Based on previous theoretical and experimental results on the electrochemical etching of silicon in HF‐based aqueous electrolytes, it is shown for the first time that silicon microstructures of various shapes and silicon microsystems of high complexity can be effectively fabricated in any research lab with sub‐micrometer accuracy and high aspect ratio values (about 100). This is well beyond any up‐to‐date wet or dry microstructuring approach and is achieved using a wet etching, low‐cost technology: silicon electrochemical micromachining (ECM). Dynamic control of the etching anisotropy (from 1 to 0) as the electrochemical etching progresses allows the silicon dissolution to be switched in real‐time from the anisotropic to the isotropic regime and enables advanced silicon microstructuring to be achieved through the use of high‐aspect‐ratio functional and sacrificial structures, the former being functional to the microsystem operation and the latter being sacrificed for accurate microsystem fabrication. World‐wide dissemination of the ECM technology for silicon microstructuring is envisaged in the near future, due to its low cost and high flexibility, with high‐potential impact on, though not limited to, the broad field of microelectronics and microfabrication.

Cryogenic fluorescent light microscopy of flash-frozen cells stands out by artifact-free fixation and very little photobleaching of the fluorophores used. To attain the highest level of resolution, aberration-free immersion objectives with accurately matched immersion media are required, but both do not exist for imaging below the glass-transition temperature of water. Here, we resolve this challenge by combining a cryoimmersion medium, HFE-7200, which matches the refractive index of room-temperature water, with a technological concept in which the body of the objective and the front lens are not in thermal equilibrium. We implemented this concept by replacing the metallic front-lens mount of a standard bioimaging water immersion objective with an insulating ceramic mount heated around its perimeter. In this way, the objective metal housing can be maintained at room temperature, while creating a thermally shielded cold microenvironment around the sample and front lens. To demonstrate the range of potential applications, we show that our method can provide superior contrast in Escherichia coli and yeast cells expressing fluorescent proteins and resolve submicrometer structures in multicolor immunolabeled human bone osteosarcoma epithelial (U2OS) cells at −140 • C.cryo-light microscopy | high-NA immersion objective | fluorescence imaging | cryofixation | cryofluorescence microscopy F luorescent light microscopy at cryogenic temperature presents significant advantages in itself and provides an important complement to electron cryomicroscopy (1, 2). In particular, bleaching decreases drastically at low temperature (3), while the fluorescence yield of many fluorophores increases (4), and the spectral bands narrow (5-8). The application of modern superresolution methods such as stimulated emission depletion microscopy (STED) (9), photoactivated localization microscopy (10), stochastic optical reconstruction microscopy (11), or structured illumination microscopy (12) at cryogenic temperature holds the prospect of imaging fluorescent proteins with high precision in 3D and correlating their localization with the ultrastructure seen in electron cryomicroscopy of the same sample (3, 4, 13-16). In contrast to chemical fixation, cryofixation provides an unbiased, undistorted representation of the native state. This is increasingly more important as imaging resolution approaches the nanometer scale.A long-standing challenge in cryogenic light microscopy is the lack of high-numerical-aperture (NA) microscope objectives. The NA of an objective is the primary figure of merit that dictates its light-collection efficiency and diffraction-limited resolution. Significant technological development has been devoted toward user-friendly platforms based on high-NA air objectives optimized for cryomicroscopy (17-19). However, air objectives are fundamentally limited to NA values <1.Immersion objectives can surpass this limit by making physical contact with the sample via an immersion medium of refractive index >1. At room temperature, th...

Measuring small changes in refractive index can provide both sensitive and contactless information on molecule concentration or process conditions for a wide range of applications. However, refractive index measurements are easily perturbed by non-specific background signals, such as temperature changes or non-specific binding. Here, we present an optofluidic device for measuring refractive index with direct background subtraction within a single measurement. The device is comprised of two interdigitated arrays of nanofluidic channels designed to form an optical grating. Optical path differences between the two sets of channels can be measured directly via an intensity ratio within the diffraction pattern that forms when the grating is illuminated by a collimated laser beam. Our results show that no calibration or biasing is required if the unit cell of the grating is designed with an appropriate built-in asymmetry. In proof-of-concept experiments we attained a noise level equivalent to ∼10 refractive index units (30 Hz sampling rate, 4 min measurement interval). Furthermore, we show that the accumulation of biomolecules on the surface of the nanochannels can be measured in real-time. Because of its simplicity and robustness, we expect that this inherently differential measurement concept will find many applications in ultra-low volume analytical systems, biosensors, and portable devices.

Photoluminescent nanostructured semiconductor/metal systems consisting of silicon nanocrystals and gold nanoparticles are obtained by gold-catalyzed chemical etching. The interplay between silicon and gold nanostructures is investigated by photoluminescence spectroscopy upon continuous and pulsed excitation, both at room and low temperature. Comparison with reference samples, obtained removing gold particles by selective etching, highlights an enhanced emission in samples containing silicon and gold nanoparticles, explained in terms of both surface modifications and optical coupling between emitting nanocrystals and nanoparticles featuring localized plasmon resonances. (C) 2010 American Institute of Physics. [doi:10.1063/1.3483617

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Nanofluidic devices exhibit unique, tunable transport properties that may lead to breakthroughs in molecular separations and sensing. However, the throughput of these devices is orders of magnitude too small for the processing of macroscopic samples. Here we overcome this problem by combining two technological innovations. First, nanofluidic channels are made as vertical slits connecting the two sides of a silicon nitride membrane. Arbitrary arrays of such nanoslits down to 15 nm wide with <6 Å uniformity were made by merging the idea of templating with chemical mechanical polishing to create a scalable, nanolithography-free wafer level process. Second, we provide for efficient solute transport to and from the openings of the nanoslits by incorporating the nanofluidic membrane into a microfluidic tangential-flow system, which is also fabricated at wafer level. As an exemplary application, we demonstrate charge-based continuous flow separation of small molecules with a selectivity of 100 and constant flux over more than 100 hours of operation. This proves the exciting possibility of exploiting transport phenomena governed by precision-engineered nanofluidic devices at a macroscopic scale.

In this work, we present a method for measuring the refractive index of liquids at cryogenic temperature by shearing interferometry. The method is self-calibrated and easy to use over the range from cryogenic to room temperature. We validated the accuracy of our approach by comparing the measured refractive index of liquid propane with data reported in the literature. Moreover, we measured the refractive indices of three as yet uncharacterized fluorinated liquids that are of great interest as immersion fluids in the field of cryogenic light microscopy.

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