Scanning Near-Field Optical Microscopy and Microspectroscopy of Green Fluorescent Protein in Intact Escherichia coli Bacteria

Scanning near-field optical microscopy (SNOM) yields high-resolution topographic and optical information and constitutes an important new technique for visualizing biological systems. By coupling a spectrograph to a near-field microscope, we have been able to perform microspectroscopic measurements with a spatial resolution greatly exceeding that of the conventional optical microscope. Here we present SNOM images of Escherichia coli bacteria expressing a mutant green fluorescent protein (GFP), an important reporter molecule in cell, developmental, and molecular biology. Near-field emission spectra confirm that the fluorescence detected by SNOM arises from bacterially expressed GFP molecules.     

25 nm resolution single molecular fluorescence imaging by scanning near-field optical/atomic force microscopy

High-resolution single molecular near-field fluorescence images were observed by scanning near-field optical/atomic force microscopy (SNOM/AFM). We modified the SNOM/AFM for both high-resolution fluorescence imaging and high-resolution topographic imaging. The imaged fluorophore, Alexa 532, is prepared with a poly-methyl-methacrylate (PMMA) film coating. A fluorescence resolution of 25 nm was obtained with a simultaneous topographic image of a flat surface. A sample prepared with a lower PMMA concentration exhibited a rough surface in the micro area. The results for the flat surface indicated that the fluorescence resolution is worst in the rough surface sample, that the maximum fluorescence intensities for the individual fluorophore are similar, and that the decay rate is faster. Thus, we concluded that the morphological effect is an important factor in fluorescence image resolution and the apparent lifetimes of the fluorescence molecules.     

AFM and SNOM characterization of carboxylic acid terminated silicon and silicon nitride surfaces

Silicon and silicon nitride surfaces have been successfully terminated with carboxylic acid monolayers and investigated by atomic force microscopy (AFM) and scanning near-field optical microscopy (SNOM). On clean Si surface, AFM showed topographical variations of 0.3-0.4 nm while for the clean Si₃N₄ surface the corrugation was around 3-4 nm. After material deposition, the corrugation increased in both samples with a value in topography of 1-2 nm for Si and 5-6 nm for Si₃N₄. The space distribution of specific chemical species was obtained by taking SNOM reflectivity at several infrared wavelengths corresponding to stretch absorption bands of the material. The SNOM images showed a constant contribution in the local reflectance, suggesting that the two surfaces were uniformly covered.     

Development of a “Built-in” Scanning Near Field Microscope Head for an Atomic Force Microscope System and Stress Mapping of an Al2O3/ZrO2 Eutectic Composite

We constructed a scanning near-field optical microscope (SNOM) on a commercially available atomic force microscopy (AFM) apparatus (SPM-9500J2; Shimadzu Corp.) to measure the stress distribution in ceramic composite materials. Features of our SNOM system are: (1) a compact SNOM head substituted for the original AFM head; (2) a wide scanning range (125 × 125 μm²) inherited from the original scanner; (3) use of conventional shear-force regulation; (4) an optical system for the illumination-collection (I-C) mode; (5) excitation by a 488 nm line of an Ar-ion laser, and (6) light detection by photon counting or a polychromator equipped with an electronically cooled charge coupled device (CCD). This SNOM system was used to measure the surface structure and stress distribution of an Al₂O₃/ZrO₂ eutectic composite. We simultaneously measured topographic images and fluorescence spectra of an Al₂O₃/ZrO₂ eutectic composite. We estimated its peak intensity, peak position, and peak width from the fluorescence spectrum during scanning, which respectively correspond to the abundance of Al₂O₃, stress in the grain, and the anisotropy of that stress. Mapping images showed that the stress and its anisotropy were weaker in the center of the Al₂O₃ grain than its boundary between Al₂O₃ and ZrO₂. That observation suggests that Al₂O₃ underwent intense anisotropic stress induced by volume expansion in the phase transition of ZrO₂ from the cubic phase to the monoclinic phase during preparation.     

