Imaging Proteins, Cells, and Tissues Dynamics during Embryogenesis with Two-Photon Light-Sheet Microscopy

Two-photon light sheet microscopy combines nonlinear excitation with the novel sheet-illumination, orthogonal to the detection direction, to achieve high penetration depth, high acquisition speed, and low photodamage, compared with conventional imaging techniques. These advantages allow unprecedented observation of the processes that govern embryogenesis, where the ability to image fast the dynamic three dimensional structure of the developing embryo, over extended periods of time, is critical. We present a selection of applications where two-photon light sheet microscopy is utilized to observe the dynamics of proteins, cells, and tissues, toward an understanding of the construction program of the developing embryos

Wide-field optical sectioning for live-tissue imaging by plane-projection multiphoton microscopy

Optical sectioning provides three-dimensional (3D) information in biological tissues. However, most imaging techniques implemented with optical sectioning are either slow or deleterious to live tissues. Here, we present a simple design for wide-field multiphoton microscopy, which provides optical sectioning at a reasonable frame rate and with a biocompatible laser dosage. The underlying mechanism of optical sectioning is diffuser-based temporal focusing. Axial resolution comparable to confocal microscopy is theoretically derived and experimentally demonstrated. To achieve a reasonable frame rate without increasing the laser power, a low-repetition-rate ultrafast laser amplifier was used in our setup. A frame rate comparable to that of epifluorescence microscopy was demonstrated in the 3D imaging of fluorescent protein expressed in live epithelial cell clusters. In this report, our design displays the potential to be widely used for video-rate live-tissue and embryo imaging with axial resolution comparable to laser scanning microscopy

The wide-field optical sectioning of microlens array and structured illumination-based plane-projection multiphoton microscopy

We present a theoretical investigation of an optical microscope design that achieves wide-field, multiphoton fluorescence microscopy with finer axial resolution than confocal microscopy. Our technique creates a thin plane of excitation light at the sample using height-staggered microlens arrays (HSMAs), wherein the height staggering of microlenses generate temporal focusing to suppress out-of-focus excitation, and the dense spacing of microlenses enables the implementation of structured illumination technique to eliminate residual out-of-focus signal. We use physical optics-based numerical simulations to demonstrate that our proposed technique can achieve diffraction-limited three-dimensional imaging through a simple optical design

Dynamic Three-Dimensional Imaging of Cellular Shape Changes and Protein Expression in the Developing Zebrafish Heart

We present our results in dynamic three-dimensional (3D) imaging and quantification of the cellular shape changes and gene expressions of the developing zebrafish heart, in the effort to understand the mechanisms of the embryonic construction of this critical organ. The vertebrate heart is built up through a series of steps taking two flat layers of cells to a hollow heart tube to a multi-layered, multi-chambered, chirally twisted structure of the mature organ. Additionally, the heart is the first organ in the developing embryo to function, through its beating and pumping of the blood, shortly after the formation of the heart tube. Despite this intrinsic dynamic 3D nature of the developing heart, previous works documenting its development consist of largely 2D and/or static imaging (utilizing pharmacological means to stop the beating of the heart), due to the challenges in achieving fast, high 3D-resolution with conventional imaging modalities. To overcome these challenges, we employ 2-photon light sheet microscopy and a wavelet-based synchronization and registration method to achieve the required spatial and temporal resolution to capture the 3D motion of the heart. The high speed 3D imaging and analysis is carried out on several transgenic zebrafish lines that have been recently generated in our lab where proteins important for heart development are fluorescently tagged at their endogenous loci. We thus document not only cellular morphology but also critical genes' expression, with sub-cellular resolution, of the developing heart, over its beating cycle and at different development times. These results provide the necessary groundwork to start deciphering the process where the dynamic changes in cellular shapes, gene expressions, and cellular physical properties participate, in concert with the genetic program, in the development of the vertebrate heart

Dynamic structure and protein expression of the live embryonic heart captured by 2-photon light sheet microscopy and retrospective registration

