Ultrafast two-photon fluorescence imaging of cerebral blood circulation in the mouse brain in vivo.

Characterizing blood flow dynamics in vivo is critical to understanding the function of the vascular network under physiological and pathological conditions. Existing methods for hemodynamic imaging have insufficient spatial and temporal resolution to monitor blood flow at the cellular level in large blood vessels. By using an ultrafast line-scanning module based on free-space angular chirped enhanced delay, we achieved two-photon fluorescence imaging of cortical blood flow at 1,000 two-dimensional (2D) frames and 1,000,000 one-dimensional line scans per second in the awake mouse. This orders-of-magnitude increase in temporal resolution allowed us to measure cerebral blood flow at up to 49 mm/s and observe pulsatile blood flow at harmonics of heart rate. Directly visualizing red blood cell (RBC) flow through vessels down to >800 µm in depth, we characterized cortical layer­dependent flow velocity distributions of capillaries, obtained radial velocity profiles and kilohertz 2D velocity mapping of multifile blood flow, and performed RBC flux measurements from penetrating blood vessels.

Kilohertz two-photon fluorescence microscopy imaging of neural activity in vivo.

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

890-nm-excited SHG and fluorescence imaging enabled by an all-fiber mode-locked laser.

We demonstrate second-harmonic generation (SHG) microscopy excited by the ∼890-nm light frequency-doubled from a 137-fs, 19.4-MHz, and 300-mW all-fiber mode-locked laser centered at 1780 nm. The mode-locking at the 1.7-µm window is realized by controlling the emission peak of the gain fiber, and uses the dispersion management technique to broaden the optical spectrum up to 30 nm. The spectrum is maintained during the amplification and the pulse is compressed by single-mode fibers. The SHG imaging performance is showcased on a mouse skull, leg, and tail. Two-photon fluorescence imaging is also demonstrated on C. elegans labeled with green and red fluorescent proteins. The frequency-doubled all-fiber laser system provides a compact and efficient tool for SHG and fluorescence microscopy.

PARC: ultrafast and accurate clustering of phenotypic data of millions of single cells.

MOTIVATION: New single-cell technologies continue to fuel the explosive growth in the scale of heterogeneous single-cell data. However, existing computational methods are inadequately scalable to large datasets and therefore cannot uncover the complex cellular heterogeneity. RESULTS: We introduce a highly scalable graph-based clustering algorithm PARC-Phenotyping by Accelerated Refined Community-partitioning-for large-scale, high-dimensional single-cell data (>1 million cells). Using large single-cell flow and mass cytometry, RNA-seq and imaging-based biophysical data, we demonstrate that PARC consistently outperforms state-of-the-art clustering algorithms without subsampling of cells, including Phenograph, FlowSOM and Flock, in terms of both speed and ability to robustly detect rare cell populations. For example, PARC can cluster a single-cell dataset of 1.1 million cells within 13 min, compared with >2 h for the next fastest graph-clustering algorithm. Our work presents a scalable algorithm to cope with increasingly large-scale single-cell analysis. AVAILABILITY AND IMPLEMENTATION: https://github.com/ShobiStassen/PARC. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Resolution enhancement in an extended depth of field for volumetric two-photon microscopy.

The resolution enhancement over the extended depth of field (DOF) in the volumetric two-photon microscopy (TPM) is demonstrated by utilizing multiple orders of Bessel beams. Here the conventional method of switching laser modes (SLAM) in 2D is introduced to 3D, denoted as the volumetric SLAM (V-SLAM). The equivalent scanning beam in the TPM is a thin needle-like beam, which is generated from the subtraction between the needle-like 0th-order and the straw-like 1st-order Bessel beams. Compared with the 0th-order Bessel beam, the lateral resolution of the V-SLAM is increased by 28.6% and maintains over the axial depth of 56 µm. The V-SLAM performance is evaluated by employing fluorescent beads and a mouse brain slice. The V-SLAM approach provides a promising solution to improve the lateral resolutions for fast volumetric imaging on sparsely distributed samples.

