Integration of synthetic optical holography in a commercial confocal scanning microscope unit

Abstract

This thesis is an effort to integrate a relatively new imaging technique called Synthetic Optical Holography (SOH) in commercial confocal scanning microscopes. In this work, we specifically present an integration with a Zeiss 710 confocal microscope. We explored different integration alternatives based on cost, compatibility and functionality. Also, we validated our proof of concept by using the capabilities of this integrated system on biological samples. We successfully retrieved the phase and amplitude of cheek and stem cells. Chapter 1 provides a brief introduction to classical optical holography and its evolution since it was conceived. Subsequently, we present the ideas of Quantitative Phase Imaging (QPI) and its profound relationship with Digital Holographic Microscopy (DHM), which is an imaging technique that has been gaining popularity during the past 20 years. After that, we present the theoretical basis of SOH and its development since it was conceived. Then, we make a brief review about Confocal Microscopy (CM) and its advantages compared to wide-field microscopy. Right away, we present a briefly discussion about the efforts made in the scientific community to combine DHM and CM. Those ideas represent the conceptual frame in which SOH and confocal scanning microscopy converge. Finally, some remarks about the importance of SOH are made. In Chapter 2 we present a characterization tool based on pseudoheterodyne interferometry with the motivation to have a method to characterize the z-stages implemented in non-linear SOH. Also, we propose a Mean Squared Error (MSE) optimization algorithm in order to process the data from the experiment and to be able to estimate the amplitude and drift of the z-stages. We validate the functionality of our approach by characterizing a nanopositioning piezo stage from Physik Instrumente (PI) and a ceramic piezo electric from Thorlabs. Finally, we present a discussion and some remarks about this approach. In Chapter 3 we present a successful integration of SOH in a Zeiss 710 confocal microscope. We compare different alternatives of integration in terms of cost and compatibility. Also, we describe the process followed to implement the system, installation of the devices, preparation of the samples, etc. Then, results obtained from using this new integrated system are presented in order to validate the functionality of SOH in the confocal unit. We used SOH in the Zeiss microscope to recover the amplitude and phase of biological samples. Then, we present a discussion about the several factors that should be taken into account when trying to implement SOH in commercial microscopes such as cost, compatibility, and functionality. Finally, we discuss some remarks about this implementation. Finally, in Chapter 4, we present our conclusions about the ideas developed in Chapters 2 and 3. We also discuss the advantages and disadvantages of the SOH modular system implemented and some future work to improve the technique.