PSF-Radon transform algorithm: Measurement of the point-spread function from the Radon transform of the line-spread function.

In this article, we present a new method called point spread function (PSF)-Radon transform algorithm. This algorithm consists on recovering the instrument PSF from the Radon transform (in the line direction axis) of the line spread function (i.e., the image of a line). We present the method and tested with synthetic images, and real images from macro lens camera and microscopy. A stand-alone program along with a tutorial is available, for any interested user, in Martinez (PSF-Radon transform algorithm, standalone program). RESEARCH HIGHLIGHTS: Determining the instrument PSF is a key issue. Precise PSF determinations are mandatory if image improvement is performed numerically by deconvolution. Much less exposure time to achieve the same performance than a measurement of the PSF from a very small bead. Does not require having to adjust the PSF by an analytical function to overcome the noise uncertainties.

Combining deep learning with SUPPOSe and compressed sensing for SNR-enhanced localization of overlapping emitters.

We present gSUPPOSe, a novel, to the best of our knowledge, gradient-based implementation of the SUPPOSe algorithm that we have developed for the localization of single emitters. We study the performance of gSUPPOSe and compressed sensing STORM (CS-STORM) on simulations of single-molecule localization microscopy (SMLM) images at different fluorophore densities and in a wide range of signal-to-noise ratio conditions. We also study the combination of these methods with prior image denoising by means of a deep convolutional network. Our results show that gSUPPOSe can address the localization of multiple overlapping emitters even at a low number of acquired photons, outperforming CS-STORM in our quantitative analysis and having better computational times. We also demonstrate that image denoising greatly improves CS-STORM, showing the potential of deep learning enhanced localization on existing SMLM algorithms. The software developed in this work is available as open source Python libraries.

Super-resolved edge detection in optical microscopy images by superposition of virtual point sources.

A new approach to the edge detection problem is presented which is specially designed to achieve high accuracy detection, below instrumental resolution (super resolution) in microscopy images. The method is based in a modified version of a recently published algorithm known as SUPPOSe, which performs a numerical reconstruction of an image as a superposition of virtual point sources. The method was tested in simulated and experimental optical microscopy images and compared to the standard Laplacian of Gaussian algorithm, showing huge differences when the size of the object is smaller than the lateral resolution provided by the instrument.

Device for real-time monitoring of oil-in-water and suspended solids based on thermal lens spectrometry and light scattering.

A novel system suitable for simultaneous monitoring of both oil-in-water and suspended solids based on thermal lens spectroscopy and forward light scattering is presented. The technique measures the concentration of dissolved hydrocarbons and simultaneously detects single oil droplets and suspended particles separately. The device was tested with injection water samples from an on-field water treatment plant, and hydrocarbon concentrations were measured with a precision better than 5% in the range of up to 100 ppm, reaching resolutions as low as 0.03 ppm. Particle detection was tested with model samples of dyed and undyed polystyrene spheres acting as absorption and scattering centers, which simulated oil droplets and suspended solids, respectively. We show that particles of different sizes are distinguished by the magnitude of the perturbations introduced in the signals, and their concentrations can be measured independently of dissolved components.

Characterization of absorptance homogeneity in thin-film coatings for high-power lasers by thermal lensing microscopy.

A focus error method photothermal microscope was designed for the characterization of absorptance homogeneity in thin-film coatings for high-power lasers. The technique relies on the detection of the thermal lens induced by the local absorption of a light power focused laser. The detailed design of the instrument is presented. The resolution of the system is better than 0.1 ppm and allows the realization of spatial sweeps and even measurements of the evolution of absorption as a function of time with a spatial resolution of 1 µm. These capabilities allow the location of defects and their characterization.

Three-dimensional confocal Raman temperature characterization of electrokinetically pumped microchannels.

A novel method for noninvasive, three-dimensional temperature characterization in microfluidic devices is presented. A specially designed confocal microscope was built and used to measure water temperature by sensing the Raman spectrum variations of the liquid. This is achieved by splitting the spectrum in the isosbestic point and detecting it with two photon counters. The difference between the signals of each detector divided by their sum shows a linear dependence with temperature. A fiber-coupled laser beam is used to pump the sample with 25 mW of optical power at 405 nm. This allows a 0.8 K temperature precision and a 9 µm axial resolution using a 1 s integration time. These features make temperature profiling in all dimensions possible, in contrast with previous methods, where the information present in the height of the channel is lost and the whole spectrum needs to be recovered before computing the sample temperature. Using this technique, different geometries of polydimethylsiloxane microchannels sealed with a 150 µm thick glass coverslip were studied, showing that heat flow through the glass is the dominating dissipation mechanism and defines the maximum temperature in the channel. The results show good agreement with previous work found in the literature.

