Polarization second harmonic imaging of biological samples
- Autores: Sotirios Psilodimitrakopoulos
- Directores de la Tesis: Pablo Loza Álvarez (dir. tes.), David Artigas García (codir. tes.)
- Lectura: En la Universitat Politècnica de Catalunya (UPC) ( España ) en 2012
- Idioma: inglés
- Tribunal Calificador de la Tesis: Sophie Brasselet (presid.), Niek F. Van Hulst (secret.), Marco Lombardo (voc.)
- Materias:
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- Resumen
The integrative approach of understanding how molecules collaborate to create complex biological systems requires the observation of molecular architectures, in living organisms. We need to know how components of living cells are organized, how they interact, how they are formed and eliminated at various stages of the life cycle of a whole organism. While significant technological advances have greatly improved the capabilities of ultrasound, magnetic resonance, computed tomography, and positron emission tomography imaging, these modalities are practically limited to resolutions of ~1mm. However, much higher spatial resolution (1µm or less) is required to visualize structural changes associated with diseased cells and tissues. Therefore, a clear need remains for high-resolution quantitative optical microscopy approaches to be performed either in vivo, or ex vivo on intact tissues. The contribution of this thesis to this issue is to investigate whether polarization sensitive second harmonic generation (PSHG) imaging can accurately classify the content and structure and differentiate normal and abnormal tissue. This study is aiming to revolutionize different disciplines in biology and in biomedical optics research, since it offers the means for high-resolution minimally invasive imaging of biological samples. After a brief review of the available optical microscopy techniques for biological imaging, in Chapter 2, we describe the harmonic up conversion from biological arrangements of molecules. A three dimensional PSHG biophysical model adapted to the experimental apparatus is then developed. This forms the basis for extracting structural information on the architecture of the SHG active molecules and their supporting filaments. Then, the two alternative procedures to retrieve information of the PSHG images are presented, which are based on iterative fitting algorithm and a fast Fourier transform (FF-PSHG). Finally in this chapter, the setup used to acquire all the PSHG images of this dissertation is presented. In particular, the PSHG microscopy and biophysical model allow retrieving structural information on SHG active species with pixel resolution, without any predefined alignment and without any specimen rotation. The above enables to gain information inaccessible to only intensity SHG imaging. In Chapter 3 we present applications of high resolution PSHG imaging concerning starch, collagen, muscle and axons microtubules. PSHG of starch and of commercial collagen type I from tendons provided the means of exploring the response of the setup to PSHG measurements in both forward and backwards detection geometries. PSHG imaging was applied in vivo to the model organism nematode C. elegans and in vitro to cortical neurons. In this Chapter we also demonstrate how PSHG reveals the (2) elements ratios and consequently the mean effective orientation in SHG active molecules. Finally, in Chapter 4 we start by demonstrating pixel-by-pixel discrimination between collagen and muscle in the same image. Implementation of the 3D-PSHG analysis is then presented for dry and hydrated starch granules. The PSHG results correlate shifts in the peak and changes in the width of the image histograms of the PSHG parameters with structural changes and the degree of organization of the SHG active molecules, respectively. Finally, we demonstrate for the first time the use of the PSHG technique for quantifying changes between healthy and ischemic neurons.