Controlled rotation and orientation of rubrene particles and Escherichia coli using optical tweezers

Optical trapping and rotating of suspended micro-sized rubrene particles were performed using optical tweezers with circularly polarized light. The experimental results show that the rotation speed of the rubrene particles is proportional to the laser power, and the orientation of the rubrene particles can be controlled by the optical tweezers with linearly polarized light. Interestingly, by combining with the rubrene particle, the Escherichia coli (E. coli) can be rotated and oriented by optical tweezers. However, the rotating and orientating are mainly determined by the characteristics of rubrene particles. Our experiment provides a simple and convenient way to orient biological particles even if they are not sensitive to the polarization of the laser beam. Moreover, the rubrene can emit strong fluorescence when excited by the laser at the wavelength of 532 nm, and which can be potential applied to manipulate other particles with the fluorescence characteristics.     

Self-assembly of dandelion-like Fe3O4@C@BiOCl magnetic nanocomposites with excellent solar-driven photocatalytic properties

In comparison with conventional organic dyes, quantum dots (QDs) have unique optical and electronic properties, which provide QDs with a wide scope of prospective application in biology and biomedicine. However, the toxicity of QDs and the fluorescence intensity of labeled bacteria must precede their application in bacterial imaging and tracing in vivo. Here, we show that treatment with CaCl₂ significantly improved bacterial labeling efficiency of CdSe/ZnS QDs with the CdSe core size of ~3.1 nm (relative fluorescence unit (RFU) value and ratio of fluorescent E. coli) with rising CdSe/ZnS QDs concentration in a concentration-dependent manner. At 12.5 nmol/L CdSe/ZnS QDs concentration, labeled Escherichia coli (E. coli) DH5α appeared as short rod-shaped and luminescent with normal size, and the survival rate and ultrastructure did not change in comparison to the control. But the ratio of fluorescent bacteria and RFU were very low. However, the survival rate of transformed E. coli was significantly inhibited by high CdSe/ZnS QDs concentrations (≥25 nmol/L). Moreover, internalization of CdSe/ZnS QDs resulted in ultrastructure damage of transformed E. coli in a concentration-dependent manner (≥25 nmol/L). Therefore, CdSe/ZnS QDs may not suitable for tracing of bacteria in vivo. Moreover, our study also revealed that colony-forming capability assay and transmission electron microscopy could be used to comprehensively evaluate the toxicity of QDs on labeled bacteria. Our findings do provide a new direction toward the improvement and modification of QDs for use in imaging and tracing studies in vivo.     

Investigation of the Doughnut Effect in Cy3-Labeled Protein Microarrays using Scanning Near-Field Optical Microscope

We demonstrate the fluorescence mapping of protein microarrays by the technique of scanning near-field optical microscopy (SNOM) and confocal microscopy. Micron sized spots (300 μm) of human Immunoglobulin G (hIgG) protein with and without a Cy3 dye labeling have been fabricated on glass substrates by an immobilization method which makes use of calixcrown derivatives termed Prolinker. We have also tried to probe into the well-known “doughnut effect” observed in fluorescence images of proteins using the SNOM technique. The topographic and fluorescence SNOM images revealed that the number of proteins at the boundary of the spot were more than at the center in the case of the microarray spot which showed brighter luminescence at the edge than at the center in the confocal image.     

Influence of Al-doping on the structure and optical properties of ZnO films

Al-doped ZnO (ZnO:Al) thin films with c-axis preferred orientation were deposited on glass substrates using the radio frequency reactive magnetron sputtering technique. The effect of Al concentrations on the microstructure and the luminescence properties of the ZnO:Al thin films were studied by atomic force microscopy (AFM), X-ray diffraction (XRD), and fluorescence spectrophotometer. The results showed that the crystallization of the films was promoted by appropriate Al concentrations; the photoluminescence spectra (PL) of the samples were measured at room temperature. Strong blue peak located at 437 nm (2.84 eV) and two weak green peaks located at about 492 nm (2.53 eV) and 524 nm (2.37 eV) were observed from the PL spectra of the four samples. The origin of these emissions was discussed. In addition, absorption and transmittance properties of the samples were researched by UV spectrophotometer; the UV absorption edge shifted to a shorter wavelength first as Al was incorporated, and then to a longer wavelength with the increasing Al concentrations. The optical band gaps calculated based on the quantum confinement model are in good agreement with the experimental values.     

