Bright-field and fluorescence chip-scale microscopy for biological imaging

Optical microscopy is an essential tool in biological science and one of the gold standards for medical examinations. Miniaturization of microscopes can be a crucial stepping stone towards realizing compact, cost-effective and portable platforms for biomedical research and healthcare. This thesis reports on implementations of bright-field and fluorescence chip-scale microscopes for a variety of biological imaging applications. The term “chip-scale microscopy” refers to lensless imaging techniques realized in the form of mass-producible semiconductor devices, which transforms the fundamental design of optical microscopes.

Our strategy for chip-scale microscopy involves utilization of low-cost Complementary metal Oxide Semiconductor (CMOS) image sensors, computational image processing and micro-fabricated structural components. First, the sub-pixel resolving optofluidic microscope (SROFM), will be presented, which combines microfluidics and pixel super-resolution image reconstruction to perform high-throughput imaging of fluidic samples, such as blood cells. We discuss design parameters and construction of the device, as well as the resulting images and the resolution of the device, which was 0.66 µm at the highest acuity. The potential applications of SROFM for clinical diagnosis of malaria in the resource-limited settings is discussed.

Next, the implementations of ePetri, a self-imaging Petri dish platform with microscopy resolution, are presented. Here, we simply place the sample of interest on the surface of the image sensor and capture the direct shadow images under the illumination. By taking advantage of the inherent motion of the microorganisms, we achieve high resolution (~1 µm) imaging and long term culture of motile microorganisms over ultra large field-of-view (5.7 mm × 4.4 mm) in a specialized ePetri platform. We apply the pixel super-resolution reconstruction to a set of low-resolution shadow images of the microorganisms as they move across the sensing area of an image sensor chip and render an improved resolution image. We perform longitudinal study of Euglena gracilis cultured in an ePetri platform and image based analysis on the motion and morphology of the cells. The ePetri device for imaging non-motile cells are also demonstrated, by using the sweeping illumination of a light emitting diode (LED) matrix for pixel super-resolution reconstruction of sub-pixel shifted shadow images. Using this prototype device, we demonstrate the detection of waterborne parasites for the effective diagnosis of enteric parasite infection in resource-limited settings.

Then, we demonstrate the adaptation of a smartphone’s camera to function as a compact lensless microscope, which uses ambient illumination as its light source and does not require the incorporation of a dedicated light source. The method is also based on the image reconstruction with sweeping illumination technique, where the sequence of images are captured while the user is manually tilting the device around any ambient light source, such as the sun or a lamp. Image acquisition and reconstruction is performed on the device using a custom-built android application, constructing a stand-alone imaging device for field applications. We discuss the construction of the device using a commercial smartphone and demonstrate the imaging capabilities of our system.

Finally, we report on the implementation of fluorescence chip-scale microscope, based on a silo-filter structure fabricated on the pixel array of a CMOS image sensor. The extruded pixel design with metal walls between neighboring pixels successfully guides fluorescence emission through the thick absorptive filter to the photodiode layer of a pixel. Our silo-filter CMOS image sensor prototype achieves 13-µm resolution for fluorescence imaging over a wide field-of-view (4.8 mm × 4.4 mm). Here, we demonstrate bright-field and fluorescence longitudinal imaging of living cells in a compact, low-cost configuration.

Optical transfer of periodic microstructures by interfering femtosecond laser beams

We report on an optical interference method for transferring periodic microstructures of metal film from a supporting substrate to a receiving substrate by means of five-beam interference of femtosecond laser pulses. Scanning electron microscopy and optical microscopy revealed microstructures with micrometer-order were transferred to the receiving substrate. In the meanwhile, a negative copy of the transferred structures was induced in the metal film on the supporting substrate. The diffraction characteristics of the transferred structures were also evaluated. The present technique allows one-step realization of functional optoelectronic devices. (C) 2005 Optical Society of America.

