Nonlinear Optical Microscopy Based On Bessel Beam And Phase Imaging

Nonlinear optical microscopy, which is based on the nonlinear optical process to form a contrast of signals, is an advanced technique for optical imaging of microscopic objects. This method provides functional information that comes from some specific molecules or chemical bonds. Compared with the conventional optical microscopy, the nonlinear optical microscopy has higher spatial resolution and deeper depth of imaging, due to the nonlinear dependence of the generated signal on the excitation intensity. In virtue of these merits, nonlinear optical microscopy plays substaintial role in biological and medical investigations. In the framework of this thesis, nonlinear optical microscopy with Bessel beam illumination and phase imaging mechanism is investigated for the purpose of enhancing the imaging depth and reducing the background noise. The following works have been completed during the Ph.D. study:1. Two-photon excitation fluorescence volume imaging(TPFVI) with a Bessel beam as an excitation source is theoretically investigated. A numerical tool for simulating the imaging process of TPFVI has been developed by combining the slice-by-slice diffraction propagation model and the angular spectrum method. By using this simulation tool, the generation of Bessel beam, its propagation in scattering media, the generation and collection of fluorescent signals, etc., have been simulated. The results reveal that the TPFVI has the advantages of deeper imaging depth and higher imaging speed, compared with the conventional two-photon excitation microscopy.2. Bessel beams are generated experimentally by a spatial light modulator(SLM), which provided a means to adjust flexibly the parameters of Bessel beams. Furthermore, Bessel beam’s propagation and the self-reconstruction in the scattering media are investigated by using the angular spectrum method combined with slice-by-slice propagation model. The investigation results provide a guide to the further experiment.3. Coherent anti-Stokes Raman Scattering(CARS) microscopy with a Bessel beam used as Pump-beam and a Gaussian beam used as Stokes-beam is proposed and investigated theoretically. Due to such specific illumination scheme, the CARS microscopy has a deeper imaging depth and a higher axial resolving power. Three-dimentional imaging can be obtained by obtaining complex amplitude distribution of the CARS field and propagating the field to the original position of the sample.4. Two kinds of single-beam phase imaging methods are investigated: the first one is quantitative phase contrast imaging, with which the contrast of the image is adjustable and quantitative phase imaging is realized by using phase-shifting operation. The second method is phase retrieval based on random-structured illumination, which combines the modulated illumination and the phase imaging, realizing the resolution improvement for the phase imaging of transparent samples.5. A wide-field CARS microscopy with phase imaging scheme is proposed and demonstrated, which allows for nonresonant background suppression in CARS imaging. Several CARS images at a few of consecutive planes perpendicular to the propagation direction are recorded to reconstruct a phase map utilizing the transport-intensity-equation or the iteration phase retrieval method. Experimental results verify that the CARS background is efficiently suppressed by the phase imaging approach, as compared to the traditional CARS imaging without background correction. The proposed background suppression method is robust against environmental disturbance, since the experimental implementation of the suggested detection scheme requires no additional modification of the standard CARS microscopy.