Multiphoton Microscopy for Ophthalmic Imaging

We review multiphoton microscopy (MPM) including two-photon autofluorescence (2PAF), second harmonic generation (SHG), third harmonic generation (THG), fluorescence lifetime (FLIM), and coherent anti-Stokes Raman Scattering (CARS) with relevance to clinical applications in ophthalmology. The different imaging modalities are discussed highlighting the particular strength that each has for functional tissue imaging. MPM is compared with current clinical ophthalmological imaging techniques such as reflectance confocal microscopy, optical coherence tomography, and fluorescence imaging. In addition, we discuss the future prospects for MPM in disease detection and clinical monitoring of disease progression, understanding fundamental disease mechanisms, and real-time monitoring of drug delivery

Improved resolution and speed in nonlinear microscopy

Department Head: Anthony A. Maciejewski.2010 Spring.Includes bibliographical references (pages 115-124).Optical microscopy is an important tool for biomedical research. New techniques for microscopy enable new capabilities for studying biological systems. Moreover, in optical microscopy, the polarization state of the focal field strongly influences the images formed due to the impact of focal spot size, adjusting the relative strength and phase of both transverse and longitudinal field components, and manipulating inter- action with the sample under study. In particular, coherent nonlinear microscopies, such as third harmonic generation (THG), and second harmonic generation (SHG), offer rich possibilities for new control over the imaging process. In the first part of this dissertation, I demonstrate that control over the spatial polarization state of the focal field can be used to improve the spatial resolution in a laser-scanning THG microscopy. First, we show a detailed design of our nonlinear scanning microscope, then we introduce a non-iterative algorithm for measurement of spatially inhomogeneous polarization distributions in third-harmonic generation microscopy. We also, show control of spatial polarization state of the focal field through imaging of a spatial light modulator to the focus of a microscope objective. Then, we introduced a novel technique for enhancing resolution in THG microscopy, through spatial polarization shaping at the focal field. In the second part of this dissertation, we show an alternative method to laser- scanning nonlinear microscopy in biological tissue, namely, nonlinear holographic microscopy. First, we introduce the foundation of nonlinear holographic microscopy by reviewing linear off-axis holography. We start by introducing digital recording in off- axis holography, its limitations, and show how through holography we can obtain 3-D images from 2-D data. We then explore numerical reconstruction of the object field from the recorded holograms. Finally, we expand this technique to SHG nonlinear holographic microscopy to construct 3-dimensional images of biological tissues

Differential modulation of the molecular dynamics of the type IIa and IIc sodium phosphate cotransporters by parathyroid hormone.

The kidney is a key regulator of phosphate homeostasis. There are two predominant renal sodium phosphate cotransporters, NaPi2a and NaPi2c. Both are regulated by parathyroid hormone (PTH), which decreases the abundance of the NaPi cotransporters in the apical membrane of renal proximal tubule cells. The time course of PTH-induced removal of the two cotransporters from the apical membrane, however, is markedly different for NaPi2a compared with NaPi2c. In animals and in cell culture, PTH treatment results in almost complete removal of NaPi2a from the brush border (BB) within 1 h whereas for NaPi2c this process in not complete until 4 to 8 h after PTH treatment. The reason for this is poorly understood. We have previously shown that the unconventional myosin motor myosin VI is required for PTH-induced removal of NaPi2a from the proximal tubule BB. Here we demonstrate that myosin VI is also necessary for PTH-induced removal of NaPi2c from the apical membrane. In addition, we show that, while at baseline the two cotransporters have similar diffusion coefficients within the membrane, after PTH addition the diffusion coefficient for NaPi2a initially exceeds that for NaPi2c. Thus NaPi2c appears to remain "tethered" in the apical membrane for longer periods of time after PTH treatment, accounting, at least in part, for the difference in response times to PTH of NaPi2a versus NaPi2c