Deformability considerations in filtration of biological cells.

Biological cells are highly sensitive to variation in local pressure because cellular membranes are not rigid. Unlike microbeads, cells deform under pressure or even lyse. In isolating or enriching cells by mechanical filtration, pressure-induced lysis is exacerbated when high local fluidic velocity is present or when a filter reaches its intended capacity. Microfabrication offers new possibilities to design fluidic environments to reduce cellular stress during the filtration process. We describe the underlying biophysics of cellular stress and general solutions to scale up filtration processes for biological cells.

Prediction of phenotype and gene expression for combinations of mutations.

Molecular interactions provide paths for information flows. Genetic interactions reveal active information flows and reflect their functional consequences. We integrated these complementary data types to model the transcription network controlling cell differentiation in yeast. Genetic interactions were inferred from linear decomposition of gene expression data and were used to direct the construction of a molecular interaction network mediating these genetic effects. This network included both known and novel regulatory influences, and predicted genetic interactions. For corresponding combinations of mutations, the network model predicted quantitative gene expression profiles and precise phenotypic effects. Multiple predictions were tested and verified.

Using polarization-shaped optical vortex traps for single-cell nanosurgery.

Single-cell nanosurgery and the ability to manipulate nanometer-sized subcellular structures with optical tweezers has widespread applications in biology but so far has been limited by difficulties in maintaining the functionality of the transported subcellular organelles. This difficulty arises because of the propensity of optical tweezers to photodamage the trapped object. To address this issue, this paper describes the use of a polarization-shaped optical vortex trap, which exerts less photodamage on the trapped particle than conventional optical tweezers, for carrying out single-cell nanosurgical procedures. This method is also anticipated to find broad use in the trapping of any nanoparticles that are adversely affected by high-intensity laser light.

Monitoring cell survival after extraction of a single subcellular organelle using optical trapping and pulsed-nitrogen laser ablation.

This paper characterizes cell viability in three different cell lines--Chinese hamster ovary cells (CHO), neuroblastoma cells fused with glialoma cells (NG108-15) and murine embryonic stem cells (ES-D3)--after N2 laser disruption of the cell membrane and removal, via optical trapping, of a single subcellular organelle. Morphological changes and viability (as determined by live/dead fluorescent stains) of the cell were monitored every half hour over a 4-h period postsurgery. The ability of the cell to survive organelle extraction was found to depend both on the conditions under which surgery was performed and on the cell type. The average viability after surgery for CHO cells was approximately 80%, for NG 108 cells it was approximately 30% and for ES-D3 cells postsurgery viability was approximately 10%. From over 600 surgeries we found the survival of the cell is determined almost exclusively within the first hour postsurgery regardless of cell line. The optimal pulse energy for N2 laser ablation was approximately 0.7 microJ. The N2 pulse produced an approximately 1-3 microm hole in the cell membrane and proved to be the primary source of cell death in those cells that did not survive the procedure.

Selective encapsulation of single cells and subcellular organelles into picoliter- and femtoliter-volume droplets.

This paper describes a method, which combines optical trapping and microfluidic-based droplet generation, for selectively and controllably encapsulating a single target cell or subcellular structure, such as a mitochondrion, into a picoliter- or femtoliter-volume aqueous droplet that is surrounded by an immiscible phase. Once the selected cell or organelle is encased within the droplet, it is stably confined in the droplet and cannot be removed. We demonstrate in droplet the rapid laser photolysis of the single cell, which essentially "freezes" the state that the cell was in at the moment of photolysis and confines the lysate within the small volume of the droplet. Using fluorescein di-beta-d-galactopyranoside, which is a fluorogenic substrate for the intracellular enzyme beta-galactosidase, we also assayed the activity of this enzyme from a single cell following the laser-induced lysis of the cell. This ability to entrap individual selected cells or subcellular organelles should open new possibilities for carrying out single-cell studies and single-organelle measurements.

Direct manipulation and observation of the rotational motion of single optically trapped microparticles and biological cells in microvortices.

