ABSTRACT
Stimulated Emission Depletion (STED) microscopy enables subdiffraction resolution in the imaging plane. However, STED's lateral improvement in resolution is generally better than the enhancement in the axial direction. Here, we combine conventional STED superresolution imaging with Double Helix Point Spread Function (PSF) modulation for axial localization with a precision better than the classical Rayleigh limit. To demonstrate the capability of the method we resolve in a STED microscope sub-diffraction fluorescent bead assemblies, and localize them axially with better than 25nm precision. We also show that the same setup allows straightforward implementation of wide field phase contrast by imaging larger beads with spiral and dark field phase filtering.
Subject(s)
Algorithms , Image Enhancement/methods , Image Interpretation, Computer-Assisted/methods , Microscopy/methods , Refractometry/methods , Microscopy/instrumentation , Phantoms, ImagingABSTRACT
Hollow tubular microfluidic channels were fabricated on quartz substrates using sacrificial layer, planar micromachining processes. The channels were created using a bottom-up fabrication technique, namely patterning a photoresist/aluminum sacrificial layer and depositing SiO(2) over the substrate. The photoresist/aluminum layer was removed by etching first with HCl/HNO(3), followed by etching in Nano-Strip, a more stable form of piranha (H(2)SO(4)/H(2)O(2)) stripper. Rapid separation of fluorescently labeled amino acids was performed on a device made with these channels. The fabrication process presented here provides unique control over channel composition and geometry. Future work should allow the fabrication of highly complex and precise devices with integrated analytical capabilities essential for the development of micro-total analysis systems.