We present an imaging and image reconstruction pipeline that captures the dynamic three-dimensional beating motion of the live embryonic zebrafish heart at subcellular resolution. Live, intact zebrafish embryos were imaged using 2-photon light sheet microscopy, which offers deep and fast imaging at 70 frames per second, and the individual optical sections were assembled into a full 4D reconstruction of the beating heart using an optimized retrospective image registration algorithm. This imaging and reconstruction platform permitted us to visualize protein expression patterns at endogenous concentrations in zebrafish gene trap lines

Imaging the Beating Heart with Macroscopic Phase Stamping

We present a novel approach for imaging the beating embryonic heart, based on combining two independent imaging channels to capture the full spatio-temporal information of the moving 3D structure. High-resolution, optically-sectioned image recording is accompanied by simultaneous acquisition of low-resolution, whole-heart recording, allowing the latter to be used in post-acquisition processing to determine the macroscopic spatio-temporal phase of the heart beating cycle. Once determined, or 'stamped', the phase information common to both imaging channels is used to reconstruct the 3D beating heart. We demonstrated our approach in imaging the beating heart of the zebrafish embryo, capturing the entire heart over its full beating cycle, and characterizing cellular dynamic behavior with sub-cellular resolution

Decade-Spanning High-Precision Terahertz Frequency Comb

The generation and detection of a decade-spanning terahertz (THz) frequency comb is reported using two Ti:sapphire femtosecond laser oscillators and asynchronous optical sampling THz time-domain spectroscopy. The comb extends from 0.15 to 2.4 THz, with a tooth spacing of 80 MHz, a linewidth of 3.7 kHz, and a fractional precision of 1.8×10^(−9). With time-domain detection of the comb, we measure three transitions of water vapor at 10 mTorr between 1–2 THz with an average Doppler-limited fractional accuracy of 6.1×10^(−8). Significant improvements in bandwidth, resolution, and sensitivity are possible with existing technologies

A decade-spanning high-resolution asynchronous optical sampling terahertz time-domain and frequency comb spectrometer

We present the design and capabilities of a high-resolution, decade-spanning ASynchronous OPtical Sampling (ASOPS)-based TeraHertz Time-Domain Spectroscopy (THz-TDS) instrument. Our system employs dual mode-locked femtosecond Ti:Sapphire oscillators with repetition rates offset locked at 100 Hz via a Phase-Locked Loop (PLL) operating at the 60th harmonic of the ∼80 MHz oscillator repetition rates. The respective time delays of the individual laser pulses are scanned across a 12.5 ns window in a laboratory scan time of 10 ms, supporting a time delay resolution as fine as 15.6 fs. The repetition rate of the pump oscillator is synchronized to a Rb frequency standard via a PLL operating at the 12th harmonic of the oscillator repetition rate, achieving milliHertz (mHz) stability. We characterize the timing jitter of the system using an air-spaced etalon, an optical cross correlator, and the phase noise spectrum of the PLL. Spectroscopic applications of ASOPS-THz-TDS are demonstrated by measuring water vapor absorption lines from 0.55 to 3.35 THz and acetonitrile absorption lines from 0.13 to 1.39 THz in a short pathlength gas cell. With 70 min of data acquisition, a 50 dB signal-to-noise ratio is achieved. The achieved root-mean-square deviation is 14.6 MHz, with a mean deviation of 11.6 MHz, for the measured water line center frequencies as compared to the JPL molecular spectroscopy database. Further, with the same instrument and data acquisition hardware, we use the ability to control the repetition rate of the pump oscillator to enable THz frequency comb spectroscopy (THz-FCS). Here, a frequency comb with a tooth width of 5 MHz is generated and used to fully resolve the pure rotational spectrum of acetonitrile with Doppler-limited precision. The oscillator repetition rate stability achieved by our PLL lock circuits enables sub-MHz tooth width generation, if desired. This instrument provides unprecedented decade-spanning, tunable resolution, from 80 MHz down to sub-MHz, and heralds a new generation of gas-phase spectroscopic tools in the THz region