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.

High-speed laser-scanning biological microscopy using FACED.

Laser scanning is used in advanced biological microscopy to deliver superior imaging contrast, resolution and sensitivity. However, it is challenging to scale up the scanning speed required for interrogating a large and heterogeneous population of biological specimens or capturing highly dynamic biological processes at high spatiotemporal resolution. Bypassing the speed limitation of traditional mechanical methods, free-space angular-chirp-enhanced delay (FACED) is an all-optical, passive and reconfigurable laser-scanning approach that has been successfully applied in different microscopy modalities at an ultrafast line-scan rate of 1-80 MHz. Optimal FACED imaging performance requires optimized experimental design and implementation to enable specific high-speed applications. In this protocol, we aim to disseminate information allowing FACED to be applied to a broader range of imaging modalities. We provide (i) a comprehensive guide and design specifications for the FACED hardware; (ii) step-by-step optical implementations of the FACED module including the key custom components; and (iii) the overall image acquisition and reconstruction pipeline. We illustrate two practical imaging configurations: multimodal FACED imaging flow cytometry (bright-field, fluorescence and second-harmonic generation) and kHz 2D two-photon fluorescence microscopy. Users with basic experience in optical microscope operation and software engineering should be able to complete the setup of the FACED imaging hardware and software in ~2-3 months.

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.

Parallelized volumetric fluorescence microscopy with a reconfigurable coded incoherent light-sheet array.

Parallelized fluorescence imaging has been a long-standing pursuit that can address the unmet need for a comprehensive three-dimensional (3D) visualization of dynamical biological processes with minimal photodamage. However, the available approaches are limited to incomplete parallelization in only two dimensions or sparse sampling in three dimensions. We hereby develop a novel fluorescence imaging approach, called coded light-sheet array microscopy (CLAM), which allows complete parallelized 3D imaging without mechanical scanning. Harnessing the concept of an "infinity mirror", CLAM generates a light-sheet array with controllable sheet density and degree of coherence. Thus, CLAM circumvents the common complications of multiple coherent light-sheet generation in terms of dedicated wavefront engineering and mechanical dithering/scanning. Moreover, the encoding of multiplexed optical sections in CLAM allows the synchronous capture of all sectioned images within the imaged volume. We demonstrate the utility of CLAM in different imaging scenarios, including a light-scattering medium, an optically cleared tissue, and microparticles in fluidic flow. CLAM can maximize the signal-to-noise ratio and the spatial duty cycle, and also provides a further reduction in photobleaching compared to the major scanning-based 3D imaging systems. The flexible implementation of CLAM regarding both hardware and software ensures compatibility with any light-sheet imaging modality and could thus be instrumental in a multitude of areas in biological research.

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.

A high-throughput all-optical laser-scanning imaging flow cytometer with biomolecular specificity and subcellular resolution.

Image-based cellular assay advances approaches to dissect complex cellular characteristics through direct visualization of cellular functional structures. However, available technologies face a common challenge, especially when it comes to the unmet need for unraveling population heterogeneity at single-cell precision: higher imaging resolution (and thus content) comes at the expense of lower throughput, or vice versa. To overcome this challenge, a new type of imaging flow cytometer based upon an all-optical ultrafast laser-scanning imaging technique, called free-space angular-chirp-enhanced delay (FACED) is reported. It enables an imaging throughput (>20 000 cells s-1 ) 1 to 2 orders of magnitude higher than the camera-based imaging flow cytometers. It also has 2 critical advantages over optical time-stretch imaging flow cytometry, which achieves a similar throughput: (1) it is widely compatible to the repertoire of biochemical contrast agents, favoring biomolecular-specific cellular assay and (2) it enables high-throughput visualization of functional morphology of individual cells with subcellular resolution. These capabilities enable multiparametric single-cell image analysis which reveals cellular heterogeneity, for example, in the cell-death processes demonstrated in this work-the information generally masked in non-imaging flow cytometry. Therefore, this platform empowers not only efficient large-scale single-cell measurements, but also detailed mechanistic analysis of complex cellular processes.

Multi-MHz laser-scanning single-cell fluorescence microscopy by spatiotemporally encoded virtual source array.

Apart from the spatial resolution enhancement, scaling of temporal resolution, equivalently the imaging throughput, of fluorescence microscopy is of equal importance in advancing cell biology and clinical diagnostics. Yet, this attribute has mostly been overlooked because of the inherent speed limitation of existing imaging strategies. To address the challenge, we employ an all-optical laser-scanning mechanism, enabled by an array of reconfigurable spatiotemporally-encoded virtual sources, to demonstrate ultrafast fluorescence microscopy at line-scan rate as high as 8 MHz. We show that this technique enables high-throughput single-cell microfluidic fluorescence imaging at 75,000 cells/second and high-speed cellular 2D dynamical imaging at 3,000 frames per second, outperforming the state-of-the-art high-speed cameras and the gold-standard laser scanning strategies. Together with its wide compatibility to the existing imaging modalities, this technology could empower new forms of high-throughput and high-speed biological fluorescence microscopy that was once challenged.

