Nm In Axial Direction Research Articles

Three dimensional drift control at nano-scale in single molecule localization microscopy.

Super-resolution imaging based on single molecule localization of cellular structures on nanometer scale requires to record a series of wide-field or TIRF images resulting in a considerable recording time (typically of minutes). Therefore, sample drift becomes a critical problem and will lower the imaging precision. Herein we utilized morphological features of the specimen (mammalian cells) itself as reference markers replacing the traditionally used markers (e.g., artificial fiduciary markers, fluorescent beads, or metal nanoparticles) for sample drift compensation. We achieved sub-nanometer localization precision <1.0 nm in lateral direction and <6.0 nm in axial direction, which is well comparable with the precision achieved with the established methods using artificial position markers added to the specimen. Our method does not require complex hardware setup, extra labelling or markers, and has the additional advantage of the absence of photobleaching, which caused precision decrease during the course of super-resolution measurement. The achieved improvement of quality and resolution in reconstructed super-resolution images by application of our drift-correction method is demonstrated by single molecule localization-based super-resolution imaging of F-actin in fixed A549 cells.

Spatial filter and its application in three-dimensional single molecule localization microscopy

Single molecule localization microscopy (SMLM) allows the imaging of cellular structures with resolutions five to ten times below the diffraction limit of optical microscopy. It was originally introduced as a two-dimensional technique based on the localization of single emitters as projection onto the x-y imaging plane. The determination of the axial position of a fluorescent emitter is only possible by additional information. Here we report a method (spatial filter SMLM (SFSMLM)) that allows to determine the axial positions of fluorescent molecules and nanoparticles on the nanometer scale by the usage of two spatial filters, which are placed in two otherwise identical emission detection channels. SFSMLM allows axial localization in a range of ca. 1.5 μm with a localization precision of 15 - 30 nm in axial direction. The technique was utilized for localizing and imaging small cellular structures - e.g. actin filaments, vesicles and mitochondria - in three dimensions.

Focusing characteristics of a 4 \u03c0parabolic mirror light-matter interface

BackgroundFocusing with a 4 π parabolic mirror allows for concentrating light from nearly the complete solid angle, whereas focusing with a single microscope objective limits the angle cone used for focusing to half solid angle at maximum. Increasing the solid angle by using deep parabolic mirrors comes at the cost of adding more complexity to the mirror’s fabrication process and might introduce errors that reduce the focusing quality.MethodsTo determine these errors, we experimentally examine the focusing properties of a 4 π parabolic mirror that was produced by single-point diamond turning. The properties are characterized with a single 174Yb + ion as a mobile point scatterer. The ion is trapped in a vacuum environment with a movable high optical access Paul trap.ResultsWe demonstrate an effective focal spot size of 209 nm in lateral and 551 nm in axial direction. Such tight focusing allows us to build an efficient light-matter interface.ConclusionOur findings agree with numerical simulations incorporating a finite ion temperature and interferometrically measured wavefront aberrations induced by the parabolic mirror. We point at further technological improvements and discuss the general scope of applications of a 4 π parabolic mirror.

Double fiber probe with a single fiber Bragg grating based on the capillary-driven self-assembly fabrication method for dimensional measurement of micro parts

Focusing on the ultra-precision dimensional measurement of parts with micro-scale dimensions and high aspect ratios, a two-dimensional double fiber probe with a single fiber Bragg grating (DS-FBG probe) is investigated in detail in this paper. The theoretical analysis of the sensing principle is verified by spectrum simulations of the DS-FBG probe with a modified transfer matrix method using the strain distribution within the DS-FBG probe. The fabrication process and physical principle of the capillary-driven self-assembly of double fibers in the UV adhesive with a low viscosity are demonstrated. Experimental results indicate that resolutions of 30 nm in radial direction and 15 nm in axial direction can be achieved, and the short-term displacement drifts within 90 seconds are 28.0 nm in radial direction and 7.9 nm in axial direction, and the long-term displacement drifts within 1 hour are 61.3 nm in radial direction and 17.3 nm in axial direction. The repeatability of the probing system can reach 60 nm and the measurement result of a standard nozzle is 300.49 μm with a standard deviation of 20 nm.

Nano-level position resolution for particle tracking in digital in-line holographic microscopy.

Three-dimensional particle tracking in biological systems is a quickly growing field, many techniques have been developed providing tracking characters. Digital in-line holographic microscopy is a valuable technique for particle tracking. However, the speckle noise, out-of-focus signals and twin image influenced the particle tracking. Here an adaptive noise reduction method based on bidimensional ensemble empirical mode decomposition is introduced into digital in-line holographic microscopy. It can eliminate the speckle noise and background of the hologram adaptively. Combined with the three-dimensional deconvolution approach in the reconstruction, the particle feature would be identified effectively. Tracking the fixed beads on the cover-glass with piezoelectric stage through multiple holographic images demonstrate the tracking resolution, which approaches 2 nm in axial direction and 1 nm in transverse direction. This would facilitate the development and use in the biological area such as living cells and single-molecule approaches.

Three-dimensional nanometre localization of nanoparticles to enhance super-resolution microscopy.

Meeting the nanometre resolution promised by super-resolution microscopy techniques (pointillist: PALM, STORM, scanning: STED) requires stabilizing the sample drifts in real time during the whole acquisition process. Metal nanoparticles are excellent probes to track the lateral drifts as they provide crisp and photostable information. However, achieving nanometre axial super-localization is still a major challenge, as diffraction imposes large depths-of-fields. Here we demonstrate fast full three-dimensional nanometre super-localization of gold nanoparticles through simultaneous intensity and phase imaging with a wavefront-sensing camera based on quadriwave lateral shearing interferometry. We show how to combine the intensity and phase information to provide the key to the third axial dimension. Presently, we demonstrate even in the occurrence of large three-dimensional fluctuations of several microns, unprecedented sub-nanometre localization accuracies down to 0.7 nm in lateral and 2.7 nm in axial directions at 50 frames per second. We demonstrate that nanoscale stabilization greatly enhances the image quality and resolution in direct stochastic optical reconstruction microscopy imaging.