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Reports on the use of modeless laser as the stokes source for multiplex coherent anti-stokes Raman scattering in molecular hydrogen. Single-shot temperature recording; Chemical vapor deposition plasma diagnostics; Multiplex thermometry.

Labelfree nanoscopy encompasses optical imaging with resolution in the 100 nm range using visible wavelengths. Here, we present a labelfree nanoscopy method that combines coherent imaging techniques with waveguide microscopy to realize a super-condenser featuring maximally inclined coherent darkfield illumination with artificially stretched wave vectors due to large refractive indices of the employed Si 3 N 4 waveguide material. We produce the required coherent plane wave illumination for Fourier ptychography over imaging areas 400 μm 2 in size via adiabatically tapered single-mode waveguides and tackle the overlap constraints of the Fourier ptychography phase retrieval algorithm two-fold: firstly, the directionality of the illumination wave vector is changed sequentially via a multiplexed input structure of the waveguide chip layout and secondly, the wave vector modulus is shortend via step-wise increases of the illumination light wavelength over the visible spectrum. We test the method in simulations and in experiments and provide details on the underlying image formation theory as well as the reconstruction algorithm. While the generated Fourier ptychography reconstructions are found to be prone to image artefacts, an alternative coherent imaging method, rotating coherent scattering microscopy (ROCS), is found to be more robust against artefacts but with less achievable resolution.

We compare the performance of linear and nonlinear methods for aligning the excitation and detection planes throughout volumes of large specimens in digitally scanned light sheet microscopy. An effective nonlinear method involves the registration of four corner extrema of the imaging volume via a projective transform. We show that this improves the light collection efficiency of the commonly used three-point affine registration by an average of 42% over a typical specimen volume. Accurate illumination/detection registration methods are now pertinent to biological research in view of current trends towards imaging large or expanded samples, at depth, with diffraction limited resolution.

A theoretical framework for widefield structured illumination microscopy (SIM) reconstruction from fewer than the commonly used nine raw frame acquisitions is introduced and applied in silico and in vitro. The proposed scheme avoids the recording of redundant spatial frequency components, which was necessary in previous SIM algorithms. This allows for gentler superresolution imaging at faster speeds. A doubling of frame rates is possible solely via changes in the computational reconstruction procedure. Furthermore, we explore numerically the effect of the sample movement on the reconstruction quality and the number of raw frames recordable. Our results show that there exists a limit above which deconvolution microscopy becomes superior to SIM.

Spectrally resolved fluorescence lifetime imaging microscopy (λFLIM) has powerful potential for biochemical and medical imaging applications. However, long acquisition times, low spectral resolution and complexity of λFLIM often narrow its use to specialized laboratories. Therefore, we demonstrate here a simple spectral FLIM based on a solid-state detector array providing in-pixel histrogramming and delivering faster acquisition, larger dynamic range, and higher spectral elements than state-of-the-art λFLIM. We successfully apply this novel microscopy system to biochemical and medical imaging demonstrating that solid-state detectors are a key strategic technology to enable complex assays in biomedical laboratories and the clinic.

Localization-based super-resolution microscopy image quality depends on several factors such as dye choice and labeling strategy, microscope quality and user-defined parameters such as frame rate and number as well as the image processing algorithm. Experimental optimization of these parameters can be time-consuming and expensive so we present TestSTORM, a simulator that can be used to optimize these steps. TestSTORM users can select from among four different structures with specific patterns, dye and acquisition parameters. Example results are shown and the results of the vesicle pattern are compared with experimental data. Moreover, image stacks can be generated for further evaluation using localization algorithms, offering a tool for further software developments.

Localization based super-resolution microscopy techniques require precise drift correction methods because the achieved spatial resolution is close to both the mechanical and optical performance limits of modern light microscopes. Multi-color imaging methods require corrections in addition to those dealing with drift due to the static, but spatially-dependent, chromatic offset between images. We present computer simulations to quantify this effect, which is primarily caused by the high-NA objectives used in super-resolution microscopy. Although the chromatic offset in well corrected systems is only a fraction of an optical wavelength in magnitude (<50 nm) and thus negligible in traditional diffraction limited imaging, we show that object colocalization by multi-color super-resolution methods is impossible without appropriate image correction. The simulated data are in excellent agreement with experiments using fluorescent beads excited and localized at multiple wavelengths. Finally we present a rigorous and practical calibration protocol to correct for chromatic optical offset, and demonstrate its efficacy for the imaging of transferrin receptor protein colocalization in HeLa cells using two-color direct stochastic optical reconstruction microscopy (dSTORM).

We present a numerical study of interactions between dispersive waves (DWs) and solitons during supercontinuum generation in photonic crystal fibers pumped with picosecond laser pulses. We show how the soliton-induced trapping potential evolves along the fiber and affects the dynamics of a DW-soliton pair. Individual frequency components of the DW periodically interact with the soliton resulting in stepwise frequency blue shifts. In contrast, the ensemble blue shifts of all frequency components in the DW appear to be quasi-continuous. The step size of frequency up-conversion and the temporal separation between subsequent soliton-DW interactions are governed by the potential well which confines the soliton-DW pair and which changes in time.