Chitosan Nanoparticles: Preparation and Application in Antibacterial Paper

Chitosan nanoparticles (CSNP) were obtained by H₂O₂ degradation of chitosan. Their morphology and size were determined by atomic force microscope (AFM), and the particles were found smooth and approximately 36 nm in size. CSNP-0.5% HAc solutions (1, 2, 5, and 10 mg/mL) were used in antibacterial paper by addition in pulp, impregnation, dispersion coating on the handsheets, and insufflation. The antibacterial activity of CSNP against Escherichia coli and Staphylococcus aureus was measured by the inhibition zone and bacterial reduction methods. Results showed that the antibacterial activity of CSNP was improved with the increase in concentration. For E. coli, at a CSNP concentration of 5 mg/mL, the antibacterial rate reached approximately 95%. However, for S. aureus, the antibacterial rate only reached 81%. In addition, the antibacterial activity of the antibacterial paper was determined by inhibition zone method. The paper prepared by insufflation had the greatest activity. For E. coli, at a CSNP concentration of 10 mg/mL, the inhibition zone reached 8.0 mm. For S. aureus, the inhibition zone was 6.8 mm.     

Analysis of sterilization effect by pulsed dielectric barrier discharge

Atmospheric pressure (AP) plasmas can sterilize against almost all kinds of bacteria because many ions and reactive species, such as oxygen atoms and ozone, etc., are generated during AP plasmas. So AP plasmas are proper processes for application to air cleaners and sterilizers. The aim of this paper is to evaluate a germicidal effect caused by pulsed plasma system in air utilizing a dielectric barrier discharge (DBD) type reactor incorporating alumina, glass, etc. Escherichia coli, Bacillus subtilis and Pseudomonas aeruginosa bacteria were used for this sterilization experiment. For analysis of the relationship between sterilization results and chemical species generated in the discharge, we used optical emission spectroscopy and we checked emission spectra by atomic oxygen (394.2 and 436.8 nm) and second positive system of nitrogen (337.1 nm). Experimental results showed that DBD treatment during 70 s sterilized E. coli with 99.99% effectively and ozone molecules were the dominant germicidal species. From these results we concluded that the pulsed DBD system is very effective for sterilization.     

Physical properties of vacuum evaporated CdTe thin films with post-deposition thermal annealing

This paper presents the physical properties of vacuum evaporated CdTe thin films with post-deposition thermal annealing. The thin films of thickness 500 nm were grown on glass and indium tin oxide (ITO) coated glass substrates employing thermal vacuum evaporation technique followed by post-deposition thermal annealing at temperature 450 °C. These films were subjected to the X-ray diffraction (XRD),UV-Vis spectrophotometer, source meter and atomic force microscopy (AFM) for structural, optical, electrical and surface morphological analysis respectively. The X-ray diffraction patterns reveal that the films have zinc-blende structure of single cubic phase with preferred orientation (111) and polycrystalline in nature. The crystallographic and optical parameters are calculated and discussed in brief. The optical band gap is found to be 1.62 eV and 1.52 eV for as-grown and annealed films respectively. The I–V characteristics show that the conductivity is decreased for annealed thin films. The AFM studies reveal that the surface roughness is observed to be increased for thermally annealed films.     

Structural characterization and optical properties of ZnSe thin films

Zinc selenide (ZnSe) thin films (d = 0.11-0.93 μm) were deposited onto glass substrates by the quasi-closed volume technique under vacuum. Their structural characteristics were studied by X-ray diffraction (XRD) and atomic force microscopy (AFM). The experiments showed that the films are polycrystalline and have a zinc blende (cubic) structure. The film crystallites are preferentially oriented with the (1 1 1) planes parallel to the substrate surface. AFM images showed that the films have a grain like surface morphology. The average roughness, R_a = 3.3-6.4 nm, and the root mean square roughness, R_rms = 5.4-11.9 nm, were calculated and found to depend on the film thickness and post-deposition heat treatment.The spectral dependence of the absorption coefficient was determined from transmission spectra, in the range 300-1400 nm.The values of optical bandgap were calculated from the absorption spectra, E_g = 2.6-2.7 eV.The effect of the deposition conditions and post-deposition heat treatment on the structural and optical characteristics was investigated.     

