Microns In Depth Research Articles

SiC, Si and diamond detectors for comparison of laser-generated plasma in TNSA regime

Monocrystalline SiC, Si and Diamond detectors have been used to monitor the radiations emitted from TNSA laser-generated plasma using a high-intensity fs laser. The comparison of their spectra was performed monitoring in time-of-flight the forward emitted TNSA plasma radiation. Carbon, aluminum, copper and gold targets were irradiated with an intensity of the order of 1019 W/cm2. SiC detector, with 80 microns depth active region, gave the best response to detect fast UV and X-rays, relativistic and cold electrons, and energetic protons and light accelerated ions. The experiments demonstrate the advantages to use semiconductor detectors to characterize the plasma enhancing the importance of the thickness of the target with respect to its electronic density.

Plasma-liquid interfacial layer detected by in situ Raman light sheet microspectroscopy

In situ Raman microscopy has been adapted to study the plasma–water interface by applying a light sheet technique. The Raman modes of the –OO stretch of H2O2, symmetric stretch (v 1) of NO3 −, and –OH bend of water were measured simultaneously. By modulating the volume of water under detection, both the bulk liquid and interface regions have been probed with micrometer depth resolution. The plasma was a DC glow discharge generated in atmospheric-pressure air with a water cathode. In the bulk liquid, the molar concentration of aqueous NO3 − increased at a linear rate of 48 µM min−1, whereas aqueous H2O2 growth stopped at about 5 mM. The concentrations of H2O2 and NO3 − both increased when measuring at depths less than about 20 µm from the interface. The depth profile of NO3 − concentration was reconstructed, showing that the interfacial layer of NO3 − has a depth of 28 µm. The shape of the meniscus may influence the interpretation of this depth. Previous models of plasma-water interfaces have predicted interfacial layers of similar depth for short-lived aqueous species such as OH but not for long-lived species such as NO3 −.

The Effects of Double Antibiotic Paste and Amoxicillin-clavulanate Paste Used in Endodontic Regeneration on Microhardness of Radicular Dentine: An In vitro Study.

Objective:Double antibiotic paste is a root canal medicament used in regenerative endodontics and its long-term exposure to dental hard tissues was shown to reduce its mechanical properties. The amoxicillin-clavulanate paste is a medicament effective against endodontic pathogens and is effective in endodontic regeneration. Its effect on radicular dentine is yet to be investigated. The purpose of this study is to investigate and compare the effects of double antibiotic paste and amoxicillin-clavulanate paste on the microhardness of radicular dentine.Materials and Methods:The root canal of 45 premolars was mechanically prepared and divided into three groups for the placement of intracanal medicament: Group 1 (n = 15) double antibiotic paste, Group 2 (n = 15) amoxicillin-clavulanate paste, and Group 3 control group (n = 15). All the specimens were subject to microhardness testing at 500 and 1000 microns depth for 1 week, 1 month, and 3 months and analyzed.Results:There is no significant reduction in microhardness of radicular dentin at 1 week on all the three groups. At 1 month and 3 month period, the amoxicillin-clavulanate paste is found to have no significant effect on dentin microhardness when compared to double antibiotic paste.Conclusion:The use of amoxicillin-clavulanate paste as an intracanal medicament does not cause significant weakening of dentin even after its long-term use.

Fabrication of Micro-channels on Polymethyl Methacrylate (PMMA) Plates by Thermal Softening Process Using Nichrome Wire: Tool Design and Surface Property Evaluation

Microfluidics, emerging system of fluid-handling technology by using micro-channels are being applied to diverse fields due to everyday demand. To overcome high volume market needs; low cost and fast processing methods are necessary to fabricate microfluidic devices. There are many ways to produce the necessary channel and feature dimensions using photolithography to milling. This study focus on the fabrication of narrow cylindrical channels having a dimension of microns (width and depth) in a poly(methyl methacrylate) substrate by a thermal softening process using the Joule heating effect of nichrome wires. The channel fabrication tool is designed and developed to generate localized heat on the substrate with a high repetition rate by using the moving hot nichrome wire as a function of feed rate (600-9000 mm/min) in a CNC machine. 3D optical profilometer study reveals that the channels are cylindrical and smooth. By increasing the feed rate, the width and depth of the microchannels are controlled from 230-80 μm, and 60 to 17 μm, respectively. These results suggest that the thermal softening process using a nichrome wire is a promising tool for the fabrication of micro-channels on flexible polymeric substrates.

