Femtosecond Pulse Temporal Overlap Estimation and Adjustment in SSFS-Based CARS System

This work is licensed under a Creative Commons Attribution 4.0 International License.We present and verify a residual-pump-based temporal overlap estimation method in soliton self-frequency shift-based coherent anti-Stokes Raman scattering system. The residual pump light, output by a highly nonlinear photonic crystal fiber, acts as a crucial link between the pump and Stokes pulses during the temporal overlap estimation. The wavelength-dependent optical delay is estimated to be 0.141 ps/nm when the Stokes wavelength is 900 nm ~1050 nm according to the temporal overlap estimation method. The actual measurement result is 0.138 ps/nm based on the nonresonant signal from a microscope slide, which is very close to the estimated result. In addition, the Raman resonant signals of liquid cyclohexane at 2853 cm -1 , 2923 cm -1 and 2938 cm -1 have also been successfully detected at the predicted optical delays 427.27 ps and 428.17 ps

Fiber Laser Based Nonlinear Spectroscopy

To date, nonlinear spectroscopy has been considered an expensive technique and confined mostly to experimental laboratory settings. Over recent years, optical-fiber lasers that are highly reliable, simple to operate and relatively inexpensive have become commercially available, removing one of the major obstacles to widespread utilization of nonlinear optical measurement in biochemistry. However, fiber lasers generally offer relatively low output power compared to lasers traditionally used for nonlinear spectroscopy, and much more careful design is necessary to meet the excitation power thresholds for nonlinear signal generation. On the other hand, reducing the excitation intensity provides a much more suitable level of user-safety, minimizes damage to biological samples and reduces interference with intrinsic chemical processes. Compared to traditional spectroscopy systems, the complexity of nonlinear spectroscopy and imaging instruments must be drastically reduced for them to become practical. A nonlinear spectroscopy tool based on a single fiber laser, with electrically controlled wavelength-tuning and spectral resolution enhanced by a pulse shaping technique, will efficiently produce optical excitation that allows quantitative measurement of important nonlinear optical properties of materials. The work represented here encompasses the theory and design of a nonlinear spectroscopy and imaging system of the simplest architecture possible, while solving the difficult underlying design challenges. With this goal, the following report introduces the theories of nonlinear optical propagation relevant to the design of a wavelength tunable system for nonlinear spectroscopy applications, specifically Coherent Anti-Stokes Spectroscopy (CARS) and Förster Resonance Energy Transfer (FRET). It includes a detailed study of nonlinear propagation of optical solitons using various analysis techniques. A solution of the generalized nonlinear Schrödinger equation using the split-step Fourier method is demonstrated and investigation of optical soliton propagation in fibers is carried out. Other numerical methods, such as the finite difference time domain approach and spectral-split step Fourier methods are also described and compared. Numerical results are contrasted with various measurements of wavelength shifted solitons. Both CARS and FRET test-bed designs and experiments are presented, representing two valuable biochemical measurement applications. Two-photon excitation experiments with a simplified calibration process for quantitative FRET measurement were conducted on calmodulin proteins modified with fluorescent dyes, as well as modified enhanced green fluorescent protein. The resulting new FRET efficiency measurements showed agreement with those of alternative techniques which are slower and can involve destruction of the sample. In the second major application of the nonlinear spectroscopy system, CARS measurement with enhanced spectral resolution was conducted on cyclohexane as well as on samples of mouse brain tissue containing lipids with Raman resonances. The measurements of cyclohexane verified the ability of the system to precisely determine its Raman resonances, thus providing a benchmark within a similar spectral range for biological materials which have weaker Raman signal responses. The improvement of spectral resolution (resonance frequency selectivity), was also demonstrated by measuring the closely-spaced resonances of cyclohexane. Finally, CARS measurements were also made on samples of mouse brain tissue which has a lipids-based Raman signature. The CARS spectrum of the lipid resonances matched well with other cited studies. The imaging of mouse brain tissue with Raman resonance contrast was also partially achieved, but it was hindered by low signal to noise ratio and limitations of the control hardware that led to some dropout of the CARS signal due to power coupling fluctuations. Nevertheless, these difficulties can be straightforwardly addressed by refinement of the wavelength tuning electronics. In conclusion, it is hoped that these efforts will lead to greater accessibility and use of CARS, FRET and other nonlinear spectral measurement instruments, in line with the promising advances in optics and laser technology

