Daniel B. Holland

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.

A direct digital synthesis chirped pulse Fourier transform microwave spectrometer

Chirped pulse Fourier transform microwave (CP-FTMW) spectrometers have become the instrument of choice for acquiring rotational spectra, due to their high sensitivity, fast acquisition rate, and large bandwidth. Here we present the design and capabilities of a recently constructed CP-FTMW spectrometer using direct digital synthesis (DDS) as a new method for chirped pulse generation, through both a suite of extensive microwave characterizations and deep averaging of the 10-14 GHz spectrum of jet-cooled acetone. The use of DDS is more suited for in situ applications of CP-FTMW spectroscopy, as it reduces the size, weight, and power consumption of the chirp generation segment of the spectrometer all by more than an order of magnitude, while matching the performance of traditional designs. The performance of the instrument was further improved by the use of a high speed digitizer with dedicated signal averaging electronics, which facilitates a data acquisition rate of 2.1 kHz.

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.

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.

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.

Selective volume illumination microscopy offers synchronous volumetric imaging with high contrast

Light field microscopy provides an efficient means to collect 3D images in a single acquisition, as its plenoptic detection captures an extended image volume in one snapshot. The ability of light field microscopy to simultaneously capture image data from a volume of interest, such as a functioning brain or a beating heart, is compromised by inadequate contrast and effective resolution, due, in large part, to light scattering by the tissue. Surprisingly, a major contribution to the image degradation is the signal scattered into the volume of interest by the typical wide-field illumination that excites the sample region outside the volume of interest. Here, we minimize this degradation by employing selective volume illumination, using a modified light sheet approach to illuminate preferentially the volume of interest. This minimizes the unavoidable background generated when extraneous regions of the sample are illuminated, dramatically enhancing the contrast and effective resolution of the captured and reconstructed images. Light Field Selective Volume Illumination Microscopy (LF-SVIM, SVIM for short) dramatically improves the performance of light field microscopy, and offers an unprecedented combination of synchronous z-depth coverage, lateral and axial resolution, and imaging speed.

Fiber-bundle illumination: realizing high-degree time-multiplexed multifocal multiphoton microscopy with simplicity

High-degree time-multiplexed multifocal multiphoton microscopy was expected to provide a facile path to scanningless optical-sectioning and the fast imaging of dynamic three-dimensional biological systems. However, physical constraints on typical time multiplexing devices, arising from diffraction in the free-space propagation of light waves, lead to significant manufacturing difficulties and have prevented the experimental realization of high-degree time multiplexing. To resolve this issue, we have developed a novel method using optical fiber bundles of various lengths to confine the diffraction of propagating light waves and to create a time multiplexing effect. Through this method, we experimentally demonstrate the highest degree of time multiplexing ever achieved in multifocal multiphoton microscopy (~50 times larger than conventional approaches), and hence the potential of using simply-manufactured devices for scanningless optical sectioning of biological systems.