High-speed Volumetric Imaging Research Articles

Inverted selective plane illumination microscopy ( i SPIM) enables coupled cell identity lineaging and neurodevelopmental imaging in Caenorhabditis elegans

The Caenorhabditis elegans embryo is a powerful model for studying neural development, but conventional imaging methods are either too slow or phototoxic to take full advantage of this system. To solve these problems, we developed an inverted selective plane illumination microscopy (iSPIM) module for noninvasive high-speed volumetric imaging of living samples. iSPIM is designed as a straightforward add-on to an inverted microscope, permitting conventional mounting of specimens and facilitating SPIM use by development and neurobiology laboratories. iSPIM offers a volumetric imaging rate 30× faster than currently used technologies, such as spinning-disk confocal microscopy, at comparable signal-to-noise ratio. This increased imaging speed allows us to continuously monitor the development of C, elegans embryos, scanning volumes every 2 s for the 14-h period of embryogenesis with no detectable phototoxicity. Collecting ∼25,000 volumes over the entirety of embryogenesis enabled in toto visualization of positions and identities of cell nuclei. By merging two-color iSPIM with automated lineaging techniques we realized two goals: (i) identification of neurons expressing the transcription factor CEH-10/Chx10 and (ii) visualization of their neurodevelopmental dynamics. We found that canal-associated neurons use somal translocation and amoeboid movement as they migrate to their final position in the embryo. We also visualized axon guidance and growth cone dynamics as neurons circumnavigate the nerve ring and reach their targets in the embryo. The high-speed volumetric imaging rate of iSPIM effectively eliminates motion blur from embryo movement inside the egg case, allowing characterization of dynamic neurodevelopmental events that were previously inaccessible.

Real-time high-speed volumetric imaging using compressive sampling optical coherence tomography

Volumetric imaging of the Optic Nerve Head (ONH) morphometry with Optical Coherence Tomography (OCT) requires dense sampling and relatively long acquisition times. Compressive Sampling (CS) is an emerging technique to reduce volume acquisition time with minimal image degradation by sparsely sampling the object and reconstructing the missing data in software. In this report, we demonstrated real-time CS-OCT for volumetric imaging of the ONH using a 1060nm Swept-Source OCT prototype. We also showed that registration and averaging of CS-recovered volumes enhanced visualization of deep structures of the sclera and lamina cribrosa. This work validates CS-OCT as a means for reducing volume acquisition time and for preserving high-resolution in volume-averaged images. Compressive sampling can be integrated into new and existing OCT systems without changes to the optics, requiring only software changes and post-processing of acquired data.

High-speed 2D and 3D fluorescence microscopy of cardiac myocytes

Oblique plane microscopy (OPM) is a light sheet microscopy technique that uses a single high numerical aperture microscope objective to both illuminate a tilted plane within the specimen and to obtain an image of the tilted illuminated plane. In this paper, we present a new OPM configuration that enables both the illumination and detection focal planes to be swept simultaneously and remotely through the sample volume, enabling high speed volumetric imaging. We demonstrate the high speed imaging capabilities of the system by imaging calcium dynamics in cardiac myocytes in 2D at 926 frames per second and in 3D at 21 volumes per second. In the future, higher frame rate CCD cameras will enable volumetric imaging at much greater rates, leading to new capabilities to study dynamic events in cells at high speeds in two and three dimensions.

High-speed volumetric imaging of cone photoreceptors with adaptive optics spectral-domain optical coherence tomography

We report the first observations of the three-dimensional morphology of cone photoreceptors in the living human retina. Images were acquired with a high-speed adaptive optics (AO) spectral-domain optical coherence tomography (SD-OCT) camera. The AO system consisted of a Shack-Hartmann wavefront sensor and bimorph mirror (AOptix) that measured and corrected the ocular and system aberrations at a closed-loop rate of 12 Hz. The bimorph mirror was positioned between the XY mechanical scanners and the subject's eye. The SD-OCT system consisted of a superluminescent diode and a 512 pixel line scan charge-coupled device (CCD) that acquired 75,000 A-scans/s. This rate is more than two times faster than that previously reported. Retinal motion artifacts were minimized by quickly acquiring small volume images of the retina with and without AO compensation. Camera sensitivity was sufficient to detect reflections from all major retinal layers. The regular distribution of bright spots observed within C-scans at the inner segment / outer segment (IS/OS) junctions and at the posterior tips of the OS were found to be highly correlated with one another and with the expected cone spacing. No correlation was found between the posterior tips of the OS and the other retinal layers examined, including the retinal pigment epithelium.

