Julie X. Yun

Laser-patterned stem-cell bridges in a cardiac muscle model for on-chip electrical conductivity analyses

Following myocardial infarction there is an irreversible loss of cardiomyocytes that results in the alteration of electrical propagation in the heart. Restoration of functional electrical properties of the damaged heart muscle is essential to recover from the infarction. While there are a few reports that demonstrate that fibroblasts can form junctions that transmit electrical signals, a potential alternative using the injection of stem cells has emerged as a promising cellular therapy; however, stem-cell electrical conductivity within the cardiac muscle fiber is unknown. In this study, an in vitro cardiac muscle model was established on an MEA-based biochip with multiple cardiomyocytes that mimic cardiac tissue structure. Using a laser beam, stem cells were inserted adjacent to each muscle fiber (cell bridge model) and allowed to form cell-cell contact as determined by the formation of gap junctions. The electrical conductivity of stem cells was assessed and compared with the electrical conductivities of cardiomyocytes and fibroblasts. Results showed that stem cell-myocyte contacts exhibited higher and more stable conduction velocities than myocyte-fibroblast contacts, which indicated that stem cells have higher electrical compatibility with native cardiac muscle fibers than cardiac fibroblasts.

Laser-guidance-based cell deposition microscope for heterotypic single-cell micropatterning

Cell patterning methods enable researchers to control specific homotypic and heterotypic contact-mediated cell-cell and cell-ECM interactions and to impose defined cell and tissue geometries. To micropattern individual cells to specific points on a substrate with high spatial resolution, we have developed a cell deposition microscope based on the laser-guidance technique. We discuss the theory of optical forces for generating laser guidance and the optimization of the optical configuration (NA ≈ 0.1) to manipulate cells with high speed in three dimensions. Our cell deposition microscope is capable of patterning different cell types onto and within standard cell research devices and providing on-stage incubation for long-term cell culturing. Using this cell deposition microscope, rat mesenchymal stem cells from bone marrow were micropatterned with cardiomyocytes into a substrate microfabricated with polydimethylsiloxane on a 22 mm × 22 mm coverglass to form a single-cell coculturing microenvironment, and their electrophysiological property changes were investigated during the coculturing days.

Laser-guided cell micropatterning system.

Employing optical force, our laser-guided cell micropatterning system, is capable of patterning different cell types onto and within standard cell research devices, including commercially available multielectrode arrays (MEAs) with glass culture rings, 35 mm Petri dishes, and microdevices microfabricated with polydimethylsiloxane on 22 mm × 22 mm cover glasses. We discuss the theory of optical forces for generating laser guidance and the calculation of optimal beam characteristics for cell guidance. We describe the hardware design and software program for the cell patterning system. Finally, we demonstrate the capabilities of the system by (1) patterning neurons to form an arbitrary pattern, (2) patterning neurons onto the electrodes of a standard MEA, and (3) patterning and aligning adult cardiomyocytes in a polystyrene Petri dish.

Megahertz streak-mode Fourier domain optical coherence tomography

Here we present an ultrahigh-speed Fourier-domain optical coherence tomography (OCT) that records the OCT spectrum in streak mode with a high-speed area scan camera, which allows higher OCT imaging speed than can be achieved with a line-scan camera. Unlike parallel OCT techniques that also use area scan cameras, the conventional single-mode fiber-based point-scanning mechanism is retained to provide a confocal gate that rejects multiply scattered photons from the sample. When using a 1000 Hz resonant scanner as the streak scanner, 1,016,000 A-scans have been obtained in 1 s. This methods effectiveness has been demonstrated by recording in vivo OCT-image sequences of embryonic chick hearts at 1000 frames/s. In addition, 2-megahertz OCT data have been obtained with another high speed camera.

4D imaging of embryonic chick hearts by streak-mode Fourier domain optical coherence tomography

Recently, we developed the streak-mode Fourier domain optical coherence tomography (OCT) technique in which an area-scan camera is used in a streak-mode to record the OCT spectrum. Here we report the application of this technique to in ovo imaging HH18 embryonic chick hearts with an ultrahigh speed of 1,016,000 axial scans per second. The high-scan rate enables the acquisition of high temporal resolution 2D datasets (1,000 frames per second or 1 ms between frames) and 3D datasets (10 volumes per second), without use of prospective or retrospective gating technique. This marks the first time that the embryonic animal heart has been 4D imaged using a megahertz OCT.

