-stretch microscopy. Our results show that this technique could enable a wider scope of applications in high-speed and high-throughput biological microscopy that were once out of reach.

Serial time-encoded amplified microscopy (STEAM) is an entirely new imaging modality that enables ultrafast continuous real-time imaging with high sensitivity. By means of optical image amplification, STEAM overcomes the fundamental tradeoff between sensitivity and speed that affects virtually all optical imaging systems. Unlike the conventional microscope systems, the performance of STEAM depends not only on the lenses, but also on the properties of other components that are unique to STEAM, namely the spatial disperser, the group velocity dispersion element, and the back-end electronic digitizer. In this paper, we present an analysis that shows how these considerations affect the spatial resolution, and how they create a trade-off between the number of pixels and the frame rate of the STEAM imager. We also quantify how STEAM's optical image amplification feature improves the imaging sensitivity. These analyses not only provide valuable insight into the operation of STEAM technology but also serve as a blue print for implementation and optimization of this new imaging technology.

One challenge in contemporary neuroscience is to achieve an integrated understanding of the large-scale brain-wide interactions, particularly the spatiotemporal patterns of neural activity that give rise to functions and behavior. At present, little is known about the spatiotemporal properties of long-range neuronal networks. We examined brain-wide neural activity patterns elicited by stimulating ventral posteromedial (VPM) thalamo-cortical excitatory neurons through combined optogenetic stimulation and functional MRI (fMRI). We detected robust optogenetically evoked fMRI activation bilaterally in primary visual, somatosensory, and auditory cortices at low (1 Hz) but not high frequencies (5-40 Hz). Subsequent electrophysiological recordings indicated interactions over long temporal windows across thalamo-cortical, cortico-cortical, and interhemispheric callosal projections at low frequencies. We further observed enhanced visually evoked fMRI activation during and after VPM stimulation in the superior colliculus, indicating that visual processing was subcortically modulated by low-frequency activity originating from VPM. Stimulating posteromedial complex thalamo-cortical excitatory neurons also evoked brain-wide bloodoxygenation-level-dependent activation, although with a distinct spatiotemporal profile. Our results directly demonstrate that lowfrequency activity governs large-scale, brain-wide connectivity and interactions through long-range excitatory projections to coordinate the functional integration of remote brain regions. This low-frequency phenomenon contributes to the neural basis of long-range functional connectivity as measured by resting-state fMRI.fMRI | optogenetic | brain connectivity | low frequency | thalamus T he brain is a highly complex, interconnected structure with parallel and hierarchical networks distributed within and between neural systems (1, 2). This integrative architecture dictates the underlying principles of how brain-wide neural connectivity supports and organizes sensory, behavioral, and cognitive processes (3). Recent advances in structural (2, 4) and functional connectivity (5-12) mapping, as well as neural circuit modulatory tools, such as optogenetics (13, 14), permit detailed, high-resolution neural examinations at unprecedented scales. In particular, functional connectivity mapping using resting-state functional MRI (rsfMRI) produces noninvasive visualization of slow and spontaneous hemodynamic fluctuations in humans (5-9) and animals (10-12). Coherent low-frequency (<1 Hz) fluctuations across functionally coupled, large-scale networks is an intriguing property, although the exact underlying neural bases and functional significance remain unclear. Previous studies integrating large-scale electrical recordings, voltage-sensitive dye (VSD), and Ca 2+ -imaging techniques (15-18) support the hypothesis that slow, oscillating neural activity constrains and elicits these hemodynamic fluctuations. Furthermore, low-frequency activity can temporally synchronize remote brain region...

Optical imaging is arguably the most effective tool to visualize living cells with high spatiotemporal resolution and in a nearly noninvasive manner. Driven by this capability, state-of-the-art cellular assay techniques have increasingly been adopting optical imaging for classifying different cell types/stages, and thus dissecting the respective cellular functions. However, it is still a daunting task to image and characterize cell-to-cell variability within an enormous and heterogeneous population - an unmet need in single-cell analysis, which is now widely advocated in modern biology and clinical diagnostics. The challenge stems from the fact that current optical imaging technologies still lack the practical speed and sensitivity for measuring thousands to millions of cells down to the single-cell precision. Adopting the wisdom in high-speed fiber-optics communication, optical time-stretch imaging has emerged as a completely new optical imaging concept which is now proven for ultrahigh-throughput optofluidic single-cell imaging, at least 1-2 orders-of-magnitude higher (up to ∼100 000 cells per second) compared to the existing imaging flow cytometers. It also uniquely enables quantification of intrinsic biophysical markers of individual cells - a largely unexploited class of single-cell signatures that is known to be correlated with the overwhelmingly investigated biochemical markers. With the aim of reaching a wider spectrum of experts specializing in cellular assay developments and applications, this paper highlights the essential basics of optical time-stretch imaging, followed by reviewing the recent developments and applications of optofluidic time-stretch imaging. We will also discuss the current challenges of this technology, in terms of providing new insights in basic biology and enriching the clinical diagnostic toolsets.

Dispersive Fourier transformation is a powerful technique in which the spectrum of an optical pulse is mapped into a time-domain waveform using chromatic dispersion. It replaces a diffraction grating and detector array with a dispersive fiber and single photodetector. This simplifies the system and, more importantly, enables fast real-time measurements. Here we describe a novel ultrafast barcode reader and displacement sensor that employs internally-amplified dispersive Fourier transformation. This technique amplifies and simultaneously maps the spectrally encoded barcode into a temporal waveform. It achieves a record acquisition speed of 25 MHz --four orders of magnitude faster than the current state-of-the-art.