Hybrid multi/single layer array transducers for increased signal-to-noise ratio

In medical ultrasound imaging, two-dimensional (2-D) array transducers are necessary to implement dynamic focusing in two dimensions, phase correction in two dimensions and high speed volumetric imaging. However, the small size of a 2-D array element results in a small clamped capacitance and a large electrical impedance, which decreases the transducer signal-to-noise ratio (SNR). We have previously shown that SNR is improved using transducers made from multi-layer PZT, due to their lower electrical impedance. In this work, we hypothesize that SNR is further increased using a hybrid array configuration: in the transmit mode, a 10 Omega electronic transmitter excites a 10 Omega multi-layer array element; in the receive mode, a single layer element drives a high impedance preamplifier located in the transducer handle. The preamplifier drives the coaxial cable connected to the ultrasound scanner. For comparison, the following control configuration was used: in the transmit mode, a 50 Omega source excites a single layer element, and in the receive mode, a single layer element drives a coaxial cable load. For a 5x102 hybrid array operating at 7.5 MHz, maximum transmit output power was obtained with 9 PZT layers according to the KLM transmission line model. In this case, the simulated pulse-echo SNR was improved by 23.7 dB for the hybrid configuration compared to the control. With such dramatic improvement in pulse-echo SNR, low voltage transmitters can be used. These can be fabricated on integrated circuits and incorporated into the transducer handle.

Two-dimensional arrays for medical ultrasound

The design, fabrication and evaluation of two-dimensional transducer arrays are described for medical ultrasound imaging. A 4 × 32, 2.8 MHz array was developed to use new signal processing techniques for improved B-scan imaging including elevation focusing, phase correction and synthetic aperture imaging. Laboratory measurements from typical array elements showed 50 Ω insertion loss of -56ddB, -6 dB fractional bandwidth of 43%, interelement crosstalk of -19 dB, and -6 dB pulse-echo angular response of 62°. Simulations of pulse-echo beam plots have shown grating lobes 20 dB below the main lobe at ±7° in the elevation direction. The complete 2-D array has been used for measurements of phase aberrations in breast, and the individual 32 element linear arrays have been used to obtain conventional B-scans. Several 16 × 16 arrays have also been developed for high speed volumetric imaging. These include 96 transmit elements and 32 receive channels. With a λ/4 matching layer, laboratory measurements show 50 Ω insertion loss of -72 dB, -6 dB fractional bandwidth of 63%, interelement crosstalk of -29 dB and -6 dB angular response of 25°. Pulse-echo sensitivity was improved by 21 dB through the use of integrated circuit preamplifiers of high impedance mounted in the transducer handle. In vivo cardiac, abdominal, and obstetric B-scans with elevation focusing, as well as high speed C-scans, have been obtained with these 2-D arrays.

Two-dimensional arrays for medical ultrasound.

The design, fabrication and evaluation of two-dimensional transducer arrays are described for medical ultrasound imaging. A 4 x 32, 2.8 MHz array was developed to use new signal processing techniques for improved B-scan imaging including elevation focusing, phase correction and synthetic aperture imaging. Laboratory measurements from typical array elements showed 50 omega insertion loss of -56 dB, -6 dB fractional bandwidth of 43%, interelement crosstalk of -19 dB, and -6 dB pulse-echo angular response of 62 degrees. Simulations of pulse-echo beam plots have shown grating lobes 20 dB below the main lobe at +/- 7 degrees in the elevation direction. The complete 2-D array has been used for measurements of phase aberrations in breast, and the individual 32 element linear arrays have been used to obtain conventional B-scans. Several 16 x 16 arrays have also been developed for high speed volumetric imaging. These include 96 transmit elements and 32 receive channels. With a lambda/4 matching layer, laboratory measurements show 50 omega insertion loss of -72 dB, -6 dB fractional bandwidth of 63%, interelement crosstalk of -29 dB and -6 dB angular response of 25 degrees. Pulse-echo sensitivity was improved by 21 dB through the use of integrated circuit preamplifiers of high impedance mounted in the transducer handle. In vivo cardiac, abdominal, and obstetric B-scans with elevation focusing, as well as high speed C-scans, have been obtained with these 2-D arrays.

High-speed ultrasound volumetric imaging system. II. Parallel processing and image display

For pt.I see ibid., vol.38, no.2, p.100-8 (1991). The authors describe the design, application, and evaluation of parallel processing to the high-speed volumetric ultrasound imaging system. The scanner produces images analogous to an optical camera or the human eye and supplies more information than conventional sonograms. Potential medical applications include improved anatomic visualization, tumor localization, and better assessment of cardiac function. The system uses pulse-echo phased array principles to steer a 2-D array transducer of 289 elements in a pyramidal scan format. Parallel processing in the receive mode produces 4992 scan lines at a rate of approximately 8 frames/s. Echo data for the scanned volume is presented online as projection images with depth perspective, stereoscopic pairs, or multiple tomographic images. The authors also describe the techniques developed for the online display of volumetric images on a conventional CRT oscilloscope and show preliminary volumetric images for each display mode.