Femtosecond laser machined microfluidic devices for imaging of cells during chemotaxis.

Microfluidic devices designed for chemotaxis assays were fabricated on fused silica substrates using femtosecond laser micromachining. These devices have built-in chemical concentration gradient forming structures and are ideally suited for establishing passive diffusion gradients over extended periods of time. Multiple gradient forming structures, with identical or distinct gradient forming characteristics, can be integrated into a single device, and migrating cells can be directly observed using an inverted microscope. In this paper, the design, fabrication, and operation of these devices are discussed. Devices with minimal structure sizes ranging from 3 to 7 lm are presented. The use of these devices to investigate the migration of Dictyostelium discoideum cells toward the chemoattractant folic acid is presented as an example of the devices' utility.

Receptor-mediated activation of heterotrimeric G-proteins in living cells.

Receptor-mediated activation of heterotrimeric GTP-binding proteins (G-proteins) was visualized in living Dictyostelium discoideum cells by monitoring fluorescence resonance energy transfer (FRET) between alpha- and beta- subunits fused to cyan and yellow fluorescent proteins. The G-protein heterotrimer rapidly dissociated and reassociated upon addition and removal of chemoattractant. During continuous stimulation, G-protein activation reached a dose-dependent steady-state level. Even though physiological responses subsided, the activation did not decline. Thus, adaptation occurs at another point in the signaling pathway, and occupied receptors, whether or not they are phosphorylated, catalyze the G-protein cycle. Construction of similar energy-transfer pairs of mammalian G-proteins should enable direct in situ mechanistic studies and applications such as drug screening and identifying ligands of newly found G-protein-coupled receptors.

Mirrored pyramidal wells for simultaneous multiple vantage point microscopy.

We report a novel method for obtaining simultaneous images from multiple vantage points of a microscopic specimen using size-matched microscopic mirrors created from anisotropically etched silicon. The resulting pyramidal wells enable bright-field and fluorescent side-view images, and when combined with z-sectioning, provide additional information for 3D reconstructions of the specimen. We have demonstrated the 3D localization and tracking over time of the centrosome of a live Dictyostelium discoideum. The simultaneous acquisition of images from multiple perspectives also provides a five-fold increase in the theoretical collection efficiency of emitted photons, a property which may be useful for low-light imaging modalities such as bioluminescence, or low abundance surface-marker labelling.

Engineered three-dimensional microfluidic device for interrogating cell-cell interactions in the tumor microenvironment.

Stromal cells in the tumor microenvironment play a key role in the metastatic properties of a tumor. It is recognized that cancer-associated fibroblasts (CAFs) and endothelial cells secrete factors capable of influencing tumor cell migration into the blood or lymphatic vessels. We developed a microfluidic device that can be used to image the interactions between stromal cells and tumor cell spheroids in a three dimensional (3D) microenvironment while enabling external control of interstitial flow at an interface, which supports endothelial cells. The apparatus couples a 200-µm channel with a semicircular well to mimic the interface of a blood vessel with the stroma, and the design allows for visualization of the interactions of interstitial flow, endothelial cells, leukocytes, and fibroblasts with the tumor cells. We observed that normal tissue-associated fibroblasts (NAFs) contribute to the "single file" pattern of migration of tumor cells from the spheroid in the 3D microenvironment. In contrast, CAFs induce a rapid dispersion of tumor cells out of the spheroid with migration into the 3D matrix. Moreover, treatment of tumor spheroid cultures with the chemokine CXCL12 mimics the effect of the CAFs, resulting in similar patterns of dispersal of the tumor cells from the spheroid. Conversely, addition of CXCL12 to co-cultures of NAFs with tumor spheroids did not mimic the effects observed with CAF co-cultures, suggesting that NAFs produce factors that stabilize the tumor spheroids to reduce their migration in response to CXCL12.

A critical period of ear development controlled by distinct populations of ciliated cells in the zebrafish.

The zebrafish (Danio rerio) is a useful model system for analyzing development of the inner ear. A number of mutations affecting the inner ear have been identified. Here we investigate the initial stages of otolith morphogenesis in wild-type embryos as well as in monolith (mnl) mutant embryos, which fail to form anterior otoliths but otherwise appear normal. Otolith growth is initiated at 18-18.5 h by localized accretion of free-moving precursor particles. This process, referred to as otolith seeding, is regulated by two classes of cilia: First, kinocilia of precociously forming hair cells (tether cells) bind seeding particles, thereby localizing otolith formation. Tether cells usually occur in pairs at the anterior and posterior ends of the ear. Despite the presence of functional kinocilia, tether cells initially appear immature and do not acquire the characteristics of mature hair cells until approximately 21.5 h. Second, beating cilia distributed throughout the ear agitate seeding particles, thereby inhibiting premature agglutination. Constraining particles with laser tweezers caused them to fuse into large untethered masses. Bringing such masses into contact with tethered otoliths caused them to fuse, greatly enhancing otolith growth. Selectively enhancing one otolith greatly inhibited growth of the second, creating an imbalance that persisted for many days. Seeding particles and beating cilia disappear soon after 24 h, and the rate of otolith growth decreases by nearly 90%. In mnl mutant embryos, tethers and beating cilia are distributed normally, but anterior otoliths fail to form in 80-85% of mutant ears. The binding properties of seeding particles appear normal, as shown by their ability to fuse when entrapped by laser tweezers and their binding to posterior tethers. We infer that anterior tethers have a weakened ability to bind seeding particles in mnl embryos. Immobilizing mnl embryos with the anterior end of the ear oriented downward effectively concentrated the dense seeding particles near the anterior tethers and permitted all to form anterior otoliths. However, immobilizing mnl embryos after 24 h when seeding particles were depleted did not facilitate anterior otolith formation. Together, these data demonstrate that the ability to initiate otolith formation is limited to a critical period, from 18.5 to 24 h, and that interfering with the functions of tether cell kinocilia or beating cilia impairs otolith seeding and subsequent otolith morphogenesis.

The conjusome: a novel structure in Tetrahymena found only during sexual reorganization.

A unique structure, the conjusome, has been identified and initially characterized in Tetrahymena thermophila. The conjusome appears only during a specific phase of conjugation. Immunofluorescence microscopy reveals that the conjusome is strongly labeled by antibodies to the protein Pdd1p. Pdd1p is a chromodomain protein and participates in the formation of chromatin-containing structures in developing macronuclear anlagen. Recent studies suggest that Pdd1p is physically associated with the elimination of specific germ-line sequences from developing macronuclei (anlagen) and may play a role in heterochromatin assembly. The conjusome contains Pdd1p, but it is devoid of any detectable DNA. The conjusome appears before DNA elimination begins in the developing anlagen and after Pdd1p is found in the parental macronucleus. Transmission electron microscopic observations reveal that the conjusome is not a membrane-bounded structure. The conjusome ranges in size from about 1 microm to sizes approaching 7 microm, depending on its maturity. It is composed of a coarse reticulum of a fibrous, electron dense material, interspersed with apparent background cytoplasm. Our initial characterization does suggest a number of possible functions for what may be a new, transient organelle.