Biophysical imaging tools exploit several properties of fluorescence to map cellular biochemistry. However, the engineering of a cost-effective and user-friendly detection system for sensing the diverse properties of fluorescence is a difficult challenge. Here, we present a novel architecture for a spectrograph that permits integrated characterization of excitation, emission and fluorescence anisotropy spectra in a quantitative and efficient manner. This sensing platform achieves excellent versatility of use at comparatively low costs. We demonstrate the novel optical design with example images of plant cells and of mammalian cells expressing fluorescent proteins undergoing energy transfer.

Interactions between supercontinuum (SC) light pulses, produced by the propagation of rapidly sequenced picosecond pump laser pulses along a photonic crystal fiber, result in spectral broadening, which we attribute to interpulse soliton collisions. This phenomenon was measured experimentally, following our observation of spectral broadening in numerical simulations that exhibit so-called "pulse wraparound" or "temporal aliasing." This occurs in simulations with narrow time grids: as early parts of the SC pulse leave the computational time domain, they "reenter" at the beginning and so interact with later parts of the evolving SC pulse. We show that this provides an effective model to predict the experimentally observed spectral changes.

We present an adaptive numerical filter for analyzing fiber-length dependent properties of optical rogue waves, which are highly intense and extremely red-shifted solitons that arise during supercontinuum generation in photonic crystal fiber. We use this filter to study a data set of 1000 simulated supercontinuum pulses, produced from 5 ps pump pulses containing random noise. Optical rogue waves arise in different supercontinuum pulses at various positions along the fiber, and exhibit a lifecycle: their intensity peaks over a finite range of fiber length before declining slowly.

A new approach to alias-free wide-field fluorescence lifetime imaging in the frequency domain is demonstrated using a supercontinuum source for fluorescence excitation and a phase-modulated image intensifier for detection. This technique is referred to as phi-squared fluorescence lifetime imaging (phi(2)FLIM). The phase modulation and square-wave gating of the image intensifier eliminate aliasing by the effective suppression of higher harmonics. The ability to use picosecond excitation pulses without aliasing expands the range of excitation sources available for frequency-domain fluorescence lifetime imaging (fd-FLIM) and improves the modulation depth of conventional homodyne fd-FLIM measurements, which use sinusoidal intensity modulation of the excitation source. The phi(2)FLIM results are analyzed using AB-plots, which facilitate the identification of mono-exponential and multi-exponential fluorescence decays and provide measurements of the fluorophore fractions in two component mixtures. The rapid acquisition speed of the technique enables lifetime measurements in dynamic systems, such as temporally evolving samples and samples that are sensitive to photo-bleaching. Rapid phi(2)FLIM measurements are demonstrated by imaging the dynamic mixing of two different dye solutions at 5.5 Hz. The tunability of supercontinuum radiation enables excitation wavelength resolved FLIM measurements, which facilitates analysis of samples containing multiple fluorophores with different absorption spectra.

We report on kilohertz-repetition-rate flame temperature measurements performed using blue diode lasers. Two-line atomic fluorescence was performed by using diode lasers emitting at around 410 and 451 nm to probe seeded atomic indium. At a repetition rate of 3.5 kHz our technique offers a precision of 1.5% at 2000 K in laminar methane/air flames. The spatial resolution is better than 150 microm, while the setup is compact and easy to operate, at much lower cost than alternative techniques. By modeling the spectral overlap between the locked laser and the probed indium lines we avoid the need for any calibration of the measurements. We demonstrate the capability of the technique for time-resolved measurements in an acoustically perturbed flame. The technique is applicable in flames with a wide range of compositions including sooting flames.

An optical gas sensor is presented, making use of a dispersed supercontinuum source, capable of acquiring broad bandwidth spectra at ultrahigh wavelength sweep and repetition rates. Wavelength sweeps from 1100 nm to 1700 nm can be performed in 800 ns at a spectral resolution of 40 pm. This is comparable to line-widths of molecular spectra at atmospheric pressure. Quantitative measurements are presented of CH(4) employing 80 nm wide sweeps over the P- Q- and R-branches of the 2nu(3) transition near 1665 nm, at rates exceeding 100 kHz. The effective acquisition rate is determined by the amount of averaging required, and the effect of this averaging on observed precision is investigated.

Evaluation schemes, e.g., least-squares fitting, are not generally applicable to any types of experiments. If the evaluation schemes were not derived from a measurement model that properly described the experiment to be evaluated, poorer precision or accuracy than attainable from the measured data could result. We outline ways in which statistical data evaluation schemes should be derived for all types of experiment, and we demonstrate them for laser-spectroscopic experiments, in which pulse-to-pulse fluctuations of the laser power cause correlated variations of laser intensity and generated signal intensity. The method of maximum likelihood is demonstrated in the derivation of an appropriate fitting scheme for this type of experiment. Statistical data evaluation contains the following steps. First, one has to provide a measurement model that considers statistical variation of all enclosed variables. Second, an evaluation scheme applicable to this particular model has to be derived or provided. Third, the scheme has to be characterized in terms of accuracy and precision. A criterion for accepting an evaluation scheme is that it have accuracy and precision as close as possible to the theoretical limit. The fitting scheme derived for experiments with pulsed lasers is compared to well-established schemes in terms of fitting power and rational functions. The precision is found to be as much as three timesbetter than for simple least-squares fitting. Our scheme also suppresses the bias on the estimated model parameters that other methods may exhibit if they are applied in an uncritical fashion. We focus on experiments in nonlinear spectroscopy, but the fitting scheme derived is applicable in many scientific disciplines.