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.
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-m widths were readily traversed by the 8 m-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-m channels; some that passed through the 4-m channels emerged from constricted space with deformations whose shape-recovery could be observed in real time. In 2-m 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-m 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-m 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.
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.
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.Despite the small size of a mammalian cell, it is an extremely heterogeneous and compartmentalized structure. Proteins and small-molecule metabolites constantly traffic among these intracellular compartments, and it has become increasingly evident that biological specificity (e.g. between proteins) relies heavily on spatial and temporal segregation and compartmentalization of molecules in addition to chemical and molecular specificity 1, 2 . Gaining information with regard to the spatial and temporal distribution and evolution of molecules within cells, therefore, is crucial to the construction of a quantitative model of cellular function. The ability to isolate selectively single subcellular compartments for chemical analysis or transplantation opens new venues for studying the spatial and temporal organization of the cell. For example, the reprogramming of the nucleus of somatic cells may be achieved via nuclear transfer 3, 4 . This paper describes and compares the use of polarizationshaped vortex traps with a Gaussian optical tweezer for performing single-cell nanosurgery.Single-beam optical gradient traps, or optical tweezers, have made significant impact on biophysical and biological research in the past two decades 5-11 . Unfortunately, while optical tweezers offer exquisite sensitivity in its ability to position microparticles and to measure the forces exerted by biological motors 12 , it suffers from one important disadvantage: the trapped particle is localized at the laser focus where light intensity is the highest, often reaching 10 7 -10 8 W/cm 2 . As a result, the laser light used to trap a particle also has a propensity to photobleach and photodamage the particle, especially when the particle is fragile and small (e.g. a subcellular organelle that is fluorescently labeled) for which high laser intensities are often required. To minimize radiation damage to the trapped biological particle, laser wavelengths between ∼800nm to ∼ 1100nm are usually used because of the low absorption cross section of water and biological molecules in this spectral range 6-8, 13-15 . Nevertheless, at the high laser intensities required for trapping and translating subcellular organelles through the dense * Corresponding author. E-mail: chiu@chem.washington.edu. ...
We describe synthetic extracellular matrix (sECM) hydrogel films composed of co-crosslinked thiolated derivatives of chondroitin 6-sulfate (CS) and heparin (HP) for controlled-release delivery of basic fibroblast growth factor (bFGF) to full-thickness wounds in genetically diabetic (db/db) mice. In this model for chronic wound repair, full-thickness wounds were treated with CS, CS-bFGF, or CS-HP-bFGF films. At 2 and 4 weeks postinjury, wound closure and formation of the new epidermis and dermis were determined. Both CS and CS-HP hydrogel films accelerated wound repair, even without bFGF. Addition of bFGF to CS films showed partial dose-dependent acceleration of wound repair. Importantly, addition of bFGF to co-crosslinked CS-HP sECM films showed a dramatic bFGF dose-dependent acceleration of wound healing, as well as improved dermis formation and vascularization. Compared with 27% wound closure in 2 weeks in the controls, 89% wound closure was observed for mice treated with the CS-HP-bFGF films. The synthetic CS-HP sECM films mimic the chemistry and biology of heparan sulfate proteoglycans, and may have clinical potential for topical delivery of growth factors to patients with compromised wound healing.
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.
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