МЕТОДИКА И РЕЗУЛЬТАТЫ МИКРОМЕТРАЖНЫХ ИССЛЕДОВАНИЙ ЭЛЕМЕНТОВ УПЛОТНИТЕЛЬНЫХ УЗЛОВ АГРЕГАТОВ ГИДРОСИСТЕМ СЕЛЬСКОХОЗЯЙСТВЕННОЙ ТЕХНИКИ

End-type sealing units based on round-section rubber rings are the most common types of seals for volumetric hydraulic drive units of various agricultural machinery. Its service life depends on the correspondence of the geometric dimensions of the elements that make them up, as well as on the combination of limit deviations of the specified elements. (Research purpose) The research purpose is in studying deviations and identifying statistical distribution of the cross-section diameters of new rings and the depth of grooves under them in order to determine the number of connections that meet the requirements of the standard. (Materials and methods) Author examined the sealing joints of the oil pipeline and the valve cover of the hydraulic distributor of the MTZ 80/82 tractor and new rubber rings that are part of repair kits intended for repair. Micrometric studies were performed using the GM-50 micrometer depth gauge and the TN 1060T thickness gauge. (Results and discussion) About 90 percent of the examined compounds do not meet the requirements of the standard, which explains to a large extent the low level of their reliability. At the same time, a significantly higher level of inconsistencies is in the metal parts of these connections. The vast majority of nonconforming joints have a combination of ring and groove sizes, which provides increased initial compression deformation of the ring in the groove. (Conclusions) Inconsistencies in the dimensions of the groove and the cross-section of the ring, which lead to an increase in the initial compression deformation of the seal, and thus the initial contact stresses, cause a certain increase in the theoretical life of the connection. To predict the connection reliability more accurate, it is necessary to study the influence on leaks not only of the geometric dimensions of the mating elements, but also other factors: surface roughness, as well as the actual operating temperature of the node and the aggressiveness of the hydraulic working fluid.

Atmospheric air oxidation of 9Cr-1Mo steel: Depth profiling of oxide layers using glow discharge optical emission spectrometry

Modified 9Cr-1Mo steel is used as a steam generator tube material of fast breeder reactors. Although air oxidation behavior of modified 9Cr-1Mo steel is qualitatively studied, the exact nature of oxide layers formed across the thickness of the film from nanometer to several microns depth is not studied systematically. In this work, the oxide scales formed on modified 9Cr-1Mo steel on air oxidation at 650 °C up to 200 h duration using Glow Discharge Optical Emission Spectrometer (GDOES) with depth profiling, X-ray Diffraction (XRD), Laser Raman Spectroscopy (LRS) and Scanning Electron Microscopy coupled with Energy Dispersive Spectroscopy(SEM-EDS) were investigated. The weight changes increase and did not follow the conventional parabolic kinetics. The top layer of the oxide scales after 25 and 50 h air oxidized samples comprised of MnCr2O4, Cr2O3 and Fe, Cr spinel oxides; however, the oxide scales of 100 and 200 h oxidized samples mainly consist of hematite. GDOES analysis indicated the presence of Fe rich oxides on the top surface of the oxide films in the case of 100 and 200 h oxidized samples. For 25 and 50 h samples, the oxide layer consists of manganese oxides in addition to Cr and Fe spinel oxides. All the samples consist of Fe and Cr spinel oxides at the interface of the oxide and alloy surfaces. SEM analysis indicated the formation and growth of oxides from small crystal and flakey type morphology to fully grown bigger size particles as the oxidation progresses. GDOES technique provided useful insights into the nature of oxide films through the thickness of the film. The intensity depth profiles of the oxide layers obtained by GDOES-DiP (Differential Interferometric Profiling) is complemented by other techniques such as XRD, LRS and SEM-EDS.