Quantitative Phase Imaging Flow Cytometry for Ultra-Large-Scale Single-Cell Biophysical Phenotyping.

Cellular biophysical properties are the effective label-free phenotypes indicative of differences in cell types, states, and functions. However, current biophysical phenotyping methods largely lack the throughput and specificity required in the majority of cell-based assays that involve large-scale single-cell characterization for inquiring the inherently complex heterogeneity in many biological systems. Further confounded by the lack of reported robust reproducibility and quality control, widespread adoption of single-cell biophysical phenotyping in mainstream cytometry remains elusive. To address this challenge, here we present a label-free imaging flow cytometer built upon a recently developed ultrafast quantitative phase imaging (QPI) technique, coined multi-ATOM, that enables label-free single-cell QPI, from which a multitude of subcellularly resolvable biophysical phenotypes can be parametrized, at an experimentally recorded throughput of >10,000 cells/s-a capability that is otherwise inaccessible in current QPI. With the aim to translate multi-ATOM into mainstream cytometry, we report robust system calibration and validation (from image acquisition to phenotyping reproducibility) and thus demonstrate its ability to establish high-dimensional single-cell biophysical phenotypic profiles at ultra-large-scale (>1,000,000 cells). Such a combination of throughput and content offers sufficiently high label-free statistical power to classify multiple human leukemic cell types at high accuracy (~92-97%). This system could substantiate the significance of high-throughput QPI flow cytometry in enabling next frontier in large-scale image-derived single-cell analysis applied in biological discovery and cost-effective clinical diagnostics. © 2019 International Society for Advancement of Cytometry.

Depth-resolved volumetric two-photon microscopy based on dual Airy beam scanning.

We demonstrate dual-Airy-beam-scanning-based volumetric two-photon microscopy (TPM) with depth-resolving capability. A pair of Airy beams with opposite acceleration is used as the excitation lights to sequentially illuminate the sample, and depth information can be resolved based on the deflection of the Airy beam. The depth-resolving range of the volumetric TPM is up to 32 µm. The advantages of the depth-resolved volumetric TPM are the depth-resolving capability over Bessel-beam-based TPM and less scanning times over traditional Gaussian-beam-based TPM. The depth-resolved volumetric TPM provides a promising fast imaging tool to study the dynamics in neural biology.

Volumetric two-photon microscopy with a non-diffracting Airy beam.

We demonstrate a volumetric two-photon microscopy (TPM) using the non-diffracting Airy beam as illumination. Direct mapping of the imaging trajectory shows that the Airy beam extends the axial imaging range around six times longer than a traditional Gaussian beam does along the propagation direction, while maintaining a comparable lateral width. Benefiting from its non-diffracting nature, the TPM with Airy beam illumination is able not only to capture a volumetric image within a single frame, but also to acquire image structures behind a strongly scattered medium. The volumetric specimen is mapped layer by layer under Gaussian mode, while the three-dimensional structure is projected to a single two-dimensional image under Airy mode, leading to a significantly increased acquisition speed. The performance of the TPM is evaluated employing a phantom of agarose gel imbedding fluorescent beads as well as a mouse brain slice. Finally, we showcase the penetration ability of the developed Airy TPM by imaging through a scattering environment.

Long-range projections coordinate distributed brain-wide neural activity with a specific spatiotemporal profile.

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 blood-oxygenation-level-dependent activation, although with a distinct spatiotemporal profile. Our results directly demonstrate that low-frequency 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.

Short pulse generation from a passively mode-locked fiber optical parametric oscillator with optical time-stretch.