Superresolution method for a single wide-field image deconvolution by superposition of point sources.

In this work, we present a new algorithm for wide-field fluorescent micrsocopy deconvolution from a single acquisition without a sparsity prior, which allows the retrieval of the target function with superresolution, with a simple approach that the measured data are fit by the convolution of a superposition of virtual point sources (SUPPOSe) of equal intensity with the point spread function. The cloud of virtual point sources approximates the actual distribution of sources that can be discrete or continuous. In this manner, only the positions of the sources need to be determined. An upper bound for the uncertainty in the position of the sources was derived, which provides a criteria to distinguish real facts from eventual artefacts and distortions. Two very different experimental situations were used for the test (an artificially synthesized image and fluorescent microscopy images), showing excellent reconstructions and agreement with the predicted uncertainties, achieving up to a fivefold improvement in the resolution for the microscope. The method also provides the optimum number of sources to be used for the fit. LAY DESCRIPTION: A new method is presented that allows the reconstruction of an image with superresolution from a single frame taken with a standard fluorescent microscope. An improvement in the resolution of a factor between 3 and 5 is achieved depending on the noise of the measurement and how precisely the instrument response function (point spread function) is measured. The complete mathematical description is presented showing how to estimate the quality of the reconstruction. The method is based in the approximation of the actual intensity distribution of the object being measured by a superposition of point sources of equal intensity. The problem is converted from determining the intensity of each point to determining the position of the virtual sources. The best fit is found using a genetic algorithm. To validate the method several results of different nature are presented including an artificially generated image, fluorescent beads and labelled mitochondria. The artificial image provides a prior knowledge of the actual system for comparison and validation. The beads were imaged with our highest numerical aperture objective to show method capabilities and also acquired with a low numerical aperture objective to compare the reconstructed image with that acquired with a high numerical aperture objective. This same strategy was followed with the biological sample to show the method working in real practical situations.

Investigation of laser annealing mechanisms in thin film coatings by photothermal microscopy.

We study the evolution of the absorptance of amorphous metal oxide thin films when exposed to intense CW laser radiation measured using a photothermal microscope. The evolution of the absorptance is characterized by a nonexponential decay. Different models that incorporate linear and nonlinear absorption, free carrier absorption, and defect diffusion are used to fit the results, with constraints imposed on the fit parameters to scale with power and intensity. The model that best fits is that two types of interband defects are passivated independently, one by a one-photon process and the other one by a two-photon process.

Thin film absorption characterization by focus error thermal lensing.

A simple, highly sensitive technique for measuring absorbed power in thin film dielectrics based on thermal lensing is demonstrated. Absorption of an amplitude modulated or pulsed incident pump beam by a thin film acts as a heat source that induces thermal lensing in the substrate. A second continuous wave collimated probe beam defocuses after passing through the sample. Determination of absorption is achieved by quantifying the change of the probe beam profile at the focal plane using a four-quadrant detector and cylindrical lenses to generate a focus error signal. This signal is inherently insensitive to deflection, which removes noise contribution from point beam stability. A linear dependence of the focus error signal on the absorbed power is shown for a dynamic range of over 105. This technique was used to measure absorption loss in dielectric thin films deposited on fused silica substrates. In pulsed configuration, a single shot sensitivity of about 20 ppm is demonstrated, providing a unique technique for the characterization of moving targets as found in thin film growth instrumentation.

Efficacy of a vaccine formula against tuberculosis in cattle.

"Test-and-slaughter" has been successful in industrialized countries to control and eradicate tuberculosis from cattle; however, this strategy is too expensive for developing nations, where the prevalence is especially high. Vaccination with the Calmette-Guérin (BCG) strain has been shown to protect against the development of lesions in vaccinated animals: mouse, cattle and wildlife species. In this study, the immune response and the pathology of vaccinated (BCG-prime and BCG prime-CFP-boosted) and unvaccinated (controls) calves were evaluated under experimental settings. A 10(6) CFU dose of the BCG strain was inoculated subcutaneously on the neck to two groups of ten animas each. Thirty days after vaccination, one of the vaccinated groups was boosted with an M. bovis culture filtrate protein (CFP). Three months after vaccination, the three groups of animals were challenged with 5×10(5) CFU via intranasal by aerosol with a field strain of M. bovis. The immune response was monitored throughout the study. Protection was assessed based on immune response (IFN-g release) prechallenge, presence of visible lesions in lymph nodes and lungs at slaughter, and presence of bacilli in lymph nodes and lung samples in histological analysis. Vaccinated cattle, either with the BCG alone or with BCG and boosted with CFP showed higher IFN-g response, fewer lesions, and fewer bacilli per lesion than unvaccinated controls after challenge. Animals with low levels of IFN-g postvaccine-prechallenge showed more lesions than animals with high levels. Results from this study support the argument that vaccination could be incorporated into control programs to reduce the incidence of TB in cattle in countries with high prevalence.