Surface Integrity of TA19 Notched Simulated Blades with Laser Shock Peening and Its Effect on Fatigue Strength

To reveal the fatigue strengthening mechanism of TA19 notched simulated blades with laser shock peening (LSP), surface integrity and fatigue strength were investigated. The surface morphology, residual stress, near-surface microstructure, fatigue strength and fatigue fracture morphology were analyzed by surface profiler, x-ray diffraction (XRD), transmission electron microscopy (TEM), QRG-100 servo-hydraulic fatigue test machine and scanning electron microscope (SEM). Results indicated that LSP induced surface micro-dents plastic deformations with a few microns in depth, surface compressive residual stresses and surface nano-grains of TA19 notched simulated blades. Compared with the as-received material, fatigue strengths of TA19 notched simulated blades were improved by 162% for LSP-1 and 218% for LSP-3. In addition, fatigue crack initiation (FCI) locations reduced after LSP, fatigue crack growth (FCG) rate decreased after LSP, and secondary cracks were observed in the FCI and FCG regions of fatigue fracture morphologies after LSP. Fatigue strengthening mechanism of TA19 notched simulated blades with LSP was compressive residual stresses and refined grains. Results could be beneficial to the application of LSP process in civil engines blades.

Topical vitamin C, vitamin E, and acetylcysteine as corneal wound healing agents: A comparative study.

Purpose:The purpose of this study was to evaluate wound healing abilities and efficacy of topical Vitamin C, Vitamin E, and acetylcysteine for their possible clinical use.Methods:The study was conducted on 36 eyes of 18 single-breed rabbits, Oryctolagus cuniculus, of both sexes. A 7.5 mm calibrated vacuum corneal trephine was used to induce a defect of 100 micron depth in center of both the corneas. The right eye of rabbits was selected as the control eye and the left eye as the trial eye, which received eyedrops as Group 1–10% Vitamin C eyedrops, Group 2–3% acetylcysteine eye drops, and Group 3–1% Vitamin E eye drops. Control eyes received ringer lactate. Evaluation was done for fluorescein stain positivity, epithelial defect size, and corneal haze on Day 2, Day 7, and Day 14 post induction of the epithelial defect.Results:On day 14, three eyes of control group, all Vitamin C and acetylcysteine treated eyes, and four Vitamin E treated eyes were fluorescein stain negative. The mean defect area on day 14 was 0, 0, 0.13, and 1.86 mm2 in Vitamin C, Vitamin E, acetylcysteine, and control eyes, respectively. Vitamin C and Vitamin E control corneal haze better than acetylcysteine in experimentally induced corneal wounds in rabbits.Conclusion:The three trial drugs with different mechanism of action showed similar effect on healing of the experimentally created corneal wounds in rabbits, with comparison showing statistical insignificance.

On the Interplay of DLIP and LIPSS Upon Ultra-Short Laser Pulse Irradiation.

Controlling laser induced surface morphology is essential for developing specialized functional surfaces. This work presents novel, multi-scale periodic patterns with two-dimensional symmetry generated on stainless steel, polyimide and sapphire. The microstructures were realized by combining Direct Laser Interference Patterning with the generation of Laser Induced Periodic Surface Structures in a one-step process. An industrial, fiber femtosecond laser source emitting at 1030 nm with a pulse duration of 500 fs was utilized for the experiments. In the case of stainless steel, it was possible to create line-like or pillar-like surface patterns by rotating the polarization orientation with respect to the interference pattern. In the case of polyimide and sapphire, the absorption of the laser radiation was promoted by a multiphoton mechanism. In polyimide, grooves and pillars of several microns in depth were produced over an area much larger than the spot size. Finally, for sapphire, the simultaneous generation of interference-like pattern and laser induced periodic surface structures was realized. The results reported here provide valuable data on the feasibility to combine two state-of-the-art techniques with an industrial apparatus, to control the induced surface morphology.

Optimization of ethosomes for topical thymoquinone delivery for the treatment of skin acne

The current study involved the development and optimization of ethosomes formulation for topical delivery of thymoquinone for improved therapy in skin acne. Formulation was optimized based on vesicles size, entrapment efficiency and flux by applying “3-factor 3-level Box-Behnken design”. The optimized formulation was further evaluated for “anti-microbial, anti-inflammatory, anti-acne and skin irritation activity”. The optimized formulation had shown the vesicles size of 105.2 ± 8.0 nm, entrapment efficiency of 85.30 ± 6.30% and flux of 65.40 ± 7.6 μg/cm2/h. The penetration depth of rhodamine loaded ethosomes across rat skin was traced with confocal laser microscopy and it was noted that the plain rhodamine solution (control) remained confined to few micrometers (14.4 μm) depth only into rat skin, while the rhodamine loaded ethosomes formulation penetrate deeper up to 103.5 μm depth into rat skin. Antimicrobial study exhibited that the marketed allopathic gel, prepared ethosomes formulation, and marketed herbal gel have presented zone of inhibition of 29.0 ± 1.00 mm, 20.33 ± 0.57 mm, and 15.00 ± 1.73 mm respectively against S. Epidermidis. The in vivo anti-inflammatory activity demonstrated that the application of prepared ethosomes and marketed diclofenac gel formulation resulted in an inhibition of rat paw edema by 80.74% and 84.61% respectively in Wistar rats. Anti-acne study demonstrated that the developed ethosomes formulation had shown considerable effect on sebaceous glands units by reducing its glands number and size. Further skin irritation study revealed that the developed formulation was safe, less irritant and well tolerable formulation for topical delivery. It was concluded that the developed thymoquinone loaded ethosomes formulation can be used as an effective treatment option for acne vulgaris and various skin disorders.