We propose a passively mode-locked fiber optical parametric oscillator assisted with optical time-stretch. Thanks to the lately developed optical time-stretch technique, the onset oscillating spectral components can be temporally dispersed across the pump envelope and further compete for the parametric gain with the other parts of onset oscillating sidebands within the pump envelope. By matching the amount of dispersion in optical time-stretch with the pulse width of the quasi-CW pump and oscillating one of the parametric sidebands inside the fiber cavity, we numerically show that the fiber parametric oscillator can be operated in a single pulse regime. By varying the amount of the intracavity dispersion, we further verify that the origin of this single pulse mode-locking regime is due to the optical pulse stretching and compression.

Video-rate centimeter-range optical coherence tomography based on dual optical frequency combs by electro-optic modulators.

Imaging speed and range are two important parameters for optical coherence tomography (OCT). A conventional video-rate centimeter-range OCT requires an optical source with hundreds of kHz repetition rate and needs the support of broadband detectors and electronics (>1 GHz). In this paper, a type of video-rate centimeter-range OCT system is proposed and demonstrated based on dual optical frequency combs by leveraging electro-optic modulators. The repetition rate difference between dual combs, i.e. the A-scan rate of dual-comb OCT, can be adjusted within 0~6 MHz. By down-converting the interference signal from optical domain to radio-frequency domain through dual comb beating, the down-converted bandwidth of the interference signal is less than 22.5 MHz which is at least two orders of magnitude lower than that in conventional OCT systems. A LabVIEW program is developed for video-rate operation, and the centimeter imaging depth is proved by using 10 pieces of 1-mm thick glass stacked as the sample. The effective beating bandwidth between two optical comb sources is 7 nm corresponding to ~108 comb lines, and the axial resolution of the dual-comb OCT is 158 µm. Dual optical frequency combs provide a promising solution to relax the detection bandwidth requirement in fast long-range OCT systems.

102-nm, 44.5-MHz inertial-free swept source by mode-locked fiber laser and time stretch technique for optical coherence tomography.

A swept source with both high repetition-rate and broad bandwidth is indispensable to enable optical coherence tomography (OCT) with high imaging rate and high axial resolution. However, available swept sources are commonly either limited in speed (sub-MHz) by inertial or kinetic component, or limited in bandwidth (<100 nm) by the gain medium. Here we report an ultrafast broadband (over 100 nm centered at 1.55-µm) all-fiber inertial-free swept source built upon a high-power dispersion-managed fiber laser in conjunction with an optical time-stretch module which bypasses complex optical amplification scheme, which result in a portable and compact implementation of time-stretch OCT (TS-OCT) at A-scan rate of 44.5-MHz, axial resolution of 14 µm in air (or 10 µm in tissue), and flat sensitivity roll-off within 4.3 mm imaging range. Together with the demonstration of two- and three-dimensional OCT imaging of a mud-fish eye anterior segment, we also perform comprehensive studies on the imaging depth, receiver bandwidth, and group velocity dispersion condition. This all-fiber inertia-free swept source could provide a promising source solution for SS-OCT system to realize high-performance volumetric OCT imaging in real time.

Flexible pulse-stretching for a swept source at 2.0 µm using free-space angular-chirp-enhanced delay.

Dispersive pulse-stretching at 2.0 µm has long been hindered by the high intrinsic optical loss from conventional dispersive media. Here a flexible pulse-stretching technique at 2.0 µm is demonstrated over a broad bandwidth with large-scale dispersion and low intrinsic optical loss. The technique employs the newly proposed pulse-stretching scheme, namely, free-space angular-chirp-enhanced delay. Both normal and anomalous temporal dispersion (up to ±500 ps/nm) with low intrinsic loss (<6 dB) over a spectral bandwidth of ∼84 nm at 2.0 µm is obtained with low nonlinear effects. Based on this method, an optical wavelength-swept source at 2.0 µm is realized and applied to spectrally encoded imaging at a line scan rate of ∼19 MHz, proving the potential of this pulse-stretching technique for continuous single-shot measurements at the 2.0 µm wavelength regime, particularly for optical microscopy and spectroscopy.

Ultrafast optical imaging at 2.0 µm through second-harmonic-generation-based time-stretch at 1.0 µm.