Detection of low quantum yield fluorophores and improved imaging times using metallic nanoparticles.

The behavior of a fluorophore near a gold nanoparticle is rationalized by a theoretical description of the parameters that modify the fluorescence emission: nanoparticle-fluorophore distance, fluorescence quantum yield (φ(0)), and fluorophore absorption and emission spectra, to find optimum conditions for designing fluorophore-nanoparticle probes. The theoretical maximum gain in brightness of the nanoparticle-fluorophore system with respect to the isolated molecule increases almost inversely proportional to φ(0). The brightness enhancement in imaging experiments in vitro was assessed by using Au-SiO(2) core-shell nanoparticles deposited on glass. A ~13-fold emission brightness enhancement for weakly fluorescent molecules was observed. A significant increase in fluorophore photostability, rendering longer imaging times, was obtained for fluorophores interacting with gold nanoparticles incorporated by endocytosis in cells. Our results illustrate a way to increase imaging times and to study molecules in the vicinity of a metallic nanoparticle after photobleaching of background fluorescence.

Inhibitors of MAPK pathway ERK1/2 or p38 prevent the IL-1{beta}-induced up-regulation of SRP72 autoantigen in Jurkat cells.

Phosphorylation is the most important post-translational event at a cellular level that is regulated by protein kinases. MAPK is a key player in the important cellular signaling pathway. It has been hypothesized that phosphorylation might have a role in the induction of break tolerance against some autoantigens such as SRP72. The aim of this study was to explore the pathways of phosphorylation and overexpression of the SRP72 polypeptide, using an in vitro model of Jurkat cells stimulated by recombinant human (rh)IL-1ß in the presence of MAPK inhibitors. We used Jurkat cells as a substrate stimulated with rhIL-1ß in the presence of MAPK inhibitors at different concentrations in a time course in vitro experiment by immunoprecipitation, immunoprecipitation-Western blotting, and real time PCR. Our results showed that rhIL-1ß causes up-regulation of protein expression and phosphorylation of SRP72 in Jurkat cells. Inhibitors of the MAPK pathway ERK1/2 or p38α/ß down-regulate the expression of SRP72 autoantigen in Jurkat cells stimulated by rhIL-1ß. Our results highlight the importance of studying the pathways of activation and overexpression of autoantigens. It will be necessary to perform careful research on various kinases pathways, including MAPK in dermatomyositis and other rheumatic diseases, to help to explain the routes of activation and inhibition of autoantigens. The understanding of this process may help to develop new therapies to prevent and control the loss of tolerance toward own normal proteins.

Single photon fluorescent microlithography for live-cell imaging.

Using fluorescent dyes to trigger the polymerization of a commercial polyurethane resin allows a rapid fabrication of micrometer and submicrometer sized fluorescent structures by one-photon absorption. Here, we show that standard He-Ne lasers emitting at 632.8 nm can be used to start the photopolymerization and that very low laser power is required. This procedure allows the fabrication of fiduciary fluorescent references on standard glass coverslips, mica sheets, or gold-coated coverslips for laser scanning or standard fluorescent microscopy. The biocompatibility of the polymerized resin with cells in culture was tested by growing Xenopus melanophores and a standard laser scanning microscope was used to demonstrate that it is possible to use equipment readily available in several laboratories. We show that fluorescent structure with less than 10 nm in height may be used as references in fluorescence microscopy allowing a smooth environment for cell growth. Different dyes were tested and the conditions for one-photon polymerization were outlined.

Photothermal measurement of absorption and scattering losses in thin films excited by surface plasmons.

