Development of novel imaging tools for selected biomedical applications

Abstract

In the quest for better and faster images of cellular and subcellular structures, biology-oriented optical microscopes have advanced significantly in the last few decades. Novel microscopy techniques such as non-linear microscopy (NLM), including two-photon excited fluorescence (TPEF) and second harmonic generation (SHG) microscopy, and light-sheet fluorescence microscopy (LSFM) are emerging as alternatives that overcome some the intrinsic limitations of standard microscopy systems. In this thesis I aimed to advance such techniques even more, and combine them with other photonic technologies to provide novel tools that would help to address complex biological questions. This thesis is organized in two main parts. The first part is dedicated to applications involving femtosecond lasers that are employed for precise microsurgery. For that, damage assessment methodologies based on NLM were developed and tested in relevant biomedical models. In the second part, wavefront engineering methods were employed to enhance the imaging capabilities of light-sheet microscopy systems. These novel methodologies were tested as well in relevant biological applications. This thesis is, therefore, organized as follows: In chapter 1, a brief and comprehensive review of the basic microscopy techniques employed in this thesis is presented, together with the challenges and achievements of this thesis in sequential order. In chapter 2, a multimodal imaging methodology for the assessment of laser induced collateral damage is presented. This was specifically developed for the control of the damage in femtosecond-laser dissection of single axons within a living Caenorhabditis elegans (C. elegans). Here, it is shown that collateral damages at the level of the myosin structure of the muscles adjacent to the axon, can be readily detected. In chapter 3, the optimized multimodal methodology developed in the chapter 2 was employed for minimally invasive dissection of axons of D-type motoneurons in C elegans. Here, a microfluidic chip for C elegans immobilization and a detailed protocol was employed to evaluate the axon regeneration of such neurons. The potential of such platform for testing drugs with regeneration-enhancing capabilities is also presented. In chapter 4, a novel use of TPEF microscopy is presented to characterize and fine tune the laser for photodisruption of excised human crystalline lens samples. In chapter 5, a thorough description of the implementation of a multimodal Digital Scanned Light-Sheet Microscope (DSLM) able to work in the linear and nonlinear regimes under either Gaussian or Bessel beam excitation schemes, is presented. The enhanced capabilities of the developed system is evaluated using in vivo C. elegans samples and multicellular tumor spheroids In chapter 6, the development of a completely new concept in light sheet-based imaging is presented. This is based on the extension of the depth-of-field of the lens in the emission path of the microscope by using wavefront coding (WFC) techniques. Furthermore, I demonstrate the application of the developed methodology for fast volumetric imaging of living biological specimens and 3D particle tracking.