KEYWORDS: Signal to noise ratio, Deep learning, Two photon imaging, Image enhancement, Imaging systems, Education and training, Two photon excitation microscopy, Signal processing, Neurons, Neural networks
We developed a high-speed two-photon volumetric imaging system with hundreds of axial layers that match a deep-learning denoising model to capture millisecond-level functional changes in individual neurons with high SNR. Compare with general deep-learning methods, the spatial information-based training method not only enhances SNR by 300% but prevents temporal distortion. Our proof-of-concept experiment focused on calcium dynamics in cerebellum Purkinje cells, revealing similar responses in the parallel dendritic layers, yet significant divergence in the somatic area. This sheds light on the intricate signal processing at individual neuron levels, validating our imaging system.
The continuous rotating planar mirror-based focus-shift system can be used for various microscopes including light-sheet, confocal, fluorescent, and others. The uniform rotation provides a fast, quiet and stable operation without the normal vibration and noise produced by reciprocal motion system. The planar mirrors produce images without the aberrations produced by the Electronically Tunable Lens (ETL) systems. In a light sheet illumination system for 3D applications, high speed change in the focal plane is required such that the imaging portion of the system is synchronized to the scanning light sheet with a stationary sample. A series of 2D images at different times are captured by the digital camera, which are converted into z-axis information and displayed as 3D images.
Significance: Two-photon microscopy has become the standard platform for deep-tissue fluorescence imaging. However, the use of point scanning in conventional two-photon microscopy limits the speed of volumetric image acquisition.
Aim: To obtain fast and deep volumetric images, we combine two-photon light sheet fluorescence microscopy (2p-LSFM) and axicon imaging that yields an extended depth of field (DOF) in 2p-LSFM.
Approach: Axicon imaging is achieved by imposing an axicon lens in the detection part of LSFM.
Results: The DOF with axicon imaging is extended more than 20-fold over that of a conventional imaging lens, liberating the synchronized scanning in LSFM. We captured images of dynamic beating hearts and red blood cells in zebrafish larvae at volume acquisition rates up to 30 Hz.
Conclusions: We demonstrate the fast three-dimensional imaging capability of 2p-LSFM with axicon imaging by recording the rapid dynamics of physiological processes.
Membrane ruffling is an essential process at the leading edge of the migrating cells, which contains protrusion and retraction of plasma membrane. The extension membrane determines the direction of migration. The dynamic of membrane ruffling depends on the interaction between filament actin and motor proteins. Upon the activation of motor proteins by calcium ions, the migration process starts. Therefore, it is important to study the correlation between local calcium concentration and the membrane dynamics. To study the dynamics of the membrane ruffling, we established several stable cell lines, which contain chemical and optogenetic inducible dimerization . In addition, the proteins of interest such as actin, myosin, membrane anchors and calcium indicators were labeled with fluorescence proteins. The dynamics of membrane ruffling was investigated by lattice light-sheet microscope (LLSM), which is capable of high spatial and temporal recording over three-dimensions.
A concept using a glass-based microchip with finger electrodes, under a two-step voltage-controlled manner, to accomplish the formation and collection of liposomes is presented. By the actions of electro-osmotic and electrolytic bubbles in low and high applied voltages, respectively, the liposomes were first formed in an electrolyte reservoir; then, they were successfully moved to a collected reservoir, which is helpful for subsequent on-chip processing. This study evaluated the distribution of an electric field for different couple-finger distances using finite-element analysis software. The simulation result shows that the electric field density increases rapidly with the decrease of finger distance, with a quadratic relationship. Experiment results show that, when the applied AC voltage is 1 Vp-p, at a driving frequency of 10 Hz, the liposomes are first formed near the boundary of the electrolyte reservoir, and when the voltage is higher, the mass production of liposomes is achieved. When the voltage is higher than 4 Vp-p, the electrolytic bubbles grow cyclically to separate the liposomes from the electrolyte reservoir and therefore collect in a specific reservoir. Liposomes encapsulate doxorubicin, and their drug release to the lung cancer cell line H1299 was also implemented to verify their biointeraction characteristics. These experimental results were all identified using an inverted fluorescence microscope.
Optical imaging techniques provide much important information in understanding life science especially cellular structure and morphology because “seeing is believing”. However, the resolution of optical imaging is limited by the diffraction limit, which is discovered by Ernst Abbe, i.e. λ/2(NA) (NA is the numerical aperture of the objective lens). Fluorescence super-resolution microscopic techniques such as Stimulated emission depletion microscopy (STED), Photoactivated localization microscopy (PALM), and Stochastic optical reconstruction microscopy (STORM) are invented to have the capability of seeing biological entities down to molecular level that are smaller than the diffraction limit (around 200-nm in lateral resolution). These techniques do not physically violate the Abbe limit of resolution but exploit the photoluminescence properties and labelling specificity of fluorescence molecules to achieve super-resolution imaging. However, these super-resolution techniques limit most of their applications to the 2D imaging of fixed or dead samples due to the high laser power needed or slow speed for the localization process. Extended from 2D imaging, light sheet microscopy has been proven to have a lot of applications on 3D imaging at much better spatiotemporal resolutions due to its intrinsic optical sectioning and high imaging speed. Herein, we combine the advantage of localization microscopy and light-sheet microscopy to have super-resolved cellular imaging in 3D across large field of view. With high-density labeled spontaneous blinking fluorophore and wide-field detection of light-sheet microscopy, these allow us to construct 3D super-resolution multi-cellular imaging at high speed (~minutes) by light-sheet single-molecule localization microscopy.