The design of an apochromatic optical spectrograph for astronomical purposes

An optical spectrograph for use in the OGS (Optical Ground Station), a 1 m telescope in Izaña Observatory (Tenerife, Spain) and the GTC telescope at Islas Canarias observatory has been designed, built and tested. The dispersive part was designed and built at the Centro de Investigaciones en Óptica, A.C. Due to requirements from Instituto Astrofísico de Canarias this Spectrograph has an f/5 collimator and camera with a plate factor of 6.55 nm/mm. Since the resolution of the system has to have a high resolution of the order of 15 μm or less, an apochromatic design was selected. This article describes this design and its main characteristics.     

Near field optical spectroscopy of colored centers stripes written in lithium fluoride by electron-beam lithography

Electron-beam lithography has been used to define color center stripes of about one hundred micrometers length and variable width (100 v nm to 5 v m) in lithium fluoride. These structures have for the first time been illustrated and spectrally characterized in near field optical microscopy (SNOM) operating in local illumination mode with an optical fiber probe and far field fluorescence detection.     

Strong Surface Enhanced Florescence of Carbon Dot Labeled Bacteria Cells Observed with High Contrast on Gold Film

Strong surface (metal) enhanced fluorescence (SEF or MEF) is observed from clusters and single E coli bacteria cells labeled with Carbon nanodots (CDs), which were synthesized from date pits. The enhancement factor (EF) for SEF of the cell clusters were close to 50 for both 533 and 633 nm laser excitation wavelength. Those EFs are ratios of emission peak areas from CD labeled cell clusters on gold film to the peak areas of the same batch cell clusters on glass substrate. SEF with 633 nm excitation performed better than SEF with 532 nm excitation, achieving higher fluorescence intensity and much higher contrast. The contrast as high as 66 for cell clusters on gold film is a ratio of fluorescent emission peak area measured at the CD labeled cell clusters to the fluorescent peak area measured at unlabeled cell clusters (autofluorescence) on the same substrate. The contrast with the background (S/N) or the ratio of fluorescent peak area measured at bacteria cells to area measured at bare substrate was as high as 200. This report may pave a way for the broader application of surface enhanced fluorescence and especially metal enhanced fluorescence imaging of CD labeled cells and other biological objects.

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Graphical abstract Carbon dots, synthesized from dates, are used for direct staining of E coli cells. Emission fluorescent spectroscopy of those CD labelled cells on gold film and glass, demonstrated enhancement factor about 50 for emission on gold as compared to glass, Excitation at 633 nm appears far superior to excitation at 532 nm in terms of contrast (up to 67) with unlabeled cells /control due to decrease in auto fluorescence of cells. Maximum Signal to noise ratio is 200.

     

AFM, SEM and GIXRD studies of thin films of red polycarbazolyldiacetylenes

In this paper we report the results of a morphological and structural investigation on film properties of a soluble polydiacetylene, the poly[1,6-bis(3,6-dihexadecyl-N-carbazolyl)-2,4-hexadiyne] (polyDCHD-HS). The red films of this polymer, prepared by standard spin-coating techniques, revealed absence of linear dichroism and birefringence in contrast with the ordered mesophases detected by powder X-ray studies. In order to interpret the optical behavior of this polymer, we performed AFM and SEM studies of polyDCHD-HS films spun on hydrophylic and hydrophobic glass substrates. We found the presence of surfaces organized in rod-like particles, more regularly oriented on the hydrophylic substrate. GIXRD studies, carried out on films sufficiently thick to allow the observation of the diffraction pattern, reveled the presence of a lamellar structure with a spacing of 3.22 nm. The low intensity of the diffraction peaks and the isotropic linear optical properties of the films show that the lamellar mesophases are not extended over large areas. These findings were compared with the data obtained from AFM and SEM studies on films of two other polydiacetylenes, the poly[1-(3,6-dihexadexyl-N-carbazolyl)-6-(N-carbazolyl)-2,4-hexadyine] (polya-DCHD) and the poly[1,6-bis(3,6-dipalmitoyl-N-carbazolyl)-2,4-hexadyine] (polyDPCHD), spun on hydrophylic glass substrate. The results confirmed the presence of nodular morphologies which seem to be a general characteristic of this class of materials. The particles organization appears instead related to the chemical nature of the substituents on the carbazolyl rings.     

