Graduate Thesis Or Dissertation
 

Applications of Holographic Optical Tweezers: Multiplexed Fluorescence Spectroscopy and the Micromechanics of Type-I Collagen

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  • The development and some applications of holographic optical tweezers (HOT) are presented. Our HOT system uses a spatial light modulator (SLM) to control the location and properties of the optical trap. We have developed a method for optimizing the diffraction efficiency of a SLM that can be applied in situ and addresses the issues of nonlinear phase modulation and phase modulation less than 2π. The method employs a one-dimensional blazed phase grating written on the SLM. For an ideal SLM, the phase shift is linear and covers 0-2π, yielding a first-order diffraction efficiency of unity. For a realistic SLM with nonlinear or reduced phase shift, the efficiency is approximately η =1 − σ², where σ² is the variance of the phase error from the ideal case. Because each pixel contributes to the phase error independently, this suggests a method to maximize the efficiency by adjusting the phase encoding of the SLM pixel-by-pixel. In practice, we do this by adjusting the gray-scale of each pixel while measuring the first-order diffracted power. The collection of optimal gray values comprises the optimized gray-scale lookup table, which exhibits the nonlinearity required to produce a linear phase grating and the saturated phase encoding that maximizes the efficiency of phase limited SLMs. The optimized SLM enables strong trapping power, even when distributed among multiple traps, which is essential to enable our system to trap multiple nanosensors and simultaneously detect the sensors’ fluorescence spectra with an imaging spectrometer. Such nanosensors are capable of detecting changes in their environment such as pH, ion concentration, temperature, and voltage by monitoring changes in the nanosensors’ emitted fluorescence spectra. We have used streptavidin labeled quantum dots bound to the surface of biotin labeled polystyrene microspheres to measure temperature changes by observing a corresponding shift in the wavelength of the spectral peak, which is excited with a 532 nm wide field laser source. Particles with diameter greater than the wavelength of light exhibit Mie resonances in their fluorescence spectrum whose spectral locations are dependent on the size of the particle and the relative index of refraction between the particle and the surrounding medium. HOT also provides a useful platform to study the micromechanical properties of elastic materials such as collagen. Collagen gels are widely used in experiments on cell mechanics because collagen is the most abundant protein in the mammalian extracellular matrix and is the primary source of its mechanical properties. Collagen gels are often approximated as homogeneous elastic materials; however, variations in the collagen fiber microstructure and cell adhesion forces cause the mechanical propertiesto be inhomogeneous at the cellular scale. We study the mechanics of type I collagen on the scale of tens to hundreds of microns by using HOT to apply picoNewton forces to micron-sized particles embedded in the collagen fiber network. We measure the local compliance and elastic modulus of the collagen network and find that particle displacements are inhomogeneous, anisotropic and asymmetric. Confocal reflection microscopy is used to reveal the local fiber structure and a simulation treating the network as a triangular lattice is used for comparison to the HOT measurements. Collagen samples prepared at 21◦C and 37◦C show that gels formed at lower temperature are more inhomogeneous, anisotropic, and compliant than those formed at high temperature, and cellularized samples allow us to characterize the effects of cell adhesion forces on the network mechanics.
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