We present a novel noncontact, photothermal technique, based on the focus error signal of a commercial CD pickup head that allows direct determination of absorption in thin films. Combined with extinction methods, this technique yields the scattering contribution to the losses. Surface plasmon polaritons are excited using the Kretschmann configuration in thin Au films of varying thickness. By measuring the extinction and absorption simultaneously, it is shown that dielectric constants and thickness retrieval leads to inconsistencies if the model does not account for scattering.

Femtosecond soliton source with fast and broad spectral tunability.

We present a complete set of measurements and numerical simulations of a femtosecond soliton source with fast and broad spectral tunability and nearly constant pulse width and average power. Solitons generated in a photonic crystal fiber, at the low-power coupling regime, can be tuned in a broad range of wavelengths, from 850 to 1200 nm using the input power as the control parameter. These solitons keep almost constant time duration (approximately 40 fs) and spectral widths (approximately 20 nm) over the entire measured spectra regardless of input power. Our numerical simulations agree well with measurements and predict a wide working wavelength range and robustness to input parameters.

10000 times volume reduction for fluorescence correlation spectroscopy using nano-antennas.

We present an experimental and theoretical study of a new scheme for Near-Field Fluorescence Correlation Spectroscopy that, using the field enhancement by optical nanoantennas, allows the reduction of the observation volume 4 orders of magnitude below the diffraction limit. This reduction can be used in two different ways: to increase the sample concentration and to improve the spatial resolution of the dynamics under study. Our experimental results using individual gold nanoparticles and a 150 microM Rose Bengal solution in glycerol confirm the volume reduction.

Distance and orientation measurement in the nanometric scale based on polarization anisotropy of metallic dimers.

We show that the orientation of a dimer and the distance between the nanoparticles that form it can be determined by measuring the scattering under polarized light illumination. Scattering microscopy has shown to be an alternative to fluorescence as it provides nonbleaching and highly biocompatible probes, that can be manufactured in different sizes with different ligands. We propose a method based on measuring the polarization anisotropy of metallic dimers to determine distances in the range from 10 nm to 200 nm, thus filling the gap between fluorescence resonance energy transfer (FRET) and conventional microscopy. By calculating the scattering cross section of metallic dimers we show that it is also possible to gather orientation information, relevant to understand many biological processes.

Two-photon fluorescent microlithography for live-cell imaging.

Fluorescent dyes added to UV-cure resins allow the rapid fabrication of fluorescent micropatterns on standard glass coverslips by two-photon optical lithography. We use this lithographic method to tailor fiduciary markers, focal references, and calibration tools, for fluorescence and laser scanning microscopy. Fluorescent microlithography provides spatial landmarks to quantify molecular transport, cell growth and migration, and to compensate for focal drift during time-lapse imaging. We show that the fluorescent patterned microstructures are biocompatible with cultures of mammalian cell lines and hippocampal neurons. Furthermore, the high-relief topology of the lithographed substrates is utilized as a mold for poly(dimethylsiloxane) stamps to create protein patterns by microcontact printing, representing an alternative to the current etching techniques. We present two different applications of such protein patterns for localizing cell adhesion and guidance of neurite outgrowth.

Impulsive stimulated Raman scattering: comparison between phase-sensitive and spectrally filtered techniques.

Impulsive stimulated Raman scattering is used to generate and control coherent phonons and other low-frequency modes. In transparent materials, pump-probe experiments are usually performed by spectrally resolving the probe beam and measuring the spectral shift as a function of pump-probe time delay. By measuring the optical phase of the probe pulse as a function of time delay, we find that the phonon signal can be increased by a factor alpha(omegadelta)(-1), where omega is the phonon frequency and delta is the pulse duration.

Calibration of subnanometer motion with picometer accuracy.

We have developed a method for calibrating subnanometer movements of a piezoelectric actuator with picometer accuracy and for a wide range of frequencies. This range make this calibration useful for scanning probe microscopes, particularly for an apertureless scanning near-field optical microscope in which the tip is dithered to modulate the optical signal. The setup consists of a Michelson interferometer that has a mobile arm capable of moving more than one fringe. The piezoelectric actuator to be calibrated vibrates at the desired frequency in the other arm. Net displacement can be calculated by simultaneous measurement of an interferometric signal and its derivative. Hysteresis of the system can be also measured. It will be shown that the actuator response is linear only for the low-frequency region (in our case as much as approximately 10 kHz). Above that frequency range, higher harmonics appear and cannot be neglected to obtain real displacement. Finally, it will be shown that the use of higher harmonics in calibration or detection schemes (that rely on the linearity of the response) must be validated, and this technique has proved adequate for that purpose.