The performance of ultrafast time-stretch imaging at long wavelengths (beyond 1.5 µm) has suffered from low detection sensitivity due to the increasing loss of optical dispersive fibers. Here, we report an ultrafast optical imaging system with a line scan rate of ∼19 MHz at the 2.0-µm wavelength window by combining second-harmonic generation (SHG) with the highly sensitive time-stretch detection at 1.0 µm. In this imaging system, the sample is illuminated by the pulsed laser source at 2.0 µm in the spectrally encoding manner. After SHG, the encoded spectral signal at 2.0 µm is converted to 1.0 µm and then mapped to the time domain through a highly dispersive fiber at 1.0 µm, which provides a superior dispersion-to-loss ratio of ∼53 ps/nm/dB, ∼50 times larger than that of the standard fibers at 2.0 µm (typically ∼1.1 ps/nm/dB). These efforts make it possible for time-stretch technology not only being translated to longer wavelengths, where unique optical absorption contrast exists, but also benefitting from the high detection sensitivity at shorter wavelengths.

Real-time observation of round-trip resolved spectral dynamics in a stabilized fs fiber laser.

Fiber-stretcher based phase-lock loop (PLL) is a mature technique in fiber mode-locked lasers for repetition-rate stabilization. However, undesired side effects may be induced if not properly handled, which is easily overlooked owing to the lack of single-shot spectral analyzers. Thanks to the ultrafast spectral analyzing capability of optical time-stretch, an intriguing spectral dynamics is observed in a repetition-rate-stabilized nonlinear polarization rotation (NPR) mode-locked laser. Under the dynamic state, the optical spectra of pulses undergo dramatic evolution in every round trip while the pulse energy is relatively constant. Indicated by estimated cross-spectral densities, such spectral dynamics results in noticeable degradation in optical spectral coherence. The physical origin of the round-trip evolved spectral dynamics is attributed to the local birefringence induced by the fiber stretcher. Therefore, the results are helpful for a proper use of fiber-stretcher based PLL in fiber lasers, particularly when a good spectral coherence is desired. Furthermore, our study has also provided a potentially useful optical source for applications where fast spectral modulation is desired.

High-speed wavelength-swept source at 2.0 µm and its application in imaging through a scattering medium.

We report a high-speed wavelength-swept source operating at 2.0 µm through advanced time-stretch technology. It sweeps over 30 nm at a speed of 3.3×109 nm/s and a repetition rate of ∼19 MHz. To the best of our knowledge, this is the first time that a megahertz-stable swept source has been implemented at such a long wavelength. A wide bandwidth is enabled by a simple mode-locked fiber laser that covers a wavelength range of ∼60 nm. The all-optical wavelength sweeping is realized by a chirped fiber Bragg grating (CFBG), which shows a superior temporal stability and power efficiency, compared with commonly used dispersive fibers, particularly in the 2.0 µm wavelength window. To showcase its specialties, here we employ it to perform high-speed spectrally-encoded microscopy (i.e., time-stretch imaging) through a scattering medium at a line-scan rate of up to ∼19 MHz. Better image quality is achieved, compared with a conventional imaging window at 1.0 µm. It is believed that the potential applications of this new high-speed swept source will benefit the transient diagnosis that requires deep penetration through a scattering medium.

Self-healing highly-chirped fiber laser at 1.0 µm.

We demonstrate a MHz wavelength-swept fiber laser with diffraction-free and self-healing properties at the bio-favorable wavelength window of 1.0 µm. This ultrafast wavelength sweeping at a high chirp rate is all-optically realized through a newly-designed dispersive fiber that can provide a dispersion amount up to -1.7 ns/nm. It is 8 times larger than the standard single-mode fiber at this window and by adopting a double-pass configuration, the dispersion amount can be further increased to about -3.5 ns/nm, which is 23 times larger than what has previously been demonstrated. Its beam profile, a 2D Airy function, shows no obvious diffraction within a propagation distance of 2 meters and furthermore, the self-healing property is also verified by blocking the main lobe of the laser beam. This is the first wavelength-swept fiber laser equipped with diffraction-free and self-healing properties at the bio-favorable window. We believe that such effort can enable real-time data processing and a deeper penetration for the high-speed spectroscopic applications in the turbid environment.