Single-fiber-laser-based multimodal coherent Raman system

Coherent Raman scattering (CRS) is an appealing technique for spectroscopy and microscopy due to its molecular specificity and the ability for 3D sectioning. However, the system usually has to use two laser sources with exactly the same repetition rate but different frequencies, which makes the setup expensive and the tuning procedure complicated. As presented in this thesis, my PhD research attempts to simplify the CRS system, and extend its capability into different applications with multiple modalities. Specifically, we have designed and developed a single-fiber-laser-based CRS spectroscopy system. Instead of using two separate lasers to provide the pump and the Stokes, we split the output of a single laser into two parts; one serves as the pump, and the other passing through a photonic crystal fiber (PCF) serves as the Stokes. The Stokes frequency shift is generated by soliton self-frequency shift (SSFS) within the PFC. By using a single fiber-laser as the source, the CRS spectroscopy system can automatically maintain pulse repetition rate synchronization between the pump and the Stokes beams, which dramatically simplifies the configuration. The impact of pulse chirping on the spectral resolution and signal power reduction of CRS has been investigated. Spectra of C-H stretches of cyclohexane induced by coherent anti-Stokes Raman scattering (CARS) and stimulated Raman scattering (SRS) were measured simultaneously and compared. The simulation results are verified through spectroscopy experiments. With minor modification, the CRS spectroscopy system can be extended into a multimodal microscopy system with the capability of performing CARS, SRS and photothermal microscopy, since they are all based on the same optical pump-probe configuration. CARS and SRS are ideal for detecting molecular vibrational mode without labeling, and photothermal microscopy is a sensitive technique most suitable for detection of light absorption by molecules that do not fluoresce. By combining these techniques into one microscopy system, not only different measurement modalities can be compared, but also offering complementary information of the sample. Distribution of hemoglobin in human red blood cells, and lipids distribution in sliced mouse brain have been imaged. Modulation frequency and power dependency of the photothermal signals are discussed in detail. The Raman gain or loss introduced through SRS is usually very weak, on the order of 10e-5, and direct detection of this small perturbation is challenging. A commonly used technique for SRS microscopy is modulating the intensity of the pump or the Stokes beam, and the stimulated Raman gain (SRG) or stimulated Raman loss (SRL) can be detected by using a lock-in amplifier synchronized with the modulating waveform. In this way, the resonant SRS signal is often accompanied by photothermal signal created through thermal lensing in the sample. The SRS signal can be easily overwhelmed by photothermal signal, especially when the modulation frequency is lower than 100KHz. We proposed a polarization stimulated Raman scattering (P-SRS) method to suppress the unwanted photothermal signal. Instead of using intensity modulated pump, we modulate the state of polarization (SOP) of pump, and the polarization-dependent SRG on the Stokes is measured. With different SOP settings, this allows the detection of different elements of the third-order susceptibility matrix. On the other hand, the photothermal signal is independent of the polarization of pump so that its impact can be eliminated. We have imaged both red blood cells and sliced mouse brain samples to demonstrate the capability of suppressing the photothermal signal from the resonant SRS signal