Ultrafast laser-scanning time-stretch imaging at visible wavelengths.

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.

A fast fluorescence imaging flow cytometer for phytoplankton analysis.

We report a fast fluorescence imaging flow cytometer for phytoplankton analysis that can achieve a volume flow rate up to 1ml/min. The instrument shows a high immunity to motion blur in image captured with a lateral resolution of 0.75 ± 0.06 µm for a wide size range ~1 µm to ~200 µm. This is made possible by suppressing the out-of-focus light using thin light sheet illumination and image deconvolution, and by precluding the motion-blur with a unique flow configuration. Preliminary results from untreated coastal water samples show the technique has high potential as a practical field instrument for monitoring phytoplankton abundance and species composition.

A light sheet based high throughput 3D-imaging flow cytometer for phytoplankton analysis.

This paper reports a light sheet fluorescence imaging flow cytometer for 3D sectioning of phytoplankton. The instrument developed has the inherent advantages of high cell counting throughput and high spatial resolution information derived from flow cytometry and light sheet microscopy. The throughput of the instrument is quantified by the sample volume flow rate of 0.5 µl/min with a spatial resolution as achieved by light sheet microscopy. Preliminary results from 3D morphology of the internal chlorophyll-a structure of two dinoflagellates species show promising application potentials of the method for phytoplankton taxonomy of selected species and species groups.

[Effects of laser wavelength on detection of metal elements in water solution by laser induced breakdown spectroscopy].

In the present paper, aiming at the problem of laser induced breakdown spectroscopy (LIBS) applyication in ocean detection, the effects of laser wavelength on the detection of Ca in water solution were investigated. The evolvement characteriza tion of electron density was studied by analyzing the time resolved spectra of the plasma. The experimental results show that the lifetime of plasma is about 1 200 and 600 ns respectively induced by 1 064 and 532 nm laser. Based on the optical transmission characteristic and the LIBS experimental results, the dependence of needed laser energy before incidence into water E(iopt)(r) for optimal detection effect on the detection distance in water was found, and the dependence was simulated by applying to the in situ detection in water solution. The simulated results suggest that the needed laser energy of 1 064 nm laser before incidence into water is about 100 mJ when the detection distance is no larger than 5 cm. When the detection distance increases to 10 cm, the needed laser energy of 532 nm before incidence into water is only about 30 mJ. So it should be considered to choose 532 nm laser as the ablation source with the increase in the in situ detection distance.

[Experimental investigation of Pb in soil slurries by laser induced breakdown spectroscopy].

Laser induced breakdown spectroscopy (LIBS) has been shown to be a promising technique for element analysis with many advantages including on-line, real time, standing off and multi-element detection capability. In the present paper, the LIBS experiments for Pb in slurry samples were carried out with the motivation of developing an in-situ sensor for monitoring heavy metal. A Q-switched Nd : YAG laser operating at 532 nm with repetition frequency of 10 Hz was utilized to generate plasma on the prepared slurry samples, which were doped with same weight manganese as reference and varied concentration of lead. The induced plasma emission was recorded by CCD. The LIBS signals at PbI 405.78 nm and MnI 403.07 nm from the slurry samples were investigated. It was found that the intensity ratio of I(Pb)/ I(Mn) increased as a linear function of the concentration of Pb with correlation coefficient R2 of 0.994 9. The obtained results show that LIBS with conjunction of referent element could be developed as a potential technique for contamination analysis of soil slurries. The main influence factors in LIBS signal detection were also discussed.

[Detection of metal ions in water solution by laser induced breakdown spectroscopy].

Environmental concerns about the hazardous heavy metals in seawaters have been greatly increased in these years. To evaluate the potential application of laser induced breakdown spectroscopy (LIBS) to on-line toxic metals pollution monitoring in ocean, some experimental investigations with LIBS technique to detect metal ions in CuSO4 and Pb(NO3)2 water solutions have been carried out in our laboratory. A Q-switched Nd:YAG laser operating at 532 nm with pulse width of 10 ns and repetition frequency of 10 Hz was utilized to generate plasma on a flowing liquid surface. The ensuing plasma emission was coupled by a quartz lens to a double grating monochromator and recorded with a PMT in conjunction with a computer controlled boxcar integrator. The temporal characteristic of the laser induced plasma and the power dependence of LIBS signal were investigated. The operation condition was improved with the optimal ablation pulse energy and the delay time for LIBS signal detection. The ablation location was varied to achieve better LIBS signal. The optimized ablation location for lead was found to be different from that for copper due to the breakdown of the ambient air. The detection limit of metal ion in water solution under the optimized operation conditions was found to be 31 ppm for copper and 50 ppm for lead. The experimental results proved that the flexibility of LIBS has the potential to be applied to the detection of toxic metals in seawaters, but the limits of detection for each element should be improved further to make a practical application of LIBS in this field.