Inspection of nano-sized SNOM-tips by optical far-field evaluation

High resolution optical microscopy has many interesting applications in solid state physics, low temperature physics, biology and semiconductor technology. Unfortunately, the lateral resolution of conventional microscopes is limited by the Rayleigh-limit. “Scanning nearfield optical microscopy” (SNOM) seems to be a promising new approach to characterize the properties of materials optically with a high lateral resolution of 50–100 nm. The most important part of such a microscope is the scanning probe (a special glass fiber tip). However, the quality of the optical fiber tip is of decisive importance. Since the production process of pulled and coated glass fiber tips is still highly empirical and error-prone, a technique would be useful to determine the tips’ quality before they are shipped to the user or mounted in the microscope. The tips’ apertures are smaller than λ/2 and therefore they cannot be measured in a non-destructive way by conventional optical microscopy. This paper discusses an easy and fast method for the optical characterization of common glass fiber SNOM tips. The effective aperture of the tip is measured from the far-field distribution of the emitted intensity recorded by a CCD target. A numerical model is introduced to solve this inverse task and a simple optical setup is presented to detect light emitted by the tip at an angle of up to 90° from the optical axis. Experimental investigation, near/far-field calculations and scanning electron microscope investigations show the working principle of this measurement technique for the analysis and evaluation of a typical nanostructured object.     

Optical detection of near infrared femtosecond laser-heating of Er-doped ZnO-Nb2O5-TeO2 glass by green up-conversion fluorescence of Er ions

The green up-conversion fluorescence of Er³⁺ ions doped in an nonlinear optical ZnO-Nb₂O₅-TeO₂ glass was observed by using 800 nm excitation from a regenerative femtosecond (fs) Ti:Sapphire laser. The detailed analysis on two fluorescence lines at 526 nm (²H_11/2-⁴I_15/2) and 548 nm (⁴S_3/2-⁴I_15/2) revealed the fs laser heating of the multi-component TeO₂-based glass, which was possibly due to its nonlinear absorption of the host glass via the imaginary part of the third-order optical susceptibility (χ⁽³⁾). The result was compared with that of a Er³⁺-doped aluminosilicate glass under the same irradiation condition. When the fs laser was irradiated to the multicomponent TeO₂-based glass in the power density of 150 TW/cm², the laser spot was heated up to ∼520 K, which however was still less than the glass transition temperature (T_g=688 K). This technique provides a useful sensing method of laser spot temperature even inside transparent materials.     

Pure UV photoluminescence from ZnO thin film by thermal retardation and using an amorphous SiO2 buffer layer

ZnO/SiO₂ thin films were fabricated on Si substrates by E-beam evaporation with thermal retardation. The as-prepared films were annealed for 2 h every 100 °C in the temperature range 400-800 °C under ambient air. The structural and optical properties were investigated by X-ray diffraction (XRD), atomic force microscopy (AFM) and photoluminescence (PL). The XRD analysis indicated that all ZnO thin films had a highly preferred orientation with the c-axis perpendicular to the substrate. From AFM images (AFM scan size is 1 μm×1 μm), the RMS roughnesses of the films were 3.82, 5.18, 3.65, 3.40 and 13.2 nm, respectively. PL measurements indicated that UV luminescence at only 374 nm was observed for all samples. The optical quality of the ZnO film was increased by thermal retardation and by using an amorphous SiO₂ buffer layer.     

Optical and atomic force microscopic study on step bunching in BaB2O4 crystal growth

The formation of macrostep during high-temperature phase of barium meta-borate (α-BaB 2 O 4) single crystal growth has been investigated by both optical in-situ observation system and atomic force microscopy (AFM).The insitu observation results demonstrate that the critical linear size of growing facet exceeding the size that the macrostep generates is significantly anisotropic.The critical linear sizes are around 280 μm and 620 μm for {1010} and {1010} planes,respectively.AFM study illustrates that macrostep train with a height of 150 nm～200 nm is one typical morphological feature of the as-grown crystal surface.The riser of each macrostep consists of several straight and parallel sub-steps,indicating the occurrence of step bunching.Additionally,triangular sub-steps with heights of several nanometers on the treads of the macrosteps are found to be another typical feature of surface morphology,which implies a microscopically competitive bunching of sub-steps between various crystallographic orientations.