Towards multimodal nonlinear microscopy in clinics

Multimodal nonlinear microscopy combining two photon excited fluorescence (TPEF), second harmonic generation (SHG) and coherent anti-Stokes Raman scattering (CARS) represents a promising and powerful tool for biomedical diagnostics. The method enables label-free visualization of morphology and chemical composition of complex tissues as well as disease related changes and is as such as detailed as staining histologic methods. In this work a compact microscope utilizing novel fiber laser sources and a new approach for data analysis based on colocalization have been developed and tested for detecting various disease patterns, e.g., atherosclerosis and brain tumors.Mit Hilfe der nichtlinearen Multikontrast-Mikroskopie basierend auf den Prozessen Zweiphotonenfluoreszenz (TPEF), Frequenzverdopplung (SHG) und kohärente anti-Stokes Raman-Streuung (CARS), können Morphologie, chemische Zusammensetzung sowie krankheitsbedingte Veränderungen komplexer Gewebe label-frei analog zu histologischen Färbungen dargestellt werden. Potentiell eignet sich die Methode daher für die in vivo Bildgebung und könnte die medizinische Diagnostik entscheidend verbessern. Im Rahmen dieser Arbeit wurde ein kompaktes TPEF/SHG/CARS-Forschungsmikroskop unter Verwendung neuer Faserlaserquellen speziell für die Verwendung in der Klinik entwickelt. Dabei wurde erforscht, wie sich der Bildkontrast durch nahinfrarote Laser sowie eine hohe spektrale Auflösung verbessern lässt. Zusätzlich wurde an Methoden der Datenanalyse multispektraler CARS-Daten gearbeitet, um mittels der Kolokalisationsanalyse die Verteilung verschiedener molekularer Marker in komplexen Geweben zu visualisieren. Das Potential für klinische Anwendungen wurde an verschiedenen Krankheitsbildern wie Arteriosklerose und Tumoren des Hirns demonstriert

Nonlinear microscopy for failure analysis of CMOS integrated circuits in the vectorial focusing regime

This thesis focuses on the development of techniques for enhancing the spatial resolution and localisation precision in the sub-surface microscopy for failure analysis in semiconductor integrated circuits (ICs). Highest spatial resolutions are obtained by implementing solid immersion lenses (SIL), which provide unsurpassed numerical aperture (NA) for sub-surface microscopy. These high NA conditions mean that scalar diffraction theory is no longer valid and a vectorial focusing description should be applied to accurately describe the focal plane electric field distribution. Vectorial theory predicts that under high NA conditions a linearly polarised (LP) light focuses to a spot that is extended along the electric field vector, but radially polarised (RP) light is predicted to form a circular spot whose diameter equals the narrower dimension obtained with linear polarisation. By implementing a novel liquid-crystal (LC) radial polarisation converter (RPC) this effect was studied for both two-photon optical-beam-induced current (TOBIC) microscopy and two-photon laser assisted device alteration (2pLADA) techniques, showing a resolution and localisation improvement using the RP beam. By comparing images of the same structural features obtained using linear, circular and radial polarisations imaging and localisation resolutions both approaching 100 nm were demonstrated. The obtained experimental results were in good agreement with modelling and were consistent with theoretically predicted behaviour. Certain artefacts were observed under radial polarisation, which were thought to result from the extended depth of focus and the significant longitudinal field component. In any application these effects must be considered alongside the benefits of the symmetric field distribution in the focal plane. While SIL sub-surface microscopy offers unmatched spatial resolutions, it is prone to being severely degraded by aberrations arising from inaccurate dimensions of the SIL, imprecise substrate thickness or imperfect contact between SIL and substrate. It is in this context that techniques to identify and even mitigate aberrations in the system are important. A simple approach is demonstrated for revealing the presence of chromatic and spherical aberrations by measuring the two-photon autocorrelation of the pulses at the focal plane inside the sample. In the case of aberration free imaging, it was shown both theoretically and experimentally that the planes of the maximum autocorrelation amplitude and shortest pulse duration always coincide. Therefore, the optics of the imaging system can be first adjusted to obtain the minimum autocorrelation duration and then the wavefront of incident light modified to maximise the autocorrelation intensity, iterating this procedure until the positions of minimum pulse duration and maximum autocorrelation amplitude coincide