Preparation and characterization of post-derivatives from functional polystyrene (ATRP) with p-nitroaniline-azomethine phenol and their thermal and optical study

The functional polystyrene, (Cl-PS)(2)-CHCOOCH2CH2OH ( designated as XPSt and coded P2) was prepared by ATRP at 130(0)C using CuCl and bipyridine as catalysts, 2,2-dichloro acetate-ethylene glycol (DCAG) as multifunctional initiator and THF as solvent. 4-Nitoroaniline azomethine-4' phenol (P1) as chromophores were covalently linked to the functional end groups of the polymer by using simple displacement reaction. The functional polystyrenes, namely XPSt (P2) and (PS)(2)-CHCOOCH2CH2OH, designated as X-PSt and coded P3 and their post-derivatives, namely, DXPSt (P4) and DX-PSt (P5) respectively were characterized by IR, NMR and UV spectroscopies, gel permeation chromatography (GPC) and thermogravimetric analysis (TGA) and differential scanning calorimetry (DSC), polarising optical microscopy (POM) and XRD studies. DSC showed that incorporation of chromophores in the side chains of polymers towards the polystyrene moiety increases the rigidity of the polymer and subsequently, its glass transition temperature; however the incorporation of side chain towards the alcoholic functional group decreases the glass transition temperature. The post derivatives do not play any significant role to increase the thermal stability ( TGA).

Fabrication, patterning, and optical properties of nanocrystalline YVO4 : A (A = Eu3+, Dy3+, Sm3+, Er3+) phosphor films via sol-gel soft lithography

Nanocrystalline YVO4:A (A = Eu3+, Dy3+, Sm3+, Er3+) phosphor films and their patterning were fabricated by a Pechini sol-gel process combined with soft lithography. X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FT-IR), thermogravimetric and differential thermal analysis (TG-DTA), atomic force microscopy (AFM) and optical microscopy, UV/vis transmission and absorption spectra, photoluminescence (PL) spectra, and lifetimes were used to characterize the resulting films. The results of XRD indicated that the films began to crystallize at 400 degreesC and the crystallinity increased with the increase of annealing temperatures. Transparent nonpatterned phosphor films were uniform and crack-free, which mainly consisted of grains with an average size of 90 nm. Patterned gel and crystalline phosphor film bands with different widths (5-60 mum) were obtained. Significant shrinkage and a few defects were observed in the patterned films during the heat treatment process. The doped rare earth ions (A) showed their characteristic emission in crystalline YVO4 phosphor films because of an efficient energy transfer from vanadate groups to them. The Sm3+ and Er3+ ions also showed upconversion luminescence in a YVO4 film host. Both the lifetimes and PL intensity of the rare earth ions increased with increasing annealing temperature from 400 to 800 degreesC, and the optimum concentration for Eu3+ was determined to be 7 mol % and those for Dy3+, Sm3-, and Er3+ were 2 Mol % of Y3- in YVO4 films, respectively.

THE USE OF LIGHT-MICROSCOPY FOR INVESTIGATIONS OF EPITAXIAL-GROWTH OF POLYMERS ON POLYMERIC SUBSTRATES

In order to study the oriented (epitaxial) crystallization of thermoplastic polymers on oriented polymer substrates, generally the transmission electron microscopy (TEM) is used. With this instrument, the crystallized material can easily be resolved and orientation relationships can be monitored by electron diffraction. Disadvantages are the time consuming sample preparations and difficulties in the in-situ observations of the crystallization events, because of the radiation sensitivity of the polymer crystals. It is demonstrated that these disadvantages of the TEM can be eleminated by the use of different methods of light optical contrasts under specific preparation conditions of the samples and that the optical microscopy being a supplementary method to the TEM for investigations of epitaxial crystallization.

Optical contrast in ion-implanted amorphous silicon carbide nanostructures

Topographic and optical contrasts formed by Ga+ ion irradiation of thin films of amorphous silicon carbide have been investigated with scanning near-field optical microscopy. The influence of ion-irradiation dose has been studied in a pattern of sub-micrometre stripes. While the film thickness decreases monotonically with ion dose, the optical contrast rapidly increases to a maximum value and then decreases gradually. The results are discussed in terms of the competition between the effects of ion implantation and surface milling by the ion beam. The observed effects are important for uses of amorphous silicon carbide thin films as permanent archives in optical data storage applications.

Manipulating plasmon modes in tip-enhanced Raman microscopy

The influence of polarization on the plasmon modes excited in tip-enhanced near-field optical microscopy have been investigated using the Finite Difference Time Domain Method. Analysis of the calculated results have laid particular emphasis on the ability to align local field enhancements with the orientation of molecules in order to optimize Raman signals, with particular relevance to recent experimental work on carbon nanotubes.