Development of imaging, sensing, and characterization of cells at Research Center for Applied Sciences (RCAS) of Academia Sinica in Taiwan is progressing rapidly. The research on advanced lattice light sheet microscopy for temporal visualization of cells in three dimensions at sub-cellular resolution shows novel imaging results. Label-free observation on filopodial dynamics provides a convenient assay on cancer cell motility. The newly-developed software enables us to track the movement of two types of particles through different channels and reconstruct the co-localized tracks. Surface plasmon resonance (SPR) for detecting urinary microRNA for diagnosis of acute kidney injury demonstrates excellent sensitivity. A fully automated and integrated portable reader was constructed as a home-based surveillance system for post-operation hepatocellular carcinoma. New microfluidic cell culture devices for fast and accurate characterizations prove various diagnosis capabilities.
Mitotic apparatus, which comprises hundreds of microtubules, plays an essential role in cell division, ensuring the correct segregation of chromosomes into each daughter cell. To gain insight into its regulatory mechanisms, it is essential to detect and analyze the behavior of individual microtubule filaments. However, the discrimination of discrete microtubule filaments within the mitotic apparatus is beyond the capabilities of conventional light microscopic technologies. Recently, we detected three-dimensional (3-D) microtubule growth dynamics within the cellular cytoplasmic space using lattice light-sheet microscopy in conjunction with microtubule growth marker protein end-binding 1, a microtubule plus-end-tracking protein, which was fused to green fluorescent protein (EB1-GFP). This technique enables high-resolution 3-D imaging at subsecond intervals. We adapted mathematical computing and geometric representation techniques to analyze spatial variations in microtubule growth dynamics within the mitotic spindle apparatus. Our analytical approach enabled the different dynamic properties of individual microtubules to be determined, including the direction and speed of their growth, and their growth duration within a 3-D spatial map. Our analysis framework provides an important step toward a more comprehensive understanding of the mechanisms driving cellular machinery at the whole-cell level.
We demonstrate two different coherent anti-Stokes Raman scattering (CARS) microscopy and microspectroscopy methods based on the spectral focusing mechanism. The first method uses strongly chirped broadband pulses from a single Ti:sapphire laser and generates CARS signals at the fingerprint region. Fast modulation of the time delay between the pump and Stokes laser pulses coupled with lock-in signal detection significantly reduces the nonresonant background and produces Raman-like CARS signals with a spectral resolution of 20 cm−1. The second method generates CARS signals in the CH (carbon-hydrogen) stretching region with IR supercontinuum pulses from a photonic crystal fiber. The spectral resolution of 30 cm−1 is achieved. Maximum entropy method is used to retrieve a Raman-equivalent CARS spectrum from lipid membranes. Chemical imaging and microspectroscopy are demonstrated with various samples.
We demonstrate a new CARS microscopy method based on fast switching of effective vibrational excitation frequency
from chirped femtosecond laser pulses. Broadband pump and Stokes pulses excite a single vibrational mode with a high
spectral resolution when the two pulses are identically chirped and their pulse durations are approaching the dephasing
time of the excited vibrational state. This "spectral focusing" mechanism is applied to CARS microscopy with a single
broadband Ti:Sapphire laser. The vibrational excitation frequency is controlled simply by the time delay between the
pump and Stokes pulses and fast switching of the excitation frequency (~100 kHz) is achieved with a Pockels cell and
polarization optics. Lock-in detection of the difference between the two CARS signals at nearby vibrational frequencies
not only eliminates the non-resonant background but also generates a spectral line shape similar to the spontaneous
Raman scattering. We demonstrate both micro-spectroscopy and vibrational imaging with various samples.
A new multiplex CARS (coherent anti-Stokes Raman scattering) microscopy technique with a single ultrafast laser pulse
is demonstrated. All the pump, Stokes, probe pulses are selected inside a single broadband cavity dumping Ti:Sapphire
oscillator laser pulse. The measured CARS signal is a coherent sum of the resonant and non-resonant signals, leading to
a complicated vibrational line shape due to the spectral interference. The resonant and non-resonant CARS signals,
however, have different symmetries in the time domain due to the causality principle of the vibrationally resonant
excitation. A new Fourier Transform Spectral interferometry (FTSI) is developed to extract the full complex quantity of
the vibrationally resonant signal against the non-resonant one utilizing the different time symmetry. This method can
generate Raman-like vibrational spectrum in a single experimental measurement, which can be readily applied to a
vibrational hyperspectral imaging. Current sensitivity, available CARS window and application to hyperspectral
vibrational microscopy are discussed.
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