High-throughput time-stretch imaging flow cytometry for multi-class classification of phytoplankton.

Time-stretch imaging has been regarded as an attractive technique for high-throughput imaging flow cytometry primarily owing to its real-time, continuous ultrafast operation. Nevertheless, two key challenges remain: (1) sufficiently high time-stretch image resolution and contrast is needed for visualizing sub-cellular complexity of single cells, and (2) the ability to unravel the heterogeneity and complexity of the highly diverse population of cells - a central problem of single-cell analysis in life sciences - is required. We here demonstrate an optofluidic time-stretch imaging flow cytometer that enables these two features, in the context of high-throughput multi-class (up to 14 classes) phytoplantkton screening and classification. Based on the comprehensive feature extraction and selection procedures, we show that the intracellular texture/morphology, which is revealed by high-resolution time-stretch imaging, plays a critical role of improving the accuracy of phytoplankton classification, as high as 94.7%, based on multi-class support vector machine (SVM). We also demonstrate that high-resolution time-stretch images, which allows exploitation of various feature domains, e.g. Fourier space, enables further sub-population identification - paving the way toward deeper learning and classification based on large-scale single-cell images. Not only applicable to biomedical diagnostic, this work is anticipated to find immediate applications in marine and biofuel research.

110 nm versatile fiber optical parametric amplifier at 1.0 µm.

The fiber optical parametric amplifier (FOPA) has been well investigated and widely adopted at the telecommunication window, and outstanding progress has been achieved in areas such as high gain, wide bandwidths, and even flexible gain-spectrum shape. In contrast, a FOPA at the bio-favorable window, 1.0 µm, has been largely underexploited, especially for its relatively limited bandwidth. Here, we demonstrate an all-fiber single-pump FOPA at 1.0 µm with versatile performances, including ultrahigh gain (∼52 dB), wide bandwidth (∼110 nm), and good gain-spectrum flatness (∼3 dB). To showcase the practical applications, the FOPA is utilized to amplify the broadband optical image signal from a spectrally encoded microscopy, yielding a sensitivity enhancement of 47 dB. Thus, it is promising that this all-fiber versatile FOPA works well as an add-on module in boosting sensitivity for existing optical systems at a 1.0 µm window.

Performance of megahertz amplified optical time-stretch optical coherence tomography (AOT-OCT).

Enabled by the ultrahigh-speed all-optical wavelength-swept mechanism and broadband optical amplification, amplified optical time-stretch optical coherence tomography (AOT-OCT) has recently been demonstrated as a practical alternative to achieve ultrafast A-scan rate of multi-MHz in OCT. With the aim of identifying the optimal scenarios for MHz operation in AOT-OCT, we here present a theoretical framework to evaluate its performance metric. In particular, the analysis discusses the unique features of AOT-OCT, such as its superior coherence length, and the relationship between the optical gain and the A-scan rate. More importantly, we evaluate the sensitivity of AOT-OCT in the MHz regime under the influence of the amplifier noise. Notably, the model shows that AOT-OCT is particularly promising when operated at the A-scan rate well beyond multi-MHz--not trivially achievable by any existing swept-source OCT platform. A sensitivity beyond 90 dB, close to the shot-noise limit, can be maintained in the range of 2 - 10 MHz with an optical net gain of ~10 dB. Experimental measurement also shows excellent agreement with the theoretical prediction. While distributed fiber Raman amplification is mainly considered in this paper, the theoretical model is generally applicable to any type of amplification schemes. As a result, our analysis serves as a useful tool for further optimization of AOT-OCT system--as a practical alternative to enable MHz OCT operation.