Dose efficient Compton X-ray microscopy

X-ray imaging techniques have proven invaluable to study biological systems at high resolution due to the penetration power and short wavelength of this radiation. In practice, the resolution and sensitivity of current X-ray imaging techniques are not limited by the performance of optics or image-recovery methods but by radiation damage. We propose the use of Compton (inelastic) X-ray scattering for high-resolution cellular imaging and provide a study of a scanning microscope geometry that requires a dose to achieve a given resolution that is three orders of magnitude lower than for coherent (elastic) scattering. We find that the dose per imaging signal is minimized at a photon energy of 64 keV. This corresponds to a short enough wavelength (0.02 nm) to provide nanometer transverse resolution and micrometer depth of field for tomographic imaging of whole cells. The microscope could be implemented at future high-energy and high-brightness synchrotron-radiation facilities to provide images of unsectioned and unlabeled cells in their native conditions at enough detail to bridge the techniques of super-resolution optical microscopy and cryo-electron microscopy.

Modification of Structure and Tribological Properties of the Surface Layer of Metal-Ceramic Composite under Electron Irradiation in the Plasmas of Inert Gases

Metal-ceramic composites are the main materials for high-load parts in tribomechanical systems. Modern approaches to extend the operation life of tribomechanical systems are based on increasing the strength and tribological properties of the surface layer having 100 to 200 microns in depth. The essential improvement of the properties occurs when high dispersed structure is formed in the surface layer using high-energy processing. As a result of the dispersed structure formation the more uniform distribution of elastic stresses takes place under mechanical or thermal action, the energy of stress concentrators emergence significantly increases and the probability of internal defects formation reduces. The promising method to form the dispersed structure in the surface layer is pulse electron irradiation in the plasmas of inert gases combining electron irradiation and ion bombardment in one process. The present work reports upon the effect of pulse electron irradiation in plasmas of different inert gases with different atomic mass and ionization energy on the structure and tribological properties of the surface layer of TiC/(Ni-Cr) metal-ceramic composite with the volume ratio of the component being 50:50. It is experimentally shown that high-dispersed heterophase structure with a fraction of nanosized particles is formed during the irradiation. Electron microscopy study reveals that refining of the initial coarse TiC particles occurs via their dissolution in the molten metal binder followed by the precipitation of secondary fine particles in the interparticle layers of the binder. The depth of modified layer and the fraction of nanosized particles increase when the atomic number of the plasma gas increases and ionization energy decreases. The wear resistance of metal-ceramic composite improves in accordance to the formation of nanocrystalline structure in the surface layer.

Design and numerical simulation demonstration of multi-functional holographic phase plate for large depth of field single molecular localization microscopy

