Fiber Laser Based Nonlinear Spectroscopy

Abstract

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.