We describe a simple method of obtaining optical sectioning in a conventional wide-field microscope by projecting a single-spatial-frequency grid pattern onto the object. Images taken at three spatial positions of the grid are processed in real time to produce optically sectioned images that are substantially similar to those obtained with confocal microscopes.

We report that two classes of membrane nanotubes between human monocyte-derived macrophages can be distinguished by their cytoskeletal structure and their functional properties. Thin membrane nanotubes contained only F-actin, whereas thicker nanotubes, i.e., those > approximately 0.7 microm in diameter, contained both F-actin and microtubules. Bacteria could be trapped and surf along thin, but not thick, membrane nanotubes toward connected macrophage cell bodies. Once at the cell body, bacteria could then be phagocytosed. The movement of bacteria is aided by a constitutive flow of the nanotube surface because streptavidin-coated beads were similarly able to traffic along nanotubes between surface-biotinylated macrophages. Mitochondria and intracellular vesicles, including late endosomes and lysosomes, could be detected within thick, but not thin, membrane nanotubes. Analysis from kymographs demonstrated that vesicles moved in a stepwise, bidirectional manner at approximately 1 microm/s, consistent with their traffic being mediated by the microtubules found only in thick nanotubes. Vesicular traffic in thick nanotubes and surfing of beads along thin nanotubes were both stopped upon the addition of azide, demonstrating that both processes require ATP. However, microtubule destabilizing agents colchicine or nocodazole abrogated vesicular transport but not the flow of the nanotube surface, confirming that distinct cytoskeletal structures of nanotubes give rise to different functional properties. Thus, membrane nanotubes between macrophages are more complex than unvarying ubiquitous membrane tethers and facilitate several means for distal interactions between immune cells.

Super-resolution 3D imaging reveals remodeling of the cortical actin meshwork at the natural killer cell immune synapse, which is likely to be important for secretion of lytic granules.

The main advantage of confocal microscopes over their conventional counterparts is their ability to optically ''section'' thick specimens; the thin image slices thus obtained can be used to reconstruct three-dimensional images, a capability which is particularly useful in biological applications. However, it is well known that the resolution and optical sectioning ability can be severely degraded by system or specimen-induced aberrations. The use of high aperture lenses further exacerbates the problem. Moreover, aberrations can considerably reduce the number of photons that reach the detector, leading to lower contrast. It is rather unfortunate, therefore, that in practical microscopy, aberration-free confocal imaging is rarely achieved. Adaptive optics systems, which have been used widely to correct aberrations in astronomy, offer a solution here but also present new challenges. The optical system and the source of aberrations in a confocal microscope are considerably different and require a novel approach to wavefront sensing. This method, based upon direct measurement of Zernike aberration modes, also exhibits an axial selectivity similar to that of a confocal microscope. We demonstrate an adaptive confocal fluorescence microscope incorporating this modal sensor together with a deformable membrane mirror for aberration correction. Aberration corrected images of biological specimens show considerable improvement in contrast and apparent restoration of axial resolution.

We present a high throughput microfluidic device for continuous-flow polymerase chain reaction (PCR) in water-in-oil droplets of nanoliter volumes. The circular design of this device allows droplets to pass through alternating temperature zones and complete 34 cycles of PCR in only 17 min, avoiding temperature cycling of the entire device. The temperatures for the applied two-temperature PCR protocol can be adjusted according to requirements of template and primers. These temperatures were determined with fluorescence lifetime imaging (FLIM) inside the droplets, exploiting the temperature-dependent fluorescence lifetime of rhodamine B. The successful amplification of an 85 base-pair long template from four different start concentrations was demonstrated. Analysis of the product by gel-electrophoresis, sequencing, and real-time PCR showed that the amplification is specific and the amplification factors of up to 5 x 10(6)-fold are comparable to amplification factors obtained in a benchtop PCR machine. The high efficiency allows amplification from a single molecule of DNA per droplet. This device holds promise for convenient integration with other microfluidic devices and adds a critical missing component to the laboratory-on-a-chip toolkit.