The development of nanoscale single-molecule localization and tracking technology for multiple bio-molecules in intact cells has important significance for studying the dynamic process in life process. Since most of cells are several microns in depth, but the focal depth of traditional optical microscopes are less than one micron, the limited depth of field is the main drawback of conventional single molecular localization microscopy that prevents observation and tracking of multiple molecules in intact cells. In this paper, based on the wavefront coding technique, a new type of holographic phase plate with high efficiency is proposed and designed to extend the depth of field of single molecular localization microscopy, which combines the distorted multi-value pure-phase grating (DMVPPG) with the double-helix point spread function (DH-PSF). The DMVPPG can be used to realize multiplane imaging of several tens of layers of a sample in a single detection plane. And the DH-PSF is an engineered point spread function which encodes the lateral and axial position with high precision of a molecule in the center of its two lobes and the angle between them respectively. Using the combined holographic phase plate, the molecules in dozens layers of a whole cell can be simultaneously imaged on the same detection plane with DH-PSF. Not only can the axial resolving power be improved, but the imaging depth can also be extended without scanning. Adding such a holographic phase plate to the imaging path, the limited imaging depth problem in single-molecule-localization microscopy can be solved without sacrificing the localization accuracy. The proposed new type of holographic phase plate can also be implemented with a spatial light modulator. In the following numerical simulation experiments, the designed holographic phase plate is composed of 600×600 pixels with a pixel size of 10 μm. The distance between two adjacent focal planes is designed to be 0.5 μm. Such a holographic phase plate is placed on the Fourier transform plane of the detection light path. When an emitter is located on the focal plane, it can be imaged as two lobes without rotation in a center area of the field of view. If an emitter is -6 μm away from the focal plane, the DH-PSF appears in the upper-left area of the field of view. Simulation results demonstrate that a total of 25 sample layers can be simultaneously imaged on the single detection plane and the 12 μm detection range can be achieved, thus proving the feasibility of this method.

Surface integrity in precision turning of steel

The functional properties of a technical component depend directly on the geometrical and material properties, in particular the surface and surface layer properties of a component. Since material loads are generated in the material during a machining process, which cause alterations in the surface and surface layer properties, so-called material modification, the knowledge of these relationships is of great interest for a material-oriented manufacturing. In order to elucidate this for precision machining, there are two main process-specific challenges: (1) Cutting edge radius and undeformed chip thickness are of the same order of magnitude, so the engagement ratios and cutting conditions vary from cutting to plowing depending on the undeformed chip thickness. This phenomenon is known as the so-called size effect, occurring as a nonlinear increase in the specific cutting force with decreasing undeformed chip thickness. Therefore, the consideration of deformation and chip formation mechanisms is necessary. (2) The layers influenced by a precision machining process are very thin and thus difficult to characterize. The experimental investigation shows that a surface layer zone with a plastic deformation of a few micrometer depth below the surface is generated by precision turning of steel (42CrMoS4, AISI 4140). In addition, there is another size effect with regard to the depth of the plastic deformation, showing a nonlinear increase with decreasing undeformed chip thickness. The plastic deformation is influenced by the process parameters as well as the process kinematics.

Characterization and investigation of size effect in nano-impact indentations performed using cube-corner indenter tip

Abstract

Microstructural Characterization and Mechanical Properties of Stainless Steel Inlay Welded Dissimilar Materials

This present work was carried out to characterize the microstructure and mechanical properties of austenitic stainless steel (SS) 316L to dissimilar welding of Inconel 52 and Inconel 82 fillers that were treated by ultrasonic nanocrystalline surface modification (UNSM) technique. The microstructure and hardness map of the specimens were characterized by optical microscope (OM) and micro-hardness tester. The increase in hardness and refinement in grain size after UNSM treatment were found for the SS 316L/52 and SS 316L/82. In addition, the induced compressive residual stress in the surface layer of base SS 316L was measured. It is expected that the increased hardness into several microns in depth and modified microstructure with a severe plastically deformed surface layer by UNSM technique can increase the resistance to corrosion and extend the service life of pressurized water reactors (PWRs).

Investigation of the impact of simulated solar radiation on the micro- and nanoscale morphology and mechanical properties of a sheet moulded composite surface

AbstractIn this paper we present the results of investigation of micro- and nanoscale degradation of a sheet moulded composite exposed to simulated solar radiation. Utilization of high resolution methods such as atomic force microscopy, optical profilometry and microcomputer tomography allowed us to provide the evidence of significant deterioration of the surface as well as the material few microns in depth. Additionally, the typically used macroscopic investigations, such as wettability and flexural strength, were performed to observe the impact of weathering process. It was also shown that high resolution techniques provide superior sensitivity of the material degradation detection. The particular effectiveness of the applied approach was related to the structure of investigated material, as due to its degradation, a number of voids appeared, causing a significant roughness increase. In addition, the impact of light radiation could be compared to other environmental conditions maintained in the climatic chamber. It should be underlined, that according to our knowledge, such a study has not been performed so far.