Supercontinuum radiation for ultra-high sensitivity liquid-phase sensing

The real-time detection of trace species is key to a wide range of applications such as on-line chemical process analysis, medical diagnostics, identification of environmentally toxic species and atmospheric pollutant sensing. There is a growing demand for suitable techniques that are not only sensitive, but also simple to operate, fast and versatile. Most currently available techniques, such as spectrophotometry, are neither sensitive enough nor fast enough for kinetic studies, whilst other techniques are too complex to be operated by the non-specialist. This thesis presents two techniques that have been developed for and applied to liquid-phase analysis, with supercontinuum (SC) radiation used for liquid-phase absorption for the first time. Firstly, supercontinuum cavity enhanced absorption spectroscopy (SC-CEAS) was used for the kinetic measurement of chemical species in the liquid phase using a linear optical cavity. This technique is simple to implement, robust and achieves a sensitivity of 9.1 × 10−7 cm−1 Hz−1/2 at a wavelength of 550nm for dye species dissolved in water. SC-CEAS is not calibration-free and for this purpose a second technique, a time-resolved variant called broadband cavity ring-down spectroscopy (BB-CRDS), was successfully developed. Use of a novel single-photon avalanche diode (SPAD) array enabled the simultaneous detection of ring-down events at multiple spectral positions for BB-CRDS measurements. The performance of both techniques is demonstrated through a number of applications that included the monitoring of an oscillating (Belousov-Zhabotinsky) reaction, detection of commercially important photoluminescent metal complexes (europium(III)) at trace level concentration, and the analysis of biomedical species (whole and lysed blood) and proteins (amyloids). Absorption spectra covering the entire visible wavelength range can be acquired in fractions of a second using sample volumes measuring only 1.0mL. Most alternative devices capable of achieving similar sensitivity have, up until now, been restricted to single wavelength measurements. This has limited speed and number of species that can be measured at once. The work presented here exemplifies the potential of these techniques as analytical tools for research scientists, healthcare practitioners and process engineers alike

Soliton dynamics in liquid-core optical fibers

Solitonen sind selbsterhaltende Wellenmuster, die in vielen dynamischen Systemen in der Natur auftreten. In der Optik ermöglichen die reichhaltigen Dynamiken von nicht- verbreiternden zeitlichen Solitonen die Generation von Superkontinuumsspektren über weite Wellenlängenbereiche. Diese Dissertation untersucht Solitondynamiken in Flüssigkernfasern theoretisch und experimentell. Einem streng empirischen Ansatz folgend werden hybride soliton-ähnliche Zustände als mögliche Lösung dieser Systeme vorgeschlagen. Wichtige Kenngrößen, wie optische Phasenbeziehungen und eine modifizierte Solitonzahl, werden als Werkzeuge zur Klassifizierung nichtinstantaner, nichtlinearer Systeme hinsichtlich ihrer Fähigkeiten zur Beherbergung von Hybrid-Solitonen gefunden und bestätigt. In dieser Arbeit werden außerdem realistische Materialmodelle erarbeitet, die die Identifizierung von Soliton-Regimen in einfach herstellbaren Flüssigkern-Fasern mit Stufenindex Design ermöglichen. Schließlich wird gezeigt, dass hybride solitonähnliche Zustände in simulierten Superkontinuumspektren für diese experimentell adressierbaren Faser- und Laserparameter auftreten. Daraufhin wird die Soliton-gestützte Superkontinuumserzeugung experimentell in Flüssigkernfasern unter Verwendung von modernsten Thuliumfaserlasern demonstriert. Im Zusammenhang mit numerischen Simulationen wird hervorgehoben, dass das ungewöhnliche Verbreitungs- und Kohärenzverhalten der gemessenen Spektren von dominanten, nichtinstantanen, nichtlinearen Effekten in Flüssigkeiten herrührt und somit die ersten positiven Hinweise für die Hypothese neuartiger Hybrid-Soliton-Dynamiken liefert. Die Studie schließt mit der experimentellen Demonstration der externen Soliton-Kontrolle über Temperatur, statischen Druck und flüssige Zusammensetzung. Dabei werden Flüssigkernfasern als dynamische Plattform für breitbandige und abstimmbare nichtlineare Lichterzeugung mit einem weitreichenden, wissenschaftlichenPotenzial hervorgehoben