Microfluidics

the next video shows microfluidic channel, note the slow diffusion into the neighboring channels after each new ink is introduced; also note that the different inks do not mix well but instead stay mostly separate (due to laminar flow).


and another video of a very simple microfluidic device showing separation, mixing and sorting

Don Eigler: Moving Atoms, one-by-one

Stimulated Emission Depletion (STED) Microscopy

Superresolution microscopy using stimulated emission depletion (STED) creates sub-diffraction limit features by altering the effective point spread function of the excitation beam using a second laser that suppresses fluorescence emission from fluorophores located away from the center of excitation. The suppression of fluorescence is achieved through stimulated emission that occurs when an excited-state fluorophore encounters a photon that matches the energy difference between the ground and excited state. Upon interaction of the photon and the excited fluorophore, the molecule is returned to the ground state through stimulated emission before spontaneous fluorescence emission can occur. Thus, the process effectively depletes selected regions near the focal point of excited fluorophores that are capable of emitting fluorescence.

STED microscopy operates by using two laser beams to illuminate the specimen. An excitation laser pulse (generally created by a multiphoton laser) is closely followed by a doughnut-shaped red-shifted pulse that is termed the STED beam. Excited fluorophores exposed to the STED beam are instantaneously returned to the ground state by means of stimulated emission. The non-linear depletion of the fluorescent state by the STED beam is the basis for superresolution. Even though both laser pulses are diffraction-limited, the STED pulse is modified to feature a zero-intensity point at the center of focus with strong intensity at the periphery. When the two laser pulses are superimposed, only molecules that reside in the center of the STED beam are able to emit fluorescence, thus significantly restricting emission. This action effectively narrows the point spread function and ultimately increases resolution beyond the diffraction limit. To generate a complete image, the central zero is raster-scanned across the specimen in a manner similar to single-photon confocal microscopy, as illustrated in the tutorial. STED microscopy is capable of 20 nanometer (or better) lateral resolution and 40 to 50 nanometer axial resolution.

source: zeiss

Photoactivated Localization Microscopy (PALM)

Photoactivated localization microscopy (PALM) is a superresolution technique that dramatically improves the spatial resolution of the optical microscope by at least an order of magnitude (featuring 10 to 20 nanometer resolution), which enables the investigation of biological processes at close to the molecular scale. The technique relies on the controlled activation and sampling of sparse subsets of photoconvertable fluorescent molecules, either synthetic or genetically-encoded. This interactive tutorial explores the sequential steps involved in creating a PALM image.

Using photoactivatable fluorescent proteins, it is possible to selectively switch on thousands of sparse subsets of molecules in a sequential manner. The basic principle behind PALM is to start with the vast majority of the molecules in the inactive state (in effect, not contributing fluorescence emission). A small fraction (less than 1 percent) is photoactivated or photoconverted using a brief pulse of ultraviolet or violet light to render that subset fluorescent. The activated molecules are then imaged and localized to produce nanometer-level precision coordinates, followed by removal from the larger set of unactivated molecules by photobleaching. In the next step, a second fraction of molecules is photoactivated, localized, and eliminated by photobleaching. The process is repeated many thousands of times until the molecular coordinates of all labeled molecules are obtained. The PALM image is a composite of all the single molecule coordinates. As new fluorescent probes for PALM are developed, the photoconversion and readout wavelengths are likely to ultimately span the entire ultraviolet, visible, and near-infrared spectral regions.

source: zeiss

A unique strategy for overcoming the diffraction barrier employs photoswitchable fluorescent probes to resolve spatial differences in dense populations of molecules with superresolution. This approach relies on the stochastic activation of fluorescence to intermittently photoswitch individual photoactivatable molecules to a bright state, which are then imaged and photobleached. Thus, very closely spaced molecules that reside in the same diffraction-limited volume are temporally separated. Merging all of the single-molecule positions obtained by repeated cycles of photoactivation followed by imaging and bleaching produces the final superresolution image. Techniques based on this strategy are often referred to as probe-based superresolution, and were independently developed by three groups in 2006 and given the names photoactivated localization microscopy (PALM), fluorescence photoactivation localization microscopy (FPALM), and stochastic optical reconstruction microscopy (STORM). All three methods are based on the same principles, but were originally published using different photoswitchable probes.

source: zeiss

Scanning Electron Microscopy Basics

useful link: http://virtual.itg.uiuc.edu/training/

Biomimetic Nanoadhesives