This paper describes a method for manipulating and monitoring the rotational motion of single, optically trapped microparticles and living cells in a microvortex. To induce rotation, we placed the microparticle at the center of rotation of the vortex and used the recirculating fluid flow to drive rotation. We have monitored the rotation of single beads (which ranged in diameter from a few micrometers to tens of micrometers) and living cells in a microvortex. To follow the rotation of a smooth and symmetrically shaped bead, we first ablated a small region ( approximately 1 microm) on the bead. An Ar(+) laser was then tightly focused ( approximately 0.5-microm spot size) onto the bead, and rotation was tracked by recording changes in the level of backscattered laser light as the ablated region repeatedly transited the laser focus. Using this method, we have followed bead rotation that varied in frequency from 0.15 to 100 Hz and have studied the effect of bead diameter on the rate of rotation at a given fluid flow rate. To monitor the rotation of single living cells, we selectively stained portions of B-lymphocytes with the fluorescent dye DiOC(6). We observed rotation by following changes in the fluorescence signal as the dye-stained region transited the laser focal volume. This technique provides a simple and sensitive method for controlling and monitoring the rotational motion of microparticles in a microfluidic environment.

Controlled rotation of biological micro- and nano-particles in microvortices.

Micrometer-sized re-circulating flows generated in a microfluidic system are used to drive the controlled rotation of biological particles of both micro- and nano-meter scale dimensions. This technique is independent of the intrinsic nature of the particle, and possesses the potential to rotate particles at high rates. We demonstrate in such microvortices the orientation control of single DNA molecules, and the axial rotation of biological cells in which the cellular contents were visibly affected by rotation.

A microfluidic model for single-cell capillary obstruction by Plasmodium falciparum-infected erythrocytes.

Severe malaria by Plasmodium falciparum is a potentially fatal disease, frequently unresponsive to even the most aggressive treatments. Host organ failure is associated with acquired rigidity of infected red blood cells and capillary blockage. In vitro techniques have played an important role in modeling cell deformability. Although, historically they have either been applied to bulk cell populations or to measure single physical parameters of individual cells. In this article, we demonstrate the unique abilities and benefits of elastomeric microchannels to characterize complex behaviors of single cells, under flow, in multicellular capillary blockages. Channels of 8-, 6-, 4-, and 2-microm widths were readily traversed by the 8 microm-wide, highly elastic, uninfected red blood cells, as well as by infected cells in the early ring stages. Trophozoite stages failed to freely traverse 2- to 4-microm channels; some that passed through the 4-microm channels emerged from constricted space with deformations whose shape-recovery could be observed in real time. In 2-microm channels, trophozoites mimicked "pitting," a normal process in the body where spleen beds remove parasites without destroying the red cell. Schizont forms failed to traverse even 6-microm channels and rapidly formed a capillary blockage. Interestingly, individual uninfected red blood cells readily squeezed through the blockages formed by immobile schizonts in a 6-microm capillary. The last observation can explain the high parasitemia in a growing capillary blockage and the well known benefits of early blood transfusion in severe malaria.

Microfluidic systems: high radial acceleration in microvortices.

Microfluidic systems can conveniently be used for rapid analysis of biological samples. Here we describe a single re-circulating flow, or microvortex, that can generate a maximum fluid rotational velocity of up to 12 m s(-1) and a corresponding radial acceleration in excess of 10(6)g. Such microvortices may be exploited in centrifugal microdevices to investigate the effects of high radial acceleration on biological and chemical processes.

Mapping fast flows over micrometer-length scales using flow-tagging velocimetry and single-molecule detection.

This paper describes a technique of characterizing microfluidic flow profiles from slow laminar flow to fast near-turbulent flow. Using a photo-activated fluorophore, nanosecond-duration photolysis pulses from a Nitrogen laser, and high-sensitivity single-molecule detection with Ar+ laser excitation, we report the measurement of flow speeds up to 47 m/s in a 33-microm-wide straight channel and the mapping of flow profiles in a 55-microm-wide microchamber. Sensitive single-molecule detection is necessary both because of the short time delay (submicrosecond) between laser photolysis and fluorescence detection and the fast transit times (as low as 10 ns) of the fluorescent molecules across the diffraction-limited beam waist of the Ar+ laser focus. This technique permits the high-resolution three-dimensional mapping and analysis of a wide range of velocity profiles in confined spaces that measure a few micrometers in dimension.