An approach for megahertz OCT: streak mode Fourier domain optical coherence tomography

We report a technique, which uses an area-scan camera to record the interference spectrum. Traditional point-scanning is remained in this streak-mode FDOCT so that the small aperture of the single-mode fiber functions as a confocal gate and screens multiply scattered photons very well. While the sample beam is scanning the specimen laterally, the interference spectrum is physically scanned on the area scan camera using a streak scanner. Therefore, pixels of the camera are illuminated by the spectrum of OCT signal row by row, corresponding to each A-scan at different lateral position. A unidirectional B-scan of 700 lines is obtained in 1 ms; thus, an A-scan time of 1.4 μs is achieved. A Day 4 chick embryo sampled is imaged using this method. This technique is highly potential for multi-Megahertz OCT imaging.

Laser Guidance-Based Cell Micropatterning

Due to the extreme complexity of in vivo environments, our understanding of cellular functions and cell-cell interactions is heavily dependent on cell culture. To understand the biological mechanisms at the cellular level in cell culture, cell-patterning methods have been developed to mimic in vivo patterns of cellular organization. Unlike traditional cell patterning techniques, which are not designed to pattern small numbers of cells with the accuracy desired for systematic cell-cell interaction studies, laser guidance-based cell micropatterning uses optical force to capture a cell and guide it to a specific location on a variety of substrates. In this chapter, we will discuss theories on optical force, demonstrate numerical simulations, and describe the design and implementation of an actual micropatterning system. Three cell patterns will be presented: a single-cell array with high spatial resolution (less than 1 μm), alignment of rod-shape adult cardiomyocytes, and neuronal networks with defined connectivity and single-cell resolution on a microelectrode array.

Laser guidance-based cell detection

Laser guidance is the technique that uses a weakly convergent laser beam to trap particles radially in the center of the beam and simultaneously propel them along the beam propagation axis with a travelling distance over millimeters. In this paper, we describe the applications of laser guidance to detect different cell types, including those of phenotypically transformed or gene-modified cells, especially for situations in which fluorescent markers used in flow cytometry for cell detection are not available or their application is contraindicated by clinical restriction. The optical force, which determines the guidance speed of the cell, is dependent on the characteristics, such as size, shape, composition and refractive index, of the cell being guided. Therefore, by measuring the guidance speed of the cell along the laser beam, cells with different properties can be effectively distinguished. We report two experimental results: 1) the laser-guidance system could significantly distinguish the metastatic cancer cell type 4T1 from its non-metastatic counterpart 4T07, which could not be achieved by using a high magnification microscope; 2) The laser-guidance experiment demonstrated that only one gene modification between L-10 and TC-1 cells resulted in ~40% difference in guidance speed. These experimental data indicate that laser guidance can be used to detect subtle differences between sub-cell types.

Doppler streak mode Fourier domain optical coherence tomography

Doppler Fourier domain optical coherence tomography is able to be used for in vivo blood flow measurement. In conventional methods, the highest velocity that can be measured is limited to the range the phase shift between two successively recorded depth profiles at the same probe-beam location, which cannot exceed (-π, π), otherwise phase wrapping will occur. This phase-wrapping limit is determined by the time interval between two consecutive A-scans. We present a novel approach to shorten the time interval between two consecutive A-scans and thus increase the phase-wrapping limit by using an area scan camera to record the interference spectrum in a streak mode. To demonstrate the effectiveness of this method, the blood flows in HH18 and HH19 chick hearts were imaged and phase wrapping free Doppler images were obtained.

MEMS motor-based common-path endoscopic Fourier-domain OCT

A rotational microelectromechanical(MEMS) motor based common-path Fourier-domain OCT for endoscopic imaging, which uses the interface between the index-match oil and distal-end surface of the fiber as a self-aligned reference mirror, is reported. The reference intensity is easy to be tuned by altering the index of the match oil to optimize the signal to noise ratio of the system. An external Michelson interferometer is used to compensate for the optical path difference and dispersion mismatch to the index-match oil and the GRIN lens. Due to this common-path design, the OCT signal is immune to bending or stretching of the endoscopic catheter. The outer diameter of the probe is 3 mm, and 22 circumferential-scans and 50,000 lines A-scans are obtained in one second.