Spectral Absorption Depth Profile: A Step Forward to Plasmonic Solar Cell Design

Absorption depth profile, a deterministic and key factor that defines the quality of excitons generation rate in optoelectronic devices, is numerically predicted using finite different time domain analysis. A typical model, nanoparticles array on silicon slab, was devised considering the concept of plasmonic solar cell design. The trend of spectral absorption depth profile distributions at various wavelengths of the solar spectrum, 460 nm, 540 nm, 650 nm, 815 nm, and 1100 nm, was obtained. A stronger and well-distributed absorption profile was obtained at ∼650 nm of the solar spectrum (i.e. ∼1.85 eV, c-Si bandgap), although the absorbing layer was affected more than a half micron depth at shorter wavelengths. Considering the observations obtained from this simulation, we have shown a simple two-step method in fabricating ultra-pure silver (Ag) nanoparticles that can be used as plasmonic nanoscatterers in a thin film solar cell. The morphology and elemental analysis of as-fabricated Ag nanoparticles was confirmed by field emission scanning electron microscope (FESEM) and FESEM-coupled electron diffraction spectroscopy. The size of the as-fabricated Ag nanoparticles was found to range from 50 nm to 150 nm in diameter. Further investigations on structural and optical properties of the as-fabricated specimen were carried out using ultraviolet–visible (UV–Vis) absorption, photoluminesce, and x-ray diffraction (XRD). Preferential growth of ZnO along {002} was confirmed by XRD pattern that was more intense and broadened at increasing annealing temperatures. The lattice parameter c was found to increase, whereas grain size increased with increasing annealing temperature. The optical bandgap was also observed to decrease from 3.31 eV to 3.25 eV at increasing annealing temperatures through UV–Vis measurements. This parallel investigation on optical properties by simulation is in line with experimental studies and, in fact, facilitates devising optimum process cost for efficient thin film solar cell design, as well as light trapping using plasmonic nanoscatterers incorporated within the active layer.

Antibiofilm efficacy of photoactivated curcumin, triple and double antibiotic paste, 2% chlorhexidine and calcium hydroxide against Enterococcus fecalis in vitro.

Root canal disinfection is one of the most important factors governing success of root canal treatment, especially when regenerative strategies are used. This study evaluated the efficacy of 5 intracanal medicaments against mature biofilms of Enterococcus fecalis in vitro: Light activated curcumin, triple antibiotic paste (TAP), double antibiotic paste (DAP), chlorhexidine, calcium hydroxide. Untreated teeth with biofilms served as controls. Confocal microscopy was used to analyse the biofilm mass and percentage of live/dead bacteria within the root canal as well as dentinal tubules. Dentinal shavings obtained from the root canal walls (at 200 and 400 microns depth) were used to quantify the colony forming units/mL. The results showed that light activated curcumin and triple antibiotic paste brought about complete disruption of the biofilm structure (P < 0.05) while chlorhexidine and calcium hydroxide were not significantly different from the control (P > 0.05). Light activated curcumin brought about the highest percentage of dead cells at both depths, but this was not significantly different from triple antibiotic paste (P > 0.05). Curcumin, TAP and DAP brought about a significant reduction of CFU/mL at both depths compared to the control and other groups (P < 0.05). Light activated curcumin brought about a 7 log reduction of bacteria at both depths.

3D tracking of single nanoparticles and quantum dots in living cells by out-of-focus imaging with diffraction pattern recognition

Live cells are three-dimensional environments where biological molecules move to find their targets and accomplish their functions. However, up to now, most single molecule investigations have been limited to bi-dimensional studies owing to the complexity of 3d-tracking techniques. Here, we present a novel method for three-dimensional localization of single nano-emitters based on automatic recognition of out-of-focus diffraction patterns. Our technique can be applied to track the movements of single molecules in living cells using a conventional epifluorescence microscope. We first demonstrate three-dimensional localization of fluorescent nanobeads over 4 microns depth with accuracy below 2 nm in vitro. Remarkably, we also establish three-dimensional tracking of Quantum Dots, overcoming their anisotropic emission, by adopting a ligation strategy that allows rotational freedom of the emitter combined with proper pattern recognition. We localize commercially available Quantum Dots in living cells with accuracy better than 7 nm over 2 microns depth. We validate our technique by tracking the three-dimensional movements of single protein-conjugated Quantum Dots in living cell. Moreover, we find that important localization errors can occur in off-focus imaging when improperly calibrated and we give indications to avoid them. Finally, we share a Matlab script that allows readily application of our technique by other laboratories.