Ink-Jet Printing of Color Optical Filters for LCD Applications

At the age of multi-media, portable electronic devices such as mobile phones, personal digital assistant and handheld gaming systems have increased the demand for high performance displays with low cost production. Inkjet printing color optical filters (COF) for LCD applications seem to be an interesting alternative to decrease the production costs. The advantage of inkjet printing technology is to be fast, accurate, easy to run and cheaper than other technologies. In this master thesis work, we used various disciplines such as optical microscopy, rheology, inkjet printing, profilometering and colorimetry. The specific aim of the thesis was to investigate the feasibility of using company-A pigment formulation in inkjet production of COF for active matrix LCD applications. Ideal viscosity parameters were determined from 10 to 20mPa·s for easy inkjet printing at room temperature. The red pigments used are fully dispersed into the solvent and present an excellent homogenous repartition after printing. Thickness investigations revealed that the printed COF were equal or slightly thicker than typically manufactured ones. The colorimetry investigations demonstrated color coordinates very close to the NTSC red standard. LED backlighting seems to be a valuable solution to combine with the printed COF regarding to the spectrum and color analysis. The results on this thesis will increase the understanding of inkjet printing company-A pigments to produce COF for LCD applications.

Optical and Structural Properties of Zein-Xanthan Gum Biofilms

Structural and optical characteristics of zein-based films produced with different xanthan gum concentrations have been studied in this work. Scanning electronic microscopy (SEM) and optical microscopy (OM) were performed to identify if the incorporation of the material into the matrix film, formed a homogeneous structure, as well as to characterize its constituents as the colour and shape. SEM showed a homogeneous matrix for the control (0% xanthan) with good lipid distribution. However, when the samples were investigated by OM, lipids globules in the control biofilm appeared larger and more dispersed in the matrix than the others samples. Transparency/opacity test measurements by UV-VIS analysis indicated that the addition of xanthan to the film matrix lowered significantly its transparency properties Overall, the addition of xanthan gum favoured lipid dispersion in the matrix, making biomaterials more homogeneous, although with less transparency.

In vitro study of the Nd : YAG laser effect on human dental enamel: Optical and scanning electron microscope analysis

Objective: the Nd:YAG laser irradiation of dental enamel was evaluated in enamel demineralization experiments in a Streptococcus mutans culture media. Summary Background Data: Previous studies had shown that a continuous wave Nd:YAG laser at an energy of approximately 67 mJ may induce an increased acid resistance in human dental enamel when exposed to severe demineralization conditions. Methods: Enamel windows of 3 x 4 cm in the buccal surface were irradiated with a continuous wave Nd:YAG laser at a wavelength of 1,064 Ecm using energy densities of from 83.75 to 187.50 J/cm(2), Enamel windows of 3 x 4 cm on the lingual surface served as control (without the laser irradiation). The enamel windows were then exposed to a Streptococcus mutans culture media at a temperature of 37 degrees C for 15 and 21 days. The laser effects and demineralization were examined both by optical microscopy and scanning electron microscopy (SEM), Results: A comparison between the lased and the unlased windows of enamel showed fusion and recrystalization of the enamel and increased acid-resistance in all groups irradiated with the Nd:YAG laser, on the other hand, the 3 x 4 delimited enamel surfaces from the control group (not irradiated with the Nd:YAG laser) showed 100% deminerization, Conclusions: These findings are consistent with the finding that laser irradiation of dental results in significant reduction of the effective solubility of enamel mineral.

Raman Spectroscopy and Scanning Electron Microscopy Characterizations of Fission Track Method Datable Zircon Grains

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Optical properties of a nanomatch-like plasmonic structure

The optical properties of a match-like plasmonic nanostructure are numerically investigated using full-wave finite-difference time-domain analysis in conjunction with dispersive material models. This work is mainly motivated by the developed technique enabling reproducible fabrication of nanomatch structures as well as the growing applications that utilize the localized field enhancement in plasmonic nanostructures. Our research revealed that due to the pronounced field enhancement and larger resonance tunabilities, some nanomatch topologies show potentials for various applications in the field of, e.g., sensing as well as a novel scheme for highly reproducible tips in scanning near field optical microscopy, among others. Despite the additional degrees of freedom that are offered by the composite nature of the proposed nanomatch topology, the paper also reflects on a fundamental complication intrinsic to the material interfaces especially in the nanoscale: stoichiometric mixing. We conclude that the specificity in material modeling will become a significant issue in future